Preparation, Preservation and Translational Research

#### **Chapter 3**

## Stem Cell-Derived Exosomes as New Horizon for Cell-Free Therapeutic Development: Current Status and Prospects

*Devashree Vakil, Riddhesh Doshi, Flyn Mckinnirey and Kuldip Sidhu*

#### **Abstract**

Exosomes have come a long way since they were first described in 1981 by Trams et al. as small lipid bilayer-enclosed vesicles of endocytic origin. Their ability to alter cell bioactivity combined with their advancing popularity as disease biomarkers and therapeutic delivery systems has compelled major Government institutions and regulatory authorities to invest further in this ever-growing field of research. Being relatively new, exosome research is besieged by challenges including but not limited to inefficient separation methods and preservation techniques, difficulties in characterization, and lack of standardized protocols. However, as excitement and research on exosomes increase, their relevance and capacity to elicit a distinct biological response is reinforced. Therefore, it is pertinent to further explore their potential as cell-free therapeutics. This review focuses on current difficulties and subsequent strategies to refine existing methodologies for efficient clinical translation of exosomes in a streamlined and cost-effective manner. The chapter is briefly divided into subsections, each relevant for sequential therapeutic development such as their classification, isolation, scaling up, storage, characterizations, regulatory requirements, therapeutic developments, and perspectives. Apart from literature search, we have endeavored to bring in our own experience in this field including some recent clinical developments.

**Keywords:** mesenchymal stem cells, exosomes, exosome characterization, signalosomes, assays

#### **1. Introduction**

This chapter reiterates the central dogma that mesenchymal stem cells (MSCs) ameliorate disease not just by virtue of their differentiation and self-renewal abilities, but in a paracrine manner, by secreting anti-inflammatory, immunomodulatory, and regenerative factors. Among these paracrine mediators, nanosized extracellular vesicles "exosomes" have generated supreme interest, owing to reports of their standalone therapeutic effect. Stem cell exosomes can circumvent the safety risks associated with the administration of cell therapy.

#### **2. History and evolution of exosomes**

A ground-breaking study by Chargaff and West in 1946 [1] for the first time detailed the phenomenon of plasma membrane fragments being shed off viable cells and forming "high particle weight lipoproteins." It was not until two decades later, when the vesicular particles isolated from body fluids were given some attention. Initially disregarded as artifacts of the separation technique [2], these were later believed to be associated with viruses [3]. Wolf and Prince then identified the usefulness of these serum-isolated extracellular vesicles and termed them "phospholipid-rich platelet dust" that could essentially be separated out by ultracentrifugation [4]. It was only in 1975, when particles of 30 to 60 nm diameter, containing an electron-dense core enveloped by a membrane, were recognized as microvesicles, and were firmly established as "breakdown products of normal cellular components," thus freeing them from any association with viruses' [5]. In 1981, Trams and co-workers coined the term "exosomes" for microvesicles harvested from tissue culture supernatant [6]. The exact physiological function of exosomes remained unknown, but reports of specific plasma membrane domains within sparked interest. In 1983, the phenomenon of formation and release of cellular vesicles by exocytosis was outlined by the works of Stahl and Johnstone, respectively [7, 8]. These reports individually identified exosomes as 50-nm spheres displaying receptors on their external surface and originating from a "non-lysosomal endocytic compartment." By mid to late 80s, the term exosome had caught on [9], and "exosome secretion pathway" was acknowledged for the existence of a novel intracellular trafficking pathway *via* shedding of cell membrane [10, 11]. In 1991, Johnstone identified cellular stress as the primary factor to aid internalization and shedding of archaic components of the plasma membrane in the form of exosomes, the mechanism of which was not yet known [12]. However, it was Johnstone's pivotal paper in 2005 [13], which underlined the biological significance of exosomes, establishing for the first time a fate for them that was beyond cellular waste. Following that, the field saw an exponential increase in exosome research including scale-up processes, use of direct modifications, and genetic engineering, paving the way for regenerative therapy. **Figure 1**, adapted from a landmark article in the field, depicts a timeline on exosome [14].

#### **Figure 1.**

*Evolution and timeline of research conducted on exosomes. A brief timeline of when EVs were discovered, and coining of the term "exosomes."*

*Stem Cell-Derived Exosomes as New Horizon for Cell-Free Therapeutic Development: Current… DOI: http://dx.doi.org/10.5772/intechopen.108865*

#### **3. Classification of exosomes**

An inherent size overlap between distinct EV subtypes poses a challenge for characterization solely based on size [15]; however, the presence of markers associated with EV origin can further assist classification. Based on size, either small or large EV categories comprise of five major populations: exomeres, exosomes, migrasomes, apoptotic bodies, and large oncosomes [16]. The largest among these are apoptotic bodies, with a diameter > 800nm (can go up to 5 μM), and consist of plasma membrane and cytoplasmic components of post-apoptotic (dying) cells. Smaller than these are microvesicles, or ectosomes ranging in size from 100 nm to 1 μm. These originate from an irregular blebbing of the plasma membrane. The smallest EVs, exosomes generate from multi-vesicular endosomes, and contain proteins, lipids, and nucleic acids [17, 18]. Their size is much under debate, with smallest exosomes at 30 nm and largest anywhere between 150 and 200 nm. Since exosomes are secreted only upon environmental and physiological cues like cellular stress, their selectively integrated cargo carries specific instructions for modulating target cells. Hence, this EV subclass is often referred to as "signalosomes." Interestingly, microvesicles and exosomes are both released by non-apoptotic cells (**Table 1**).

#### **4. Exosome morphology**

Dehydration during sample preparation in conventional electron microscopic techniques forces exosomes to reveal a cup-shaped structure, and they appear as flattened spheres [19, 20]. Cryo-electron microscopy helps exosomes remain fully hydrated and enables exosomes to retain a proper spherical morphology, thus it is a superior technique [21].


*Exosomes are the only EV class that originate through multivesicular body formation via endocytosis. Endosome-derived EVs are generally referred to as 'exosomes' throughout this chapter and may be interchangeably used with the term 'EVs'.*

**Table 1.** *EV classification.*

#### **5. Biogenesis, release, and uptake of exosomes**

Exosome biogenesis and their secretion involve a complex molecular pathway and exchange of material which is tightly regulated by each source cell [22, 23]. Cells secrete exosomes at different rates, depending on their type, metabolism, and other factors. The first step in this multistep pathway is invagination of the plasma membrane *via* endocytosis [24], forming endosomes, which mature to late endosome, also known as Multivesicular Bodies (MVBs), containing a population of ILVs (Intraluminal Endosomal Vesicles) [24]. The final fate of MVBs is to either i) undergo degradation *via* lysosomes or ii) fuse with plasma membrane and release the ILVs as exosomes into extracellular space [25–27]. Most crucial step is the process of channeling and deposition of a specific subset of proteins (including tetraspanins and some endosomal proteins), lipids (ceramide), and other macromolecules into the ILVs, for which the Endosomal Sorting Complex Required for Transport (ESCRT) pathway is recruited [28–30]. The ESCRT pathway is an intricate, Adenosine Tri Phosphate (ATP)-dependent process involving the use of four complexes—ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III along with associated proteins Tsg101 and Alix among others [31–33]. Alternatively, the sorting of exosomal content and biogenesis may occur *via* an ESCRT-independent pathway [34, 35] that surpasses ceramidemediated membrane budding [15]. Trafficking of exosomes to plasma membrane and their subsequent release involve binding to tether proteins mediated by Rab GTPases [36], followed by the fusion complex SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) that brings membranes into close proximity [37], while sphingomyelinase mediates release [29]. The possible outcomes after exosome release are as follows:


Target cells uptake exosomes through (i) endocytosis mediated by clathrin, claveolin, or lipid rafts; (ii) direct plasma membrane fusion; or (iii) receptor-ligand interaction on the cells surface [38], as shown in **Figure 2**. Once internalized by the target cell, exosomes will fuse with an endocytic vesicle, releasing RNA and proteins in the cytosol. All cell types including stem cells secrete exosomes, found in various body fluids such as saliva, tears, plasma, serum, cerebrospinal fluid, bronchial fluid, synovial fluid, amniotic fluid, breast milk, urine, semen, lymph, bile, gastric acids [39–49]. However, this endosomal pathway for exosome biogenesis is the one factor that distinguishes exosomes from other extracellular vesicles (EVs) [50, 51]. Contrastingly, both Apoptotic bodies and Microvesicles are formed *via* outward blebbing of plasma membrane [15, 52–54].

#### **6. Exosome composition**

Stem cell exosomes partly replicate the content of their cells of origin [26, 55]. This discovery, coupled with the revelation that they represent a very specific subcellular compartment, the components of which are selectively sequestered, led to the hypothesis that exosomes are more than just cell debris. Stem cell exosomes comprise

*Stem Cell-Derived Exosomes as New Horizon for Cell-Free Therapeutic Development: Current… DOI: http://dx.doi.org/10.5772/intechopen.108865*

#### **Figure 2.**

*MSC exosome biogenesis, release, and uptake. Pathway for stem cell exosome biogenesis, release, and three mechanisms for subsequent uptake by recipient cells (1). Direct fusion with target cell (2). Endocytosis (3). Binding to cell surface receptor on target cell and internalization.*

a specific milieu of cytoplasmic and membrane proteins including receptors, enzymes, transcription factors, extracellular matrix proteins, lipids, and nucleic acids all of which target molecular pathways and are biologically active in recipient cells [56, 57]. Proteomic databases have now made available the protein composition of exosomes [58, 59]. Irrespective of their origin, all exosomes share few proteins, for instance a specific subset of endosomal, plasma membrane, and cytosolic proteins including cell adhesion molecules (CAMs), integrins, tetraspanins (CD9, CD63, and CD81), heat shock proteins (Hsp60, Hsp70, and Hsp90), biogenesis-related proteins (ALIX and TSG101), and Major Histocompatibility Complex—MHC-I/II proteins). Other transfer and fusion proteins such as Flotillins, Annexins, Heat Shock Proteins, Rab2, Rab7 may be up- or down-regulated depending on the tissue of origin [17, 60], which also include MVB Biogenesis proteins, prostaglandins, platelet-derived growth factor, latherin, transmembrane proteins, lysosome-associated membrane protein-2B, and other phospholipases [61–63]. The lipid bilayer of exosomes (**Figure 3**) with a characteristic thickness of 5 nm [64] is enriched in cholesterol, sphingomyelin and other sphingolipids, ceramide, phosphoglycerides like phosphatidyl serine, and diacylglycerol, which are usually conserved and specific to the parent cell [65]. Since lipids are a key component forming and protecting the exosome structure, lipid content is conserved, and variations are only observed among different cell types [66]. Lastly, another essential cargo that is conserved is nucleic acids, which are relatively distinct from the cytosolic pool of the parent cell. These include single- and doublestranded deoxyribonucleic acids (ssDNA and dsDNA), mitochondrial (mtDNA), and coding and non-coding ribonucleic acid (RNA) such as mRNA and microRNAs [67]. Cholesterol and sphingomyelin along with GPI-anchored proteins and Flotillin are also enriched in "lipid-rafts," implying a role for exosomes in transport [68]. Exosomes are a source of pro-inflammatory cytokines—Interleukins such as IL-1β, IL-6, and IL-8, monocyte chemoattractant protein 1 (MCP-1), Tumor Necrosis Factor-alpha (TNF-α)

**Figure 3.**

*Mesenchymal stem cell exosome structure – A representation. Exosome structure comprises of a cargo-bearing cytosol enveloped by a bilayer membrane carrying receptors, transmembrane proteins, integrins, lipid rafts, lipid molecules, and a range of surface markers, along with associated protein corona.*

[69, 70]. **Figure 3** is a visualization of the identity and function of well-established components that make up the stem cell exosome structure, which consists of a lipid bilayer backbone, along with protein corona/glycocalyx attached to the surface (based on initial findings) [71].

#### **7. Exosome function**

The process of exosome biogenesis aids packaging and transfer of information in the form of lipids, nucleic acids, and proteins from the parent cell into the recipient [70]. Consequently, the precise biological functions of exosomes are dependent on their composition, and the reason stem cell exosomes receive tremendous attention is their biological fingerprint and functionality mirroring that of their parental cells [72]. Stem cell exosomes have generated a lot of scientific interests owing to their primary role as message and cellular cargo transporters in intercellular communication as well as involvement in processes such as coagulation, antigen presentation, immune modulation and inflammation, regeneration, cell differentiation, waste management, proliferation and apoptosis, tumor growth, and metastasis [73–85]. Owing to their specific lipid content, they can alter lipid composition, specifically cholesterol and sphingomyelin in target cells, thereby regulating target cell homeostasis [86]. Exosomes, owing to their unique composition, provide innovative opportunities as biomarkers for diagnosis and in treatments. Currently, many clinical trials have been registered for exosome-related studies [87].

*Stem Cell-Derived Exosomes as New Horizon for Cell-Free Therapeutic Development: Current… DOI: http://dx.doi.org/10.5772/intechopen.108865*

#### **8. Isolation methods**

Stem cell exosomes are multi-component vesicles with heterogeneity among populations, which necessitates the development of an effective isolation method. Many isolation techniques have been developed, which enable high yield, variable purity, and quality of isolated exosomes. The relative merits and demerits of each of these procedures are summarized in **Table 2**.



*Table lists and summarizes advantages and limitations of techniques regularly employed for isolation and enrichment of exosomes.*

#### **Table 2.**

*The merits and demerits of stem cell exosome isolation methods.*

#### **8.1 Differential ultracentrifugation**

Based on small size and low density, stem cell exosomes can easily be separated by ultracentrifugation that works on stepwise speed increments (300 x g to 2000 x g to 10,000 x g to 100,000 x g) or alternating between high and low speed (100,000 x g to 200,000 x g), ensuring that different sized particles are separated at different times. Despite its many shortcomings, ultracentrifugation is considered the gold standard for exosome isolation and accounts for 56% of all exosome isolation techniques used in research [93]. Since this process does not separate different sized EVs, the final product is more heterogeneous and may not be suitable for governing bodies [94].

#### **8.2 Density gradient ultracentrifugation**

There are two types of preconstructed density gradient media—moving-zone gradients and isopycnic gradients. Moving-zone gradients allow EVs of distinct sizes, but same density to separate out together. Once the sample is layered from top into a tube containing progressively increasing density from top to bottom, it is subjected to multiple rounds of ultracentrifugation allowing the exosomes to individually move toward the bottom, based upon their sedimentation rate. Moving gradient ultracentrifugation employs sucrose, deuterium oxide, or iodixanol-based gradients [95, 96]. When isopycnic density gradients (like caesium chloride) are employed, separation occurs because of differences in sedimentation rates/densities between exosomes and other impurities [97]. Exosomes concentrate at the density region of 1.10 and 1.21 g/cm3 , and pure exosome pellets are obtained by ultracentrifugation at 110,00 x g to 120,000 x g [98–100]. As inferred in **Table 1**, this may not be ideal, especially since the density separations of microvesicles as well as apoptotic bodies are remarkably close to exosomes.

*Stem Cell-Derived Exosomes as New Horizon for Cell-Free Therapeutic Development: Current… DOI: http://dx.doi.org/10.5772/intechopen.108865*

#### **8.3 Size exclusion chromatography (SEC)**

SepharoseCL-2B, Sepharose CL-4B, Sephacryl S-100 columns, or SEC matrices exploit the property of smaller vesicles having longer diffusion paths in the paths between porous gels, leading to their retention in the columns. Since exosomes have large hydrodynamic radii, these are excluded from entering the pores, resulting in longer retention times within the column [101–103]. Widely used in many areas of biology, size exclusion chromatography (SEC) can be used to isolate EVs based on the molecular size, for example, QEV (izon), EC SEC columns (Stem cell). This method is used in collaboration with filtration or multiple columns and is reported to alter the characteristics of the EVs to a lesser extent (than other methods like Precipitation) [104].

#### **8.4 Ultrafiltration**

One of the most popular size-based separation methods is where particles in suspension are separated on basis of their size/molecular weight. Exosomes are isolated using membrane filters of specific molecular weight, using molecular weight cut-offs (MWCO). A great example is TFF (Tangential Flow Filtration), a method that filters EV sizes 100 kDa and above, using tangential flow of the fluid across the filter surface, avoiding filter cakes and clogging of the pores. This is a preferred method compared to UC. Most commercial large-scale EV purification use either 100 kDa or 300 kDa molecular weight cut-off filters. Because TFF has a history of use in biopharma, it can be easily translated into large-scale downstream processing. Companies such as Pall and Sartorius have developed TFF systems that can be modified to work with their 3D bioreactor systems making EV scale-up easier.

#### **8.5 Asymmetric-flow field-flow fractionation (AF4)**

AF4 employs a porous rectangular channel, which is subjected to parabolic flow around its axis, and sample retention and diffusivity are controlled by a cross-flow [93].

#### **8.6 Immunoaffinity-capture**

Precise isolation, based on antigen molecules highly concentrated on exosomal surface, targeted by specific fluorescently labeled antibodies immobilized on a polystyrene substrate is purified either using a microplate (ELISA) or using submicronsize magnetic beads [89]. Coupling with mass spectrometry significantly enhances the capacity and is referred to as "Mass Spectrometric immunoassay."

#### **8.7 Polymer-based precipitation**

Hydrophilic polymers like polyethylene glycol (PEG) are employed to tie up water molecules and thereby force less soluble exosomes from stem cell-secreted culture superfluates. The resulting exosome-precipitate is separated by low-speed centrifugation or ultrafiltration [105, 106].

To meet the demands of the ever-growing field of exosome therapeutics, there is a desperate need for a robust, highly reproducible and high-throughput isolation method that is still under development.

### **9. Exosome (EXO) characterization**

#### **9.1 Characterization based on physicochemical properties**

Depending on their physiological origin, exosomes differ in the composition (quantity and quality) of their bioactive cargo capable of modulating and reprogramming recipient cells. While this property has conferred therapeutic prowess to exosomes, it also proves an impediment to the accurate assessment of their potency and efficacy. Consequently, a crucial question for clinical development of exosome therapeutics is to unravel the precise molecular composition and specific characteristics of exosomes. Unlike isolation, there is no available gold standard technique for either quantification or potency assessment of exosomes, owing mainly to inconsistencies in isolation methods and batch-to-batch variations. There are multiple guidelines available from ISEV (International Society for Extracellular Vesicles) to accurately characterize exosomes.

**Table 3** represents a comprehensive list of the different physicochemical properties such as size, shape, surface charge, density, and porosity assessed by various Exosome Characterization methods.



*Stem Cell-Derived Exosomes as New Horizon for Cell-Free Therapeutic Development: Current… DOI: http://dx.doi.org/10.5772/intechopen.108865*

#### **Table 3.**

*Exosome characterization methods.*

#### *9.1.1 Nano flow cytometry (nFCM)*

nFCM is a high-resolution flow cytometric approach that allows generic quantitative and qualitative analysis of individual EVs as well as sorting of EV subsets (including exosomes), based on antibody and/or fluorescence staining. Nano Flow requires elaborate staining protocols that efficiently eliminate confounding variables such as high background noise caused by buffers, unbound fluorophore-conjugated antibodies, unincorporated dyes, protein aggregates, and other submicrometer-sized particles that can interfere with the EV measurements [107–109].

This is perhaps the only technique that enables analysis of exosome particles in low abundance (for instance, disease-related exosomes purified from clinical samples).

nFCM is the future of quantitative and standardized measurement of therapeutically significant nanoparticles, especially EVs. In our study, we optimized conditions for antibody-labeled, precipitation-enriched exosomes to be analyzed by nFCM based on exosome-specific markers.

#### *9.1.2 Cytokine Array*

We used a cytokine array kit to determine the expression of 36 different cytokinerelated proteins in our EV/EXO preparations, using the Human Cytokine Array Kit

#### **Figure 4.**

*Stem cell exosome quality control: Methods for multi-parameter characterization. Exosomes can be characterized based on multiple parameters that include qualitative and quantitative analyses such as their morphology, physical characteristics, size, concentration, cargo content, intact-ness, origin as well as their function.*

(Proteome Profiler; R&D Systems) according to the manufacturer's instructions. This is essentially a membrane-based sandwich immunoassay, where a biotinylated antibody-stained exosome sample is incubated with an array membrane that is spotted with capture antibodies to the Exosomal target proteins and visualized using chemiluminescence. This process is semi-quantitative in that the signal produced is proportional to the amount of bound analyte. In our study, we discovered at least 106 different cytokines and growth factors bound to the Exosome membrane.

**Figure 4** enlists the methods used to characterize Exosomes based on their physical properties, concentration, cargo, and function.

#### **9.2 Characterization based on exosome potency**

Exo potency is assessed by a matrix of functional assays that are performed under a tightly regulated validation process. Some of the *in vitro* potency assays that exploit the immunomodulatory properties of exosomes including immune cell signaling, wound closure, and angiogenesis are as listed below:

#### *9.2.1* In vitro *potency assays*

#### *9.2.1.1 Angiogenic evaluation of exosomes*

Tube formation assay/vascular-like Network Formation Assay

To assay the angiogenic potential of MSC Exos, human endothelial cells are treated with exosome solution in variable ratios (1:10, 1:00, 1:1000) for 12 h in a 96 well plate, on Matrigel or Geltrex basement membrane matrices that enable vascular network formation. This assay works around the principle that MSC Exos significantly enhance formation of tube-like structures, thereby promoting angiogenesis in human endothelial *Stem Cell-Derived Exosomes as New Horizon for Cell-Free Therapeutic Development: Current… DOI: http://dx.doi.org/10.5772/intechopen.108865*

cells. Validity of the assay can be reinstated by using non-supplemented cell growth media as a negative control and complete media as positive control. Imaging of capillary network is usually acquired with regular light microscopy; total tube length is measured using the Angiogenesis Analyzer plugin of ImageJ software and plotted for different Exo concentrations [110]. MSC Exos are internalized by many cells, including human Endothelial cells, and this assay proves the potency of Exos to promote angiogenesis *in vitro.*

#### *9.2.1.2 Vascular endothelial growth factor (VEGF) immunoassay*

VEGF plays a crucial role in angiogenesis and immunomodulation. This assay investigates the proteolytic stability of VEGF in Exo preparations, by mixing them in 1:1 ratio with Trypsin and analyzing *via* a membrane-based immunoassay [111]. This assay runs on the principle that the presence of MSC Exos protect secreted factors like VEGF from protease-mediated degradation. Functional testing of the angiogenesis assay is done by blocking VEGF with an anti-VEGF antibody along with protease treatment of Exos, which will inhibit tube formation in vascular endothelial cells. This is manifested in significantly reduced vascular network formation.

#### *9.2.1.3 Immunomodulatory assessment of exosomes*

#### *9.2.1.3.1 T cell proliferation assay*

MSC Exos inhibit T cell proliferation *in vitro*, leading to impaired T cell function, and 300,000 CFSE-labeled PBMCs from donors are stimulated with 5ug/mL PHA (Phytohemagglutinin) in a CD3-coated flat bottom 96 well-plate to induce mitogenesis at 96 h (Day4), after which Exos are introduced in varying doses. Flow cytometry determines the percentage of proliferating T cells by measuring percentage of viable CD3 positive cells, which also depict lower CFSE staining compared to non-PHA stimulated cells. No Exos Negative control is used to express maximum T cell proliferation. Maximum proliferation is carried out using Flow Cytometry. Production of CD3-stimulated T cells significantly decreases when treated with exosomes *in vitro*. Exos significantly inhibit T cell proliferation in a dose-dependent manner.

#### *9.2.1.3.2 IL-10 release assay*

Another *in vitro* potency assay for MSC exosomes is based on the release of IL-10 from PBMCs following incubation with exosomes. Post-incubation with Exos at 37C for 16-18 h, PBMCs are stimulated with LPS (Lipopolysaccharide) for 5 h. The Supernatant is assayed for IL-10 using ELISA and raw absorbance values are converted to concentration. Higher IL-10 secretion indicates high exosome potency of exosomes [112].

#### *9.2.1.3.3 Macrophage polarization assay*

This assay exploits the phenomenon that macrophages maintain a proinflammatory (classically activated M1) phenotype during active infections, and then switch back to a normal, anti-inflammatory M2 phenotype. Here, macrophages are incubated for 3 hours in the presence of PKH7-stained Exos and the principle of this assay is that the majority of the macrophages (>70%) should efficiently internalize Exos in their cytoplasm. This will significantly increase cell proliferation of the Exo-recipient macrophages, which is

measured by Flow Cytometry using BrDU incorporation, as compared to Exo-untreated cells. This phenotype modulation of Macrophages from M1 to M2 can be quantified by assessing relative intensities of pro-inflammatory markers (like Ly6C, CD11b, CD40, and CD86) which are downregulated, while anti-inflammatory markers such as CD36, CD51, CD206 are upregulated [113].

Another form of potency assessment is to check the capacity of MSC Exos to suppress mRNA induction of Tumor Necrosis Factor Alpha (TNF-α) in M1 macrophages generated by LPS and IFN-γ (Interferon gamma) stimulation, in the presence or absence of MSC Exos for 24 h. After 24 hours, TNF-α mRNA levels are quantified by RT-qPCR. The functional end point of this assay is half minimal effective concentration or 50% decrease in levels of TNF-α, relative to control (EC50) during the 24 hours [114].

Exosomes play a role in macrophage phenotype modulation by triggering their proliferation and polarization to decrease inflammation.

#### *9.2.1.3.4 Exosome uptake by peripheral blood Mononucleocytes (PBMCs)*

Exosome uptake by cells gives us an inkling about their ability to alter signaling in the recipient immune cells and subsequently their potency toward immunomodulation. In this simplistic assay, PBMCs are incubated for different time intervals (maximum being 48 hours) with pre-stained Exos in a ratio of Exo: PBMC = 5:1. The percentage and intensity of Exo uptake is quantified using either Flow Cytometry or fixing and visualizing with laser scanning confocal microscopy.

#### *9.2.1.4 Wound healing scratch assay*

This assay works on the simple principle that Exosomes activate human fibroblast cells to proliferate and migrate to the site of injury. Varying concentrations of Exosomes are added to wells containing Fibroblast cells, with manual scratch where cells lift off. After 24 hours, gap closure is measured using Image J Analysis software and visualization of gap closure under light microscope.

#### *9.2.1.5 Multidrug resistance protein 1 (MRP1) assay*

This assay investigates the ability of MSC Exos to downregulate the expression of MRP1 in a dose-dependent manner.

All *in vitro* assays should be performed in triplicates, with at least two independent experiments. It would be interesting to compare potency of freshly isolated stem cells Exos with frozen Exos and lyophilized Exos stored at different time intervals (3 months, 6 months) to obtain important insights into their stability over an extended period. It would also be interesting to see the differences in potency of Exos produced under hypoxic conditions as compared to normoxia-produced Exos.

#### *9.2.2* In vivo *potency assays*

Biological *in vivo* potency assays need to be disease-specific, fit-for-purpose and should employ relevant functional end points. These cover multiple aspects of applicability, from administration route to dosing, and provide information about therapeutic effects and toxicity. Moreover, only an *in vivo* system will be able to precisely map exosome distribution and localization post-administration, circulation time, half-life, and target organs.

Most *in vivo* assays are extensions of their *in vitro* counterparts, including *in vivo* angiogenic assay (myocardial infarction model), *in vivo* macrophage modulation (M1-M2) in skeletal muscle injury model, *in vivo* wound healing assays.

#### **10. Exosome preservation and storage**

Stem cell-derived exosomes are being developed for variety of applications from primary diagnostics and drug delivery (engineered exosomes). Traditional storage method, cryopreservation has been found to cause decrease in exosome concentration and quality over extended period and is cost intensive. Herein, we discuss current developments in preservation of exosomes through techniques such as cryopreservation, freeze drying, and spray drying [115–117].

Cryopreservation involves freezing exosomes at temperatures much below the optimum required temperature for enzymatic and biochemical activity of constituent biomolecules. Common challenge is formation of ice crystals, which affects the functional and morphological properties of exosomes [118]. To overcome this, various cryoprotectants (CPAs) are used which controls the kinetics of ice formation and concentration of solute pockets, prevent aggregation of particles, and maintain osmotic balance internally and externally [119, 120]. Penetrating CPAs are permeable to the exosome membrane due to their low molecular weight (e.g., dimethyl sulfoxide, ethylene glycol, and glycerol), while non-penetrating CPAs form a glassy matrix or coating externally due to their high molecular weight (e.g., trehalose, sucrose, mannitol, and other sugars) [119, 121, 122]. Studies have shown that cocktails of these cryoprotectants aide in extending the shelf life of the exosomes at low temperatures [116]. Chung et al. [123] in their patent (US2020/0230174A1) used carboxylated Poly-lysine as cryoprotectant to avoid using DMSO. Critical parameter that needs to be optimized is the concentration of CPAs as higher concentration can have toxicity effects, while lower concentrations can cause cryoinjury [124].

Freeze-drying or lyophilization involves freezing exosome solutions followed by sublimation of ice to vapor phase under vacuum forming a dry powder/precipitate thereby maintaining biological properties [125]. Challenges with this method involve uncontrolled ice formation and stresses caused due to drying that affects the exosome stability and membrane integrity. Lyoprotectants such as trehalose, sucrose, buffers, or cocktails can be used to extend the product life [126, 127]. Driscoll et al. [128] compared sucrose, trehalose, and mannitol as lyoprotectants and found trehalose to be most economical and effective lyoprotectant in conjugation with manifold-based lyophilization. Kim et al. and Lim et al. in their patents present a technique to lyophilize exosomes in the presence of one or more lyoprotectants [129, 130]. Since lyophilized products involve storage temperatures between 4°C and room temperature as compared to cryopreservation at freezing conditions, this technique can increase dry state stability, reduce cold chain transportation, and can be used by dissolution in water.

Spray drying is a continuous drying process where wet solution is first atomized, followed by contacting with dry warm air, which leads to instant vaporization of moisture producing dry powder [131]. This technique reduces the risks associated with freezing and dehydration stresses. As this is a continuous operation, spray drying can be economical as well as scalable for large-scale manufacturing. Some critical parameters that affect the shelf life and quality of dry powder are initial feed concentration rate, atomization pressure, and outlet temperature. Behfar et al. [132] have patented a system of exosomes encapsulated in alginate by spray drying. A cyclone

separator/electrostatic precipitator can be used for better retention of dry powder. However, experiments are required to be carried out to evaluate if the final product meets required critical quality attributes.

Other than preservation techniques, storage conditions such as pH, temperature, and number of freeze thaw cycles also affect the relative particle concentration, protein content, particle diameter, shelf life, and cellular uptake of exosomes [133, 134].

In summary, trehalose, a FDA approved non-reducing disaccharide sugar, presents as the most suitable option for cryopreservation and lyophilization by stabilizing the lipid membrane preventing exosome aggregation [135].

#### **11. Exosome engineering**

#### **11.1 Targeted delivery of pay loads**

Native exosomes present therapeutic properties and cargo similar to parent cell type thereby limiting the cargo quality and delivery of exosomes to targeted tissues or cells. Engineered exosomes with targeted delivery overcome these limitations by enhancing exosome efficacy as well as avoiding possible adverse reactions. Some of the methods to modify or load cargo onto native exosomes are as follows: (a) surface modification or passive cargo loading where the exosomes are modified chemically or physically directly to deliver desired cargo and (b) genetic engineering or active cargo loading where the parent cell type is genetically modified to secrete exosomes with specific cargo [136–140]. Some drawbacks to these modifications are compromised exosomal structure and possible induction of immunogenic effects as compared to native exosomes, Challenges linked to engineered exosomes are scalability, production cost, trained professionals, and downstream processing. Hence, there is a need to conduct further studies on these shortcomings at the same time maintaining exosome therapeutic value in conjugation with delivery systems.

#### **11.2 Hydrogel and exosome engineering for tissue regeneration and enhanced secretion**

Hydrogels are 3D network of physically or chemically crosslinked polymeric materials with a high affinity for water. Moreover, hydrogels have tissue-mimicking properties such as porous hydrophilic structure, biocompatibility, tunable stiffness, response to external stimuli (pH, temperature, enzymes, cells, etc.), and controlled degradability; thus, hydrogels present a suitable choice for targeted drug delivery system and tissue engineering.

Due to their porous structure and biocompatibility, exosomes are readily encapsulated in hydrogel matrix, offering a sustained and controlled bulk exosome delivery system for therapeutics. For instance, ADSC exosomes encapsulated in GelMA promote tendon regeneration [141], UMSC exosomes laden low-stiffness HA-MA hydrogels stimulate nervous tissue regeneration [142], UMSC-exosomes encapsulated in a hydrogel wound dressing encourage diabetic wound healing [143], and MSC-Exosomes in a sprayable fibrin heart patch regulate myocardial infarction [144]. Hydrogels can be printed with exosomes providing off-the-shelf solution to customized tissue constructs [145–147].

*Stem Cell-Derived Exosomes as New Horizon for Cell-Free Therapeutic Development: Current… DOI: http://dx.doi.org/10.5772/intechopen.108865*

As hydrogels represent a native ECM such as environment and control over their functional chemistries, they can affect exosome secretion activity of hMSCs. Some examples are hydrogel composition and stiffness enhancing MSC secretome and exosome secretion profile [148–150]. Chen et al. [151] have developed a 3D bioprinting method, which augmented exosomes secretion compared to plastic cell culture.

Thus, hydrogels present an exciting drug delivery system and improve exosome secretion profile for native and engineered exosomes.

#### **12. Exosomes: clinical and regulatory guidance**

Thirty-seven clinical trials in Phase 1–4 are currently listed at clinicaltrilas.gov using the term extracellular vesicle (EV) or exosomes. The most abundant are in lung and respiratory diseases followed by graft-versus-host disease (www.clincaltrilas.gov). The rise in respiratory and lung diseases is a direct result of the current pandemic (Coronavirus-19), which has a fast track to authorization due to its disease class. Most jurisdictions look to the Food and Drug Administration (FDA) in the United States for guidance. The current guidance for industry is the same as the current guideline for mesenchymal stem cells, which requires a demonstration of safety and efficacy across multiple clinical trials and shows product purity and potency. Consumer alerts have been issued by authorities due to unregulated stem cells and exosomes. The International Society for Extracellular Vesicles (ISEV) and the European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD) have formulated certain guidelines to foster their clinical use [152]. However, three is no harmonized regulatory framework around exosomes at the international level yet but some isolated approvals provided on case-to-case basis. The current status of ongoing trails in MSC derived exosomes can be seen in **Table 4** [87, 153].



#### **Table 4.**

*Current clinical trials using MSC exosomes.*

#### **13. Exosomes: scale-up production**

#### **13.1 Upstream processing**

In order to meet demand of aforementioned EVs in clinical trials and success thereof, upscale technologies must be employed [154, 155]. One of the major challenges to cellular and non-cellular biologics is upstream scaling of manufacture to bioreactor culturing. Generally, manufacture relies on the development from T flasks to multilayer stacks; however, this method of culturing is restrictive in terms of surface-tovolume ratio. From 2D culture, the system can be scaled into roller bottles or spinner flasks. From there, lab-scale technologies can be employed which can then be

#### *Stem Cell-Derived Exosomes as New Horizon for Cell-Free Therapeutic Development: Current… DOI: http://dx.doi.org/10.5772/intechopen.108865*

transferred or scaled into high-capacity bioreactor systems. Manufacturing current Good Manufacturing Practice (cGMP) EVs to a commercial scale which are not only (i) reproduceable but (ii) cost effective remains a somewhat arduous task. Upscaling manufacture is a necessary step on route to commercialization; however, large investment is required to ensure a smooth translation from bench to bedside and gain successful regulatory acceptance. With upscaling comes less batch testing, less lot release criteria, less labor, less facility time, less consumables and reagents costs, and perhaps most importantly less impact of variation. However, there are also higher risk considerations including higher costs of failure, larger equipment costs and depreciation, more upfront research and product development, and undesired product changes [156]. Although much can be learnt from stainless steel bioreactors that are currently in use in other fields (Monoclonal antibody and vaccine production) at a scale of 20,000 Liters (L), cell and EV therapy scale-up solutions need to generate high volumes in single-use sterile bioreactor systems (SUBs). SUBs allow for less qualification and validation due to presterilization and minimal contact. Capacity of SUBs currently stands at 6000 L (Wuxi Biologics, China); however, downstream systems for purification and sensors are not necessarily compatible with the SUBs and product requirements.

#### *13.1.1 Single-use technology*

With an ever-changing landscape in bioreactor technology, it is important the technology of choice has longevity. A change of method nearing Phase 3 or commercialization could be devastating. System suitability relies on many factors including cell type, downstream processing, and carrier suitability. For EVs to reflect the expression pattern from the parent cells, it is a challenge with most historical characterization being completed on cells from 2D culture systems and most cell types being extremely sensitive to hydrodynamic conditions [157]. Hydrodynamic conditions within a bioreactor will significantly impact the biological performance of the cells and/or secreted molecules like EVs. This makes the choice of SUB of upmost importance [158]. Bioreactors enable the user to control gas, temperature, pH, and feed addition; however, these factors are reliant on which type of reactor is employed [159]. The main types of reactors used for EV manufacturing include those in which employ microcarriers or macrocarriers that are utilized in the following: 1. continuous stirred tank bioreactors, 2. hollow fiber reactors, 3. packed bed bioreactors, and 4. wave reactors [155, 160, 161]. By far the most widely used bioreactor type is the stirred tank reactor due to its high flexibility and low-operating costs, **Tables 1** and **2** outline a range of SUBs currently available and some of their limitations and advantages for EV production The Xcellerex (GE Healthcare), Allegro (Pall), and BIOSTAT (Sartorius) offer manufacturing platforms of SUBs with designs that closely match traditional reactors (**Table 5**) [162].

#### *13.1.2 Substrate technology*

Microcarriers and macrocarriers are generally used in SUB systems as the 3D surface the cells can attach and grow. Microcarriers come in many forms and can be characterized based on matrix, coating or size. This includes glass, diethylaminoethyl (DEAE)-dextran, acrylamide, polystyrene, collagen, and alginate [163]. To increase cell attachment, they are either coated (collagen) or non-coated (charged). Regulatory bodies require the culture system to be animal origin free for human use to avoid xenogeneic reactions that limits the choice. In the case of xeno-free microcarriers,


#### **Table 5.**

*Examples of SUBs commercially available.*

some examples include Hillex and Star Plus (Sartorius, USA) as well as other dissolvable carriers like Synthemax (Corning, USA). One advantage of not being required to harvest the adherent cells as with EV production is the challenge of enzymatic detachment. Using microcarriers in stirred tank or wave reactors can cause issues including aggregation, engulfment, and beaching where the microcarriers and cells are stuck together or in the case of the wave bag reactor are caught in the corners of the bag. For EV manufacture using adherent cells, it is therefore preferred to use macrocarriers, generally made as disks (fibra-cel, Eppendorf) or strips from polytetrafluoroethylene (PTFE).

#### **13.2 Downstream processing**

Selective purification is necessary to isolate exosomes from other EV subsets as well as from the heterogeneous "soup" that is the stem cell secretome. This secretome contains a myriad of analytes, including cytokines, chemokines, enzymes, growth factors, Extracellular Matrix (ECM) proteins, and factors involved in ECM remodeling, different types of Extracellular Vesicles including Exosomes, microvesicles, apoptotic bodies, and others. The downstream processing of a mixed biological like the secretome requires careful precision. From regulatory standpoint purity, potency, safety, and efficacy of derived exosomes are of upmost importance for clinical relevance [164]. A pure product without contaminants from culture media and cells is critically important. Methods for EV isolation are broad and rely on alternative characteristics of the EVs for purification; however, challenges remain on which on scale vs. purity. Many methods are starting to become available to researchers and manufacturing organizations, for example, immunocapture of CD81-, CD63-, and CD9-positive molecules and microfluidics and the methods described below include Ultracentrifugation (UC), Precipitation, Size exclusion chromatography (SEC), and Tangential Filtration Flow (TFF) are most commonly used in largerscale downstream processing. Currently, there are no well-defined methods for exosome isolation in high-efficiency and high-throughput, and the recommendation is a combination of the below methods.

*Stem Cell-Derived Exosomes as New Horizon for Cell-Free Therapeutic Development: Current… DOI: http://dx.doi.org/10.5772/intechopen.108865*

#### **14. Perspectives**

Secretion of extracellular vesicle (EVs) is a universal phenomenon and hence must have biological implications. They have come a long way since first discovered in 70's by Peter Wolf [4]. Fast forward to today, there are extensive publications and clinical trial data to promote their role in diagnostics and therapeutics, including in drug delivery. However, the jury is still out there on EVs to fulfill these roles and gain prime time.

Among the two broader categories of EVs: ectosomes and exosomes, the latter are inward budding and hence endosomal origin with a size range of 40 to 160 nm in diameter. Endosomes undergo a process of systematic invagination of the plasma membrane with the formation of multivesicular bodies that intersect with other intracellular vesicles (phagosomes) and organelles, contributing to diversity of exosome composition before they are released back to intercellular space. These subset of EVs as exosomes have attracted attention for their critical role in cell-to-cell communication and hence therapeutic values. In particular, the mesenchymal stem cells (MSCs)-derived exosomes seem to have strong anti-inflammatory and immunomodulatory roles and hence have been studied extensively.

The role of exosomes in intracellular and intercellular communication is quite apparent from many critical studies thus far. The developmental pathways for exosomes are highly regulated that are being unraveled at the molecular level; however, how they impact cells is still elusive. Nevertheless, recent studies including preclinical and clinical appraisals have demonstrated their role in mitigating symptoms of various diseases as delineated in this chapter. *Given the cellular therapy still facing huge challenges regarding cell differentiation, maturation, and integration, opportunity with exosomes to develop non-cellular active pharmaceutical ingredients (API)-based allogeneic therapy may be more appeasing to the regulatory authorities.*

This chapter has endeavored to address questions systematically by first reviewing the developments in the field and then putting perspectives on feasibility of their roles and the technical and regulatory hurdles involved. The scientific rigor behind exosomal research puts a demand on regulatory bodies to develop appropriate framework for promoting their developmental pathways toward human medicine.

#### **15. Challenges**

The current drug developmental paradigm critically requires defining the active ingredient in the product to its finest detail to eliminate heterogeneity and batch-tobatch variations before defining its pharmacology and pharmacokinetic profiling for final approval after clinical trials. This is followed up by rigor of their scale-up production under GMP from established master cell bank (MCB) with quality controlled (QC) protocols, cryopreservation if any, and shelf life for potency. This is a very arduous, time-consuming, and expensive journey in drug development and takes about 5–10 years with a projected cost of 3–4 billion USD.

#### **15.1 Regulatory challenges**

Purification of exosomes by various methods described in this chapter gives rise to a heterogeneous EV population consisting of 40–160 nm diameter vesicles, putatively classified into many subcategories based on size, Exo A, B, C; content, Exo 1, Exo 2, Exo 3; function, Exo α, Exo β, Exo γ; and source, Exo I, Exo II, Exo III [28]. However, as per societal guidelines, all EVs qualify as one based on their characterization. The major question is whether such heterogeneity matters, particularly when blood and blood-derived products are approved for therapeutic use. In fact, the pleotropic effects of exosomes are beneficial in some cases that include blood transfusion. Therefore, the current classification of exosomes API (active pharmaceutical ingredient) needs due diligence and redefining by the regulatory authorities so that it is at par with blood and blood products.

There are no harmonized protocols available as yet that endow regulatory approvals on exosome production for clinical development. Approvals that have been granted are on a case basis depending upon clinical needs, but no main stream approvals yet.

#### **15.2 Technical challenges**

Characterization: The current societal guidelines for characterizing exosomes include the following:

For size and number, Nanoparticle Tracking Analysis (NTA) or Dynamic Light Scattering (DLS) are both complementary techniques that offer different insights. DLS will generally measure a wider size range than NTA, but NTA offers greater resolution than DLS. There are inherent difficulties in both these techniques with wide variations within each sample and aggregation of exosomes affecting measurements. Stabilizing exosomes with some cryoprotectants such as trehalose can offer some respite. Alternatively, quantification using the Exo-Flow-ONE staining kit may be more accurate.

Exosome-specific surface markers such as CD9, 63, 81, 107, Alix, are generally assessed by using Western blot; however, the procedure is cumbersome and not efficient for a large number of samples. Additionally, nano-flowcytometry that is more efficient is not widely used because of the cost involved and lack of harmonized published protocols. Nevertheless, some recent publications including our own endeavour showed success with this technique [162–166]. Alternately superresolution microscopy topology could be employed though it is neither cost effective nor efficient.

Proteomics and micro-RNA profiling are generally outsourced for bulk analyses that are more cost effective but relevance of such comprehensive data for drug development is far from clear, though may be relevant for diagnostic purposes.

Exosome potency assays include inhibiting T cell proliferation, macrophage phagocytosis, fibroblast activity, vascular-like network formation assay on Matrigel [167] and scratch wound healing assays [168] that are very useful and can be optimized quickly in the lab. Emerging *in vivo* potency assays such as EV-mediated wound healing [166, 169] (assess the biological response of exosomes in a disease model [170]. Critically understanding the mechanism of how exosomes elicit a response will assist in regulatory approval.

Scale-up production involves establishing a stem cell biobank made up of MSCs extracted from biologically available sources such as umbilical cord, bone, tooth, fat; however, these MSCs have finite life and reach senescence after 6–7 passages. To circumvent this problem, immortalized cell lines were used in the past that caused regulatory and safety issues. Current approach is therefore to reprogram pluripotent stem cells like iPSC (induced pluripotent stem cell) from human blood or skin tissue *Stem Cell-Derived Exosomes as New Horizon for Cell-Free Therapeutic Development: Current… DOI: http://dx.doi.org/10.5772/intechopen.108865*

and differentiate these into MSCs so that there is an unending source of materials in the biobank. These iMSC derived from iPSCs continue to proliferate beyond passage 10–12 and do not senesce easily, likewise eMSC derived from hESC is other source of long-life stock from which MSCs can be derived easily. Producing these sources of iPSC and MSCs under GMP is very expensive proposition. We have recently moved from planar culture to 3D culture using bioreactors for improved exosome production. We observed an improvement in both quality and quantity of EVs produced. Incorporating such devices within aseptic environment is leading the way for transition to clinical trials and then clinics.

While cryopreservation of cell lines is a standardized procedure, storing exosomes is still an evolving field. Notionally, these can be stored in saline at -80°C with good keeping quality for years; however, there are inherent problem with exosomes aggregation and/or lysis happening during storage. Lyophilization is a quick and efficient way to store exosomes at room temperature for clinical purposes. Cryoprotectants like trehalose help in protecting against aggregation as discussed in this chapter.

Targeted delivery of exosome payloads by genetic engineering increases efficiency and efficacy. Considerable progress has been made toward understanding the logistics for exosome delivery. The controlled release of exosomes at the site of injury is by using various biodegradable gels, and extracellular matrices are on trials as in our labs. Particularly in this regard is 3D printing of exosomes onto bandages as therapeutics is of great relevance in this field.

#### **16. Conclusions and perspectives**

In conclusion, exosomes field has emerged as a critical area of therapeutic development as a third pillar of medicine with proof of principle and good science behind it. However, bringing it to fruition requires liaising with the regulatory authorities to harmonize framework around quality control protocols, which will further facilitate clinical trials and more importantly bring focus and excitement for continued funding in this field.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Devashree Vakil, Riddhesh Doshi, Flyn Mckinnirey and Kuldip Sidhu\* CK Cell Technologies Pty Ltd., Sydney, NSW, Australia

\*Address all correspondence to: k.sidhu@ckcelltechnologies.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Stem Cell-Derived Exosomes as New Horizon for Cell-Free Therapeutic Development: Current… DOI: http://dx.doi.org/10.5772/intechopen.108865*

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### **Chapter 4**

## Evaluation and Characterization of Human Bone Marrow Mesenchymal Stromal Cells Cryopreserved in Animal Component-Free, Chemically Defined, Serum-Free Conditions

*Suresh Kannan, Swaroop Bhagwat, Pawan Kumar Gupta and Udaykumar Kolkundkar*

#### **Abstract**

Mesenchymal stromal cells (MSCs) have the potential to treat various disease indications and are the future of cell therapy-based regenerative medicine. Typically, MSCs cryopreserved in serum-containing freezing formulation are supplied at the clinical site, which necessities that this formulation is removed before the administration. This is a cumbersome process, and there is an immediate need for identifying serum-free, xeno-free cryopreservation medium that can be readily used. Here, we analysed two commercially available serum-free, xeno-free, defined freezing media *viz.*, CryoStor 5 (CS5) and CryoStor 10 (CS10) on their effect on human bone marrow MSCs at different freezing cell densities (5, 10, 12.5, 15 and 25 million cells per ml) over a period of 6 months and compared them to the in-house PlasmaLyte A (PLA) based cryopreservation media. We found that the MSCs cryopreserved in CS5 and CS10 showed similar characteristics as compared with the in-house freezing media for the various parameters analysed including post-thaw recovery, viability, phenotypic marker expression, CFU-F ability and trilineage differentiation potential of the MSCs. Our results show that human MSC could be successfully cryopreserved using serum-free and xeno-free cryopreservation media and can be delivered to the bedside without any manipulations.

**Keywords:** bone marrow mesenchymal stromal cells, phenotypic characterization, cryopreservation, stability, apoptosis, serum-free and xeno-free cryopreservation media

#### **1. Introduction**

The potential use of Bone marrow mesenchymal stromal cells (BM-MSCs) to treating various disease indications is in steady increase [1] and demands huge availability of clinical quality BM-MSCs to meet the growing demands. Typically, MSCs frozen in cryopreservation solution were supplied to the clinical site, and the method of freezing along with the composition of freezing media itself plays an important role in determining the characteristics of MSCs before infusion [2, 3]. One essential requirement of cGMP grade formulation reagents in cryopreservation is that they are free from animal serum proteins and toxic chemicals, as the xenogeneic compounds possess the risk of transmission of animal viral, prion and zoonose contamination [4].

Presently, the common practice of cryopreserving MSCs is in PlasmaLyte A supplemented with albumin and a cryoprotectant of DMSO and/or dextran [5, 6]. Conventionally, 10% DMSO is used because they readily penetrate cell membranes and thus confer protection to the intracellular components [7, 8]. But it is proven that freezing media containing a higher concentration of DMSO is toxic to patients [8, 9]. Efforts to reduce toxicity include the removal of DMSO prior to transfusion or decreasing the amounts used in the freezing process. But these post-thaw manipulations consume lots of time, decrease the viability of the cellular product and further require aseptic zone for processing and concentration of cells at site [8]. Several groups have investigated MSCs cryopreservation using low concentrations of DMSO and or many alterative to DMSO like polyvinylpyrrolidone (PVP), methylcellulose, polyethene glycol (PEG) trehalose and polymer mimics without or with DMSO from 2.5 to 7.5% [10–13]. But it is necessary that these in-house cryopreservation formulations are prepared from USP grade or cGMP grade reagents to meet the safety requirements and may pose many regulatory challenges while filing an NDA and obtaining regulatory clearances. One of the possible alternatives is to screen the commercially available GMP compliance USP grade, serum-free cryopreservation media for MSCs. Several groups have evaluated the commercial MSC cryopreservation for a short period of time from 1 week to 1 month, but it is necessary to study the real-time stability testing for at least 6 months. In this study, we evaluated the commercially available cryopreservation solutions − CryoStor (CS) 5 and CS10 from BioLife Solutions, USA for their ability to cryopreserve BMMSCs and compared them to our in-house developed PlasmaLyte A-based cryopreservation formulation. We analysed post-thaw viability, total cell recovery, trilineage differentiation, phenotypic marker expression, CFU-F potential and apoptotic cell percentage at a testing interval of 1 week, 1, 2, 3 and 6 months time point and discussed the data here.

#### **2. Materials and methods**

#### **2.1 Isolation and culture of human bone marrow-derived MSCs**

The BM was collected from healthy donors of age group between 19 and 40 years, after obtaining approval from the Institutional Ethics Committee of the Kasturba Hospital, Manipal and signed informed consent. The mononuclear cells (MNC) were isolated following the density gradient centrifugation method using Lymphoprep (Axis-Shield PoC) as described previously [9]. The isolated MNCs were seeded at a density of 50 million cells per T-75 flask in Dulbecco's modified Eagles medium-Knock Out (DMEM-KO) supplemented with 10% FBS (Hyclone, Waltham, MA), 2 mM L-glutaMAX (Invitrogen, Carlsbad, CA) and 1X penstrep (Invitrogen,

*Evaluation and Characterization of Human Bone Marrow Mesenchymal Stromal Cells… DOI: http://dx.doi.org/10.5772/intechopen.106573*

Carlsbad, CA). The cells were passaged when they reached 80−90% confluency using trypsin (0.25%)/ Ethylenediamine tetra acetic acid (EDTA; 1 mM) (Gibco, USA). MSCs from three donors were pooled at passage 2 (P2) in equal proportion and cultured at a seeding density of 1000 cells per cm2 in bFGF (2 ng/ml) enriched KO-FBS complete medium till P5.

#### **2.2 Cryopreservation**

Cryopreservation of BM-MSCs involves freezing of cells in 1 ml of formulation medium containing 10% (v/v) Dimethyl sulfoxide (DMSO) as a cryoprotectant with 5% (v/v) human serum albumin and quantified (QS) with PlasmaLyte A (Baxter Inc) in 5 ml Daikyo Crystal Zenith (CZ) vials from West Pharma. The commercial formulation of Cryostor CS10 and CS5 (BioLife Solutions Inc) were used for cryopreservation of BM-MSCs in CZ vials for comparison. MSCs after trypsinisation were centrifuged to remove the trypsin and the pellet was dislodged by gentle tapping to the pellet, 2−8°C refrigerated cryopreservation media was slowly added. The cells were resuspended at five different concentrations (5, 10, 12.5, 15 and 25 million cells per ml) in these three formulations. These cells were aliquoted @ 1 ml per CZ vials and cryopreserved by slow freezing @ 1°C/min till 80°C by keeping them in Cryomed controlled rate freezer (CRF). During this time an aliquot of the cells was taken to measure the pre-freeze 7AAD/viability. The frozen vials were then shifted to canister racks and were stored in the vapour phase of liquid nitrogen (LN2).

#### **2.3 Thawing**

Each sample was thawed according to the internal Stempeutics SOP. Briefly, after a minimum of 1-week storage in liquid nitrogen, the vials were retrieved and thawed immediately by placing them in a water batch at 37°C till the ice crystals were just disappearing. The thawed cells were transferred into 15 ml tube and the sample was diluted at 1:9 with complete media. The cells were centrifuged at 1200 rpm for 10 minutes and the pellet was then resuspended in KO-FBS complete media.

#### **2.4 Testing frequency, cell count and manual viability assessments**

The samples from three different cryopreservation mediums at five different freezing concentrations were analysed at time points of 1 week, 1 month, 2 months, 3 months and 6 months. The total cell recovery (TCR) and viability analysis by manual method and by flow cytometry method were carried out immediately after the thawing procedure. The total cell count and manual viability were determined by the trypan blue (Fluka) exclusion method. The number of viable (non-stained) and non-viable (stained) cells were enumerated microscopically. TCR is calculated by adding the total number of stained and unstained cells. The viability percentage was calculated by dividing the total number of viable cells by TCR and multiplied by 100. For estimation of viability by flow cytometry, the cells were stained with 7AAD and were analysed following the protocol as described previously [14].

#### **2.5 Immunophenotyping**

We analysed a set of two positive (CD90-PE, CD73-PE) and two negative cell surface markers (CD14-FITC and CD19-FITC) by flow cytometry. All the antibodies used for these studies were purchased from BD Pharmingen, San Diego. The cryopreserved cells were thawed and resuspended in wash buffer containing phosphate buffer saline (PBS) supplemented with 1% (v/v) FBS and 1% (w/v) sodium azide for analysis. The cells were incubated with saturating concentrations of fluorescein isothiocyanate (FITC) or phycoerythrin-(PE) conjugated antibodies at 4°C for 30 minutes in dark. After that, the cells were washed with wash buffer three times and re-suspended in 0.5 ml of wash buffer. The labelled cells were analysed in EasyCyte (Guava Technology) flow cytometer after setting the instrument parameters with respective isotype-matched controls. For every sample, 10,000 events were captured and the data was analysed using Guava Express Pro software (Guava Technologies). Fluorescence intensity of 25% or above its isotype control is considered an antigenic event and was used for calculation.

#### **2.6 Differentiation potential**

The differentiation potential of frozen MSCs was analysed by their ability to differentiate into osteogenic, adipogenic and chondrogenic lineages. Osteogenic differentiation was induced by culturing P5 BM-MSCs in the KO-FBS supplemented with 10<sup>−</sup><sup>8</sup> M dexamethasone, 30 μg/ml ascorbic acid and 10 mM β-glycerophosphate (all Sigma-Aldrich). For adipogenic differentiation, cells were cultured in the KO-FBS supplemented with 1 μM dexamethasone, 0.5 mM isobutylmethylxanthine (IBMX), 1 μg/ml insulin and 100 μM indomethacin (all Sigma-Aldrich). The chondrogenic differentiation was induced using STEMPRO (Invitrogen) chondrogenesis differentiation medium. After 21 days of differentiation, the cells were fixed and stained with Von Kossa, Oil Red O and Safranine O, respectively, for osteo, adipo and chondro differentiation cultures. The images were captured using Nikon Eclipse 90i microscope (Nikon Corporation, Japan, www.nikon.com) and Image-Pro Express software (Media Cybernetics, Inc., Silver Spring, MD, www.mediacy.com).

#### **2.7 CFU-F assay**

For CFU-F assay, 100 MSCs from 5 different freezing concentrations of three cryopreservation media at six-month time points were plated in KO-FBS (n = 2 of each condition) on a 100 mm<sup>2</sup> cell culture dish. After 14 days in culture, the plates were stained with crystal violet and the number of colonies was counted.

#### **2.8 Apoptosis analysis**

Apoptosis analysis was carried out using Tali apoptosis kit following the manufacturer's instructions (Tali Apoptosis kit – Annexin V Alexa Fluor 488 and propidium iodide, Cat # A10788. Life technologies). Briefly, 1 million cells/ml were resuspended in annexin binding buffer (ABB) and 5 ul of Annexin V were added per 100 μl of sample. The samples were incubated for 20 minutes in dark, centrifuged and resuspended in 100 μl of ABB. After adding 1 μl of PI and incubation for 5 min, the samples were read in Tali® Image-Based Cytometer.

#### **2.9 Statistical analysis**

All values are expressed as mean ± SEM (standard error of mean). Data were analysed by using Graphpad Prism (version 5, Graphpad Software Inc., La Jolla, *Evaluation and Characterization of Human Bone Marrow Mesenchymal Stromal Cells… DOI: http://dx.doi.org/10.5772/intechopen.106573*

CA, USA). Two-way ANOVA (Analysis of variances) was performed in order to compare means between groups and Bonferroni post-tests were carried out to find out the significance of the variables tested. P value <0.05 was considered significant.

#### **3. Results**

#### **3.1 Post-thaw viability – 7 Amino-actinomycin D (7AAD)**

BM-MSCs were scaled-up to P5 in multiple numbers of ten cell stacks and the cultures were frozen in various cell concentrations ranging from 5 x 106 to 25 x 106 per ml in 5 ml CZ vials using 3 different cryopreservation media (PLA, CS10 and CS5). Pre-freeze viability of BM-MSCs in PLA, CS10 and CS5 formulation media was >98.6 ± 1.2% (mean ± standard deviation). Upon, thawing, BM-MSCs viability of PLA, CS10 and CS5 of 5 different cell concentrations (5, 10, 12.5, 15 and 25 million cells per ml) were measured by flow cytometry using 7AAD at different time points viz., 1 week, 1, 2, 3 and 6 months. All the samples have shown >92% (**Figure 1**) viability by 7AAD in all the time points. No significant difference in percentage of viable cells was observed after six-month storage when compared to one-week storage. Twoway analysis of variance (ANOVA) was performed to assay differences over different time points and among different cryopreservation media. The results indicate that CS5 is equally good to that of CS10 and PLA and there are no significant differences in comparison with either time points or different cryopreservation media.

#### **3.2 Total cell recovery and viability**

The Post-thaw cell recovery and viability of five different concentrations at 5 different time points (1st (first) week, 1st month, 2nd month, 3rd month and 6th month) in 3 different cryopreservation media were assayed using trypan blue dye

#### **Figure 1.**

*Post-thaw viability by 7AAD of BM-MSCs following cryopreservation with PlasmaLyte a based cryopreservation solution (control), CryoStor (CS) -10, and CS5 variants. Cell viability assessed at 5 different time points (1st week, 1st month, 2nd month, 3rd month and 6th month) at 5 different freezing concentration (5, 10, 12.5, 15 and 25 million cells per ml) shows no significant differences among three cryopreservation media.*

exclusion assay. The total viable and non-viable cell count was taken as total cell recovery and shown in **Figure 2A**. The results demonstrated that no cell loss was observed upon thawing the samples at all time points in 3 cryopreservation media. There are **Figure 2B** also no significant differences in total cell recovery percentage in the lowest and highest freezing densities (5 and 25 million cells/ml respectively) at 3rd and 6th month of recovery after freezing (). Simultaneously viability by dye exclusion method (DEM) was analysed in all of the samples and the results were shown in **Figure 3**. Viability by DEM was between 85 to 95% in all test samples (**Figure 2C**).

#### **Figure 2.**

*Post-thaw Total cell recovery (TCR) of BM-MSCs cryopreserved in CS10, CS5 compared to control. (A) TCR in millions as assessed by summing total viable and non-viable cell counts after trypan blue staining. (B) TCR in percentage between the lowest and highest freezing densities at 3rd and 6th month. (C) Viability percentage as calculated by trypan blue exclusion method. There is no significant differences among the three cryopreservation media.*

*Evaluation and Characterization of Human Bone Marrow Mesenchymal Stromal Cells… DOI: http://dx.doi.org/10.5772/intechopen.106573*

**Figure 3.**

*Immuno-phenotype of BM-MSCs following cryopreservation with PlasmaLyte A-based cryopreservation solution (control) (A), CryoStor (CS) 10 (B), and CS5 (C) at six month time point. Two positive markers of CD90 and CD73 and two negative markers of CD19 and CD14 were analysed. No significant differences among three cryopreservation media at 5 different freezing concentrations except for CD 73 expression variation among CS10 group.*

#### **3.3 Immunophenotyping**

The surface marker expression was evaluated and analysed by using flow cytometry for BM-MSCs cryopreserved in control, CS10 and CS5 of 5 different cell concentrations. The freeze–thaw BM-MSCs in all three cryopreservation media across all time points were positive for CD90 and CD73 and negative for CD19 and CD14. BM-MSCs showed similar expression of CD markers in CS5 compared with that of CS10 and control (**Figure 3**). There are no significant differences in positive and negative marker expression observed among different seeding densities in any of those three groups except for CD 73 expression variation in CS10 group. There is a significant difference (n = 5,\*p < 0.05) between 12.5 and 25 million per ml and this significance is even stronger (n = 5, \*\*p < 0.01) between 5 and 12.5 million per ml. Howsoever, these expression levels were more than 95% for positive markers and < 2.5% for negative markers in all three cryopreservation media at different cell densities.

#### **3.4 Differentiation potential**

We investigated the in-vitro functional tri-lineage differentiation potential of P5 BM-MSCs at the 6th month time point after cryopreservation in PLA-based cryopreservation medium (control), CS10 and CS5 at 5 different cell freezing concentrations. The differentiation towards adipocytes was evident by the formation of fatty vacuole deposits and was observed by Oil Red O staining (**Figure 4A**). The differentiation towards osteoblasts was observed by Von Kossa staining (**Figure 4B**) and that of chondrocytes was observed by safranine O staining (**Figure 4C**) after 21 days of differentiation induction. The cells cryopreserved at different cell densities in different cryopreservation media stained for all three lineages, showing that the trilineage differentiation ability is maintained in all these conditions. The results of CS5 and CS10 were comparable with that of the control cryopreservation medium for all 5 different cell freezing concentrations.

#### **3.5 CFU-F assay**

Clonogenic potential of cryopreserved MSCs at 5 different cell freezing concentrations (5, 10, 12.5, 15 and 25 million cells per ml) in each of the 3 different

#### **Figure 4.**

*Trilineage differentiation of BM-MSC in 3 different cryopreservation medium at 6th month time point. BM-MSC cryopreserved in PlasmaLyte a based cryopreservation solution (control), CryoStor (CS) 10 and CS5 were differentiated into (A) adipocytes (B) osteocytes and (C) chondrocytes. Adipocytes stained with oil red O, osteocytes with Von kossa staining and chondrocytes by safronin O stain. All pictures captured with magnification of 10X.*

cryopreservation media (control, CS10 and CS5) at 6-month time points were assessed by counting the number of colonies with more than 50 cells. There is no significant change in the number of colonies formed by cells cryopreserved at different concentrations in all the three different cryopreservation media, except for two different cell concentrations in CS5 storage (**Figure 5**). The number of colonies formed by 5 and 10 million per ml freezing concentration (n = 2, P < 0.001) was significantly lower compared to other concentrations in CS5.

#### **3.6 Apoptosis analysis**

The comparative post-thaw apoptosis assay was performed at 6th month time point of BM-MSCs cryopreserved in control, CS10 and CS5 of 5 different freezing concentrations. The percentage of apoptotic cells increased with the decrease in freezing concentration, with the lowest concentration of 5 million/ml having the

*Evaluation and Characterization of Human Bone Marrow Mesenchymal Stromal Cells… DOI: http://dx.doi.org/10.5772/intechopen.106573*

#### **Figure 5.**

*CFU-F assay of BM-MSC in 3 different cryopreservation medium at 6th month time point. The number of colonies formed in 5 different freezing concentration at 6th month time point show no statistically difference between control and CS10. There is a significant difference (\*\*\*P < 0.001) observed in CS5 at lower freezing density of 5 and 10 million cells per mL compared to other 3 freezing concentration (12.5, 15 and 25 million cells per mL).Abbreviation:CFU — colony forming unit fibroblast.*

#### **Figure 6.**

*Apoptosis assay. Percentage of apoptotic cells in 3 cryopreservation media (control, CS5, CS10) in 5 different freezing concentration at 6th month time point reveals significant increase in percentage of apoptosis from 5 million cells per ml to 25 million cells per ml in all three cryopreservation media. (\*P < 0.05,\*\*p < 0.01\*\*\*P < 0.001).*

highest value and vice versa in all the three freezing media (**Figure 6**). The differences are significant (n = 2,\*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.001) except between 25 and 15 million cells per ml of CS10 and CS5. Overall, CS10 and CS5 have a lesser percentage of apoptotic cells compared to control in all concentrations tested.

#### **4. Discussion**

The demand for MSCs in clinical application is growing and necessitates availability of good quality cryopreserved MSCs with minimal pre-transplantation manipulations at the clinical trial site or final usage of the product. Optimal cryopreservation protocols including freezing density were not yet tested or published globally and no consensus has reached for the standard freezing density of MSCs. Moreover, published efforts were guided by HSCs cryopreservation protocols [15, 16]. Cryopreservation technology for MSCs is still evolving, as MSCs seem to lose viability very rapidly and are mostly attributed due to the rapid development of apoptotic processes and cryopreservation-induced delayed-onset cell death (CIDOCD) [17]. Most commonly, MSCS were cryopreserved in formulations containing 10% DMSO that needs to be removed from the final cellular product before infusion for human use. The removal of DMSO has its own challenge and requires the sample to be handled in a GMP compliance area and any improper removal may cause several ailments like headache, nausea, vomiting, sedation, high blood pressure, bradycardia or anaphylactic shock to the patients [18].

The total substitute of DMSO is not advisable, as it affects the viability and decreases the shelf life of MSCs during cryopreservation. Though many in-house cryopreservation formulations have been reported [19, 20], it is important that all their constituents meet the bio-safety standards. The use of approved and commercially available freezing formulations will ease the complications and facilitates hassle-free filing to NDA (New Drug Application), auditing, screening and testing etc.

In the present study, we have critically evaluated 5% and 10% DMSO containing commercial cryopreservation media (CS5 & CS10, respectively) for post-thaw viability, CFU-F, phenotypic markers expression, differentiation potential and percentage apoptotic cells of cryopreserved BMMSCs and compared to in-house formulations (PlamsLyte A with 10% DMSO and 5% HSA) at different time points up to 6 months. PlamsLyte A is an isotonic solution with the physiochemical properties closely resembling human plasma and is widely used as perioperative fluid [21]. PlasmaLyte A is commonly used in cryopreservation of MSCs and in our earlier studies it is known to preserve the viability and efficiency of BM-MSCs for clinical trials [22].

We have demonstrated comparable results for all the characteristics analysed in all these formulations at different time points. Our results showed that the postthaw viability analysed by two methods viz., trypan blue dye exclusion and 7AAD were > 85% in all conditions tested up to 6 months time period. Few studies reported a reduction in post-thaw viability in time with the use of DMSO-based cryoprotectant [10, 23] but they used different freezing concentrations and DMSO percentages. The viability of >85% of what we obtained should not be a problem as there are reports stating that even with 70% viability, the cells were demonstrated to have enhanced immunosuppression within 6 months of time period [24]. We also demonstrated that the total cell recovery of BM-MSCs is >85% in all these conditions and it probably seems that higher cell freezing densities yields lower cell recovery compared to lower freezing density, but the differences in not significant in the conditions tested. It may be probably that the lower concentration cells tend to thaw faster and dilute DMSO faster during post-thawing procedures maintaining cell integrity and lower osmotic cell shock to the cells.

The phenotypic marker expression of MSCs cryopreserved in all the three different cryopreservation media was comparable with >95% expression for CD90 and CD73 and < 3% for CD19 and CD14 markers at all tested variables. There was no significant difference in expression of CD markers in all three freezing media with respect to different freezing densities at different time points except for CD 73 markers in different CS10 concentrations. It should not be a matter of concern as the expression in all of them is above 95% as stated by ISCT guidelines. Though contradictory studies showed the stable expression [25] or decreased expression [23] of phenotypic markers over different time points, we have not observed such difference in any of the time points with respect to freezing media or different freezing densities.

The trilineage differentiation ability of MSCs is also not compromised in any of these three formulations. There are not many studies, which compare the differentiation characteristics of MSCs at different freezing densities and various time points. Nevertheless, a study by Naaldijk *et al*. 2012 found a slight reduction in osteogenesis capacity of MSCs with higher DMSO concentrations [2]. In our study, we did not do any quantification but we observe that all of these conditions retain the trilineage differentiation capabilities.

Additionally, we evaluated the CFU-F ability and apoptotic cell percentage in cryopreserved BM MSC in different cryopreservation formulations at different cell *Evaluation and Characterization of Human Bone Marrow Mesenchymal Stromal Cells… DOI: http://dx.doi.org/10.5772/intechopen.106573*

densities at 6-month time point. These extended assays were done to comply with the stability testing of new drug substances and products of ICH guidelines 21 CFR 31.2.23(a) (7) (ii) which requires a minimum testing period of 6 months to confirm the functionality of the product. The CFU-F ability is one of the markers of stemness and proliferation capacity of MSCs. We observed a similar competence in the number of CFU-Fs formed at different cell concentrations in all three cryopreservation media, except for the two lowest cell densities in CS5 formulation. There are no studies that report the effect of freezing cell concentrations on CFU-F, however, a study reports no difference in number of CFU-Fs between 5% and 10% DMSO concentration in freezing media [26]. With regard to percentage of apoptotic cells at 6 months time point, we found that the lower freezing densities are prone to higher apoptotic rates compared to higher freezing densities in all the formulations. Higher level of intrinsic proteolytic activity may be higher in lower freezing concentration, as the DMSO availability per cell is high, compared to higher freezing cell density, where the DMSO availability per cell is low. Usually, post-thaw activation of caspase-3 demonstrated the proteases activity and subsequently increase intrinsic proteolytic activity following cryopreservation [20, 27]. Hence higher freezing density of BM-MSCs has a lesser apoptotic percentage compared to a lower freezing density.

#### **5. Conclusion**

In this study, we demonstrated the possibility of using reduced 5% DMSO containing cryopreservation media (CS5) for cryopreserving BM-MSCs without any impact on viability, phenotypic characteristics and functional properties of MSCs. This was the first study to provide the characterization and comparison of human BM-MSCs cryopreserved in different freezing densities ranging from as low as 5 M cells per ml to higher freezing densities as 25 M cells per ml in two commercially available variants of CS (CS10 and CS5) and comparing it to in-house formulation.

Based on our presented data, we can conclude that chemically defined reduced DMSO-based formulation of CS5 addresses challenges and minimizes the postcryopreservation manipulation of MSCs for clinical use. However, these data needs to be backed up by safety and efficacy studies, with long-term stability program up to 1 year with more intrinsic molecular, proteomics analysis and immune-suppressive ability of cryopreserved MSCs before employing the CS5 for the cryopreservation of MSCs for therapeutic applications.

#### **Acknowledgements**

This work was fully funded by Stempeutics Research Pvt. Ltd., India.

#### **Author contribution statement**

SK: Design of studies, data analysis, interpretation and manuscript writing. SB: Design of studies, perform experiment, data collection and manuscript writing. PKG: Design of studies and manuscript correction. UK: Design of studies and data analysis. Correction and final approval of the manuscript.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Ethics statement**

The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. Obtaining bone marrow from consenting healthy donors was approved by the Institutional Ethics Committee (IEC) at the Manipal Hospital, Bangalore, India.

#### **Author details**

Suresh Kannan, Swaroop Bhagwat, Pawan Kumar Gupta and Udaykumar Kolkundkar Stempeutics Research Pvt. Ltd, Bengaluru, India

\*Address all correspondence to: uday.kumar@stempeutics.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Evaluation and Characterization of Human Bone Marrow Mesenchymal Stromal Cells… DOI: http://dx.doi.org/10.5772/intechopen.106573*

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#### **Chapter 5**

## Nanotechnology-Based Stem Cell Therapy: Current Status and Perspectives

*Ponpandian Samuel, Shenbagamoorthy Sundarraj and D.N.P. Sudarmani*

#### **Abstract**

The nanoparticles or nanobots are equivalent to the size of biological molecules of the human body and this is claimed to be the massive advantage of nanotechnology. Currently, top-down and bottom-up fabrication methods are being adopted to synthesize nanomaterials. Hence, the products developed from nanotechnology can be used for assessment of several biological parameters under *in vitro* and *in vivo* conditions. Effective production of nanoparticles, accompanied by the advent of novel characterization studies, enables us to manipulate the arrangement of atoms distributed on the surface of the nanomaterials to make it functionally more effective than before. In addition to the support imparted by nanotechnology, it also plays a primary role in the field of diagnostics. Another important outcome of nanotechnology is nanomedicine, which deals with the site-specific delivery of drugs with the aid of fabricated nanosystems. The advent of technology in recent years has enabled researchers to build novel forms of drug delivery systems like liposomes, dendrimers, nanoparticles and nanocrystals, which in turn ensure the précised delivery of drugs to suitable targets. Several needbased and value-added applications of nanotechnology are enlisted in the chapter.

**Keywords:** nanoparticles, nanobots, liposomes, nanosystems, nanocrystals, dendrimers

#### **1. Introduction**

Nanotechnology and stem cell therapies are two diverse fields and recent prominent areas of research in the direction of improvement to solve challenges during the treatment. Stem cell therapy, also known as regenerative medicine, promotes the repair mechanism of dysfunctional, incapacitated or wounded tissue by stem cells or their derivatives. Stem cell therapy is a treatment that has been done with stem cells. Stem cell therapy holds promise for treating a broad spectrum of diseases, such as cancer, heart disease, diabetes and neurodegenerative diseases. Researchers are still studying various sources for stem cells, which are applied for stem-cell treatment [1]. It is fascinating that the integration of two disciplines, nanotechnology and stem cell sciences, divulges new ways to identify the role of molecular apparatus in the

mechanism of the differentiation of stem cells regulation and elucidate more about the stem cell-based treatment strategies for insight into the human disease, prevention and theranostics.

Nanotechnology-based approaches in stem cell research have been established by utilizing biocompatible, biodegradable, solubility, stability, specificity, multimodality and efficacy for undergoing attachment to cognate receptors. Researchers have already shown that the following nanoparticle has been developed for the applications in stem cells differentiation and regeneration therapy, such as superparamagnetic iron oxide nanoparticles (SPIONs)-(ferucarbotran) NPs [2, 3], auto-assembled peptide [4], magnetic NPs [5], polyelectrolyte NPs [6], cerium oxide NPs [7], graphene oxide NPs [8], poly-ε-caprolactone [9], ZnO NPs [10, 11], SiO2-NPs [12], iron oxide NP [13], collagen nanofiber [14], retinoic acid loaded with polymeric nanoparticles [15], tri-CaPSO4 (tricalcium phosphate) [16], carbon nanofiber [17, 18], graphene-oxide nanoparticles (GO-NPs) [19], AuNPs [20, 21], PANPs [22], Au@BSA@PLL [23], USPIO [24], PFCE-NPs [25] and tri-Ca-silicate [26].

In the latest time, the application of nanotechnology in stem cell research has engaged better advances, which is attractive to an emerging interdisciplinary field. Stem cell nanotechnology is developing towards stem cell isolation, lineage and differentiation, stem cell imaging, active tracking, regenerative medicine and tissue engineering of stem cells (**Figure 1**). Nevertheless, stem cell nanotechnology also faces many challenges similar to any emerging interdisciplinary field. The mechanism of interaction between nanomaterials and stem cells still needs to be elucidated well as nanomaterials and nanostructures are modified to enhance the function of stem cells, and the action of metabolizing nanomaterials inside stem cells is arduous. The fabrication of multifunctional or homogenous nanostructures developed by existing knowledge and principles has been a great challenge in synthesizing, modifying and characterizing the quality and stability of nanomaterials and the mechanism of interacting with the stem. However, stem cell nanotechnology shows great fascinating

**Figure 1.**

*Applications of nanotechnology in stem cell.*

scenarios, stem cells are emerging for the application of the drug and macromolecular delivery for degenerative diseases [27].

#### **2. Application of nanotechnology in stem cells isolation and differentiation**

A crucial point in stem cell-based therapy is the segregation of appropriate cell types. The magnetic cell isolation technique was used to isolate specific types of cells. Magnetic nanoparticles can be used to label the stem cells for identification from a pool of different cell types by magnetic-activated cell sorting (MACS) [28]. This process involves combining MNPs with monoclonal antibodies (MAB) directed against unusual cell surface antigens, which causes the magnetic field of the cells expressing these antigens to be retained. It has been demonstrated that MNPs and anti-CD34 antibodies work together to efficiently label and distinguish peripheral blood progenitor cells from the blood. An uninterrupted quadruple magnetic flow sorter consisting of a flowing carrier and a quadrupole magnet with 1.42 T maximum field loudness and optimal field strength was able to separate these cells from mononuclear cells suspension of whole blood when MNP-conjugated anti-CD34 proteins were used to label CD34-cells. The CD34 cells collected had a purity of 60–96%, a retrieval rate of 18–60%, an improvement rate of 12–169 and a throughput of (1.7–9.3) 104 cells/s [29]. The optimized cells could be employed for cell transplantation-based regenerative medicine.

For SC proliferation and differentiation, scaffold-dependent nanomaterials and associated polymers have been used. Different scaffolds have been investigated with a focus on nanotubes, nanoparticles and nanofibers to control the differentiation of SC. Carbon nanotubes (CNTs) and titanium dioxide (TiO2) are viable possibilities for scaffold creation, such as bone replacement therapy, due to their outstanding mechanical properties [30]. The impact of biological molecules and intricate interactions with scaffold substances improves SC development. Due to their exceptional electrical, mechanical and refractive indices and extensive surface topographical features, many nanomaterial-based scaffolds have been used in tissue engineering applications; nevertheless, this sector is focused on graphene and graphene oxide (GO) as non-toxic scaffolds [31, 32].

The researchers identified several peptide sequences that can firmly attach to NSCs. The new peptide (HGEVPRFHAVHL, HGE) was combined with quantum dots, Zhao et al. [30] discovered that the 48/34 kDa proteins on the membranes of NSCs produced from monkey ESCs but not human ESCs were particularly identified by this HE-quantum dot combination. According to this work, ESC-conjugated particular peptides may be used to examine the lineages they have committed to, and they may also be a mechanism for separating differentiated cells from ESCdifferentiated cell populations. iPSCs often need to be cultivated on the feeder layer cells to preserve their pluripotency. Graphene (G) and graphene oxide (GO) have lately been established as cell culture substrates due to their biocompatibility at low concentrations and 2D structure with a large surface area. G and GO can support the culture of mouse iPSCs by allowing stem cells to differentiate. The cell proliferation and differentiation properties are induced by graphene materials (**Figure 2**). While iPSCs cultured on GO surfaces exhibit faster rates of adherence and proliferation than those on glass surfaces, iPSCs cultured on G surfaces show a similar effect on cell adhesion and proliferation [34]. Another benefit of GO is that it keeps the iPSCs in the undifferentiated stage while speeding up the differentiation [33].

#### **Figure 2.**

*Schematic representative of scaffold structure fabricated from graphene–nanofiber for differentiation of neural stem cells. The illustration was created by Asil et al. [33], and published in Appl. Sci. 2020, 10(14), 4852; https:// doi.org/10.3390/app10144852. Licensed under CC by 4.0.*

#### **3. Application of nanotechnology in stem cell-based regeneration**

One of the main concerns in cell therapy is whether SCs can be guided to specific areas to repair damaged brain regions [35]. In earlier studies, cells could only be observed after the animals were sacrificed. The mechanism of stem cell homing to specific tissues was identified using novel labelling techniques or materials without compromising SC proliferation, differentiation or migration, which is vital for tissue engineering and regenerative medicine. Proper labelling enables the practical detection of transplanted cells and the tracking of cells at the defect site to ascertain their role in tissue regeneration. For example, hMSCs were labeled with different nanoparticles, including quantum dots, fluorescence-labeled silica nanoparticles, gold nanoparticles and super-paramagnetic iron oxide nanoparticles, to follow these cells during live imaging and ascertain whether SCs are taking part in repair processes. Magnetic nanoparticles (dMNPs) coated with polyamidoamine dendrimer are used to incorporate the pluripotent transcription factors Oct4, Sox2, Lin 28 and Nanog to manufacture lentiviruses that generate iPSCs [36]. The generated lentivirus was ten times more potent than viruses produced by the liposome method. After generating iPSCs, these cells were labeled with fluorescent magnetic nanoparticles. The fluorescence signals were observed using fluorescence microscopy, and the magnetic nanoparticles were located using magnetic resonance imaging. Successful cellular uptake and long-term retention of these nanoparticles in cells are advantageous for monitoring and labelling these cells after implantation. Even though these different nanoparticles can penetrate and mark cells effectively and efficiently, cytotoxicity has been raised as a concern about nanoparticle application. The cytotoxic effect of a substance is influenced by its size, shape, content, surface charge and hydrophobicity. These NP characteristics result in a rise in cytosolic reactive oxygen species, chromosomal aberrations and cell death. Therefore, it is crucial to focus on enhancing this nanoparticle's cellular absorption for monitoring and labelling while reducing their cytotoxicity and interference with cellular differentiation in the context

*Nanotechnology-Based Stem Cell Therapy: Current Status and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.109275*

#### **Figure 3.**

*Nanoparticle mediated transduction of induced pluripotent stem cells differentiate into specialized cells.*

of biocompatibility. Future research on NP applications should concentrate on advancements in tissue-specific cell labelling, imaging and tracking (**Figure 3**).

Metallic NP-induced processes stimulate the proliferation and differentiation of stem cells through several mechanisms, including the alteration of signaling pathways, the production of reactive oxygen species and the tinkering of numerous transcription factors. Metallic nanoparticles have a potential impact on stem cell differentiation and proliferation both *in vivo* and *in vitro*. The superparamagnetic properties of the IONPs (iron oxide nanoparticles), often referred to as superparamagnetic iron oxide (SPIO) NPs, enable them to travel to the injured region, making them a potentially valuable tool for the treatment of degenerative illnesses. Human MSCs (hMSCs) can multiply when treated with SPIO- (Ferucarbotran) nanoparticles, which work by reducing intracellular H2O2. They can also speed up the cell cycle by upregulating proteins like cyclin D1, cyclin B, and cyclin-dependent kinase 4. Consequently, SPIO-NPs can be exploited as a secure supply of nanomaterials to promote the proliferation of stem cells [37].

#### **4. Application of nanotechnology in stem cell imaging**

For imaging and stem cell tracing, nanoparticles such as gold nanorods, MNPs and quantum dots have been utilized due to their distinct characteristic features. In cellular imaging, immunoassays, DNA hybridization and optical barcoding, QDs have been employed successfully. A new practical platform introduced for bioanalytical sciences and biomedical engineering is provided by quantum dots. Mesenchymal stem cells (MSCs) can internalize the quantum dots coupled with an antibody against the mortalin protein to produce i-QD composites, which label the MSCs. The normal adipocyte, osteocyte and chondrocyte development that the i-QD tagged MSCs underwent *in vitro* and *in vivo* strongly suggests that i-QDs can be used for *in vivo* imaging diagnostics and tracing of stem cells in the distribution of mouse body [38]. QDs can be created nanoprobes with unique functions that can be utilized for molecular imaging, gene or medication administration or molecule tracing. These biomolecules can be added to QDs, such

as liposomes, PEG, peptides or antibodies. MNPs were used for molecular imaging, and stem cell tracing in addition to quantum dots. Superparamagnetic iron oxide nanoparticles (SPIONs) were found to be multifunctional MRI-based contrast agents and the same can be used for labelling and tracking transplanted stem cells [39, 40]. Dextran-coated iron oxide nanoparticles were covalently bounded to fluorescent molecules to define HSC labelling and the engraftment process. Fluorophores were conjugated to the dextran coat for fluorescence-activated cell sorting and purification, which removed false signals from nanoparticle contaminants that were not sequestered [27].

With no significant toxicity *in vitro* or *in vivo*, a short-term specified incubation technique was devised, effectively labeled both cycling and quiescent HSCs. Immunodeficient mice were given purified primary human cord blood cells that were CD34-positive and lineage-depleted, allowing tagged human HSCs to be found in the recipient mice's bones. The cell populations that had snatched up the nanoparticles were precisely quantified, and their destiny after transplantation was monitored using flow cytometry. The presence of MNPs-labeled human stem cells in the bone marrow was confirmed by flow cytometry analysis. There has been substantial research in stem cell treatment for various central nervous system (CNS) illnesses [41]. The Endoremlabeled GFP MSCs were transplanted to rats intravenously into the femoral vein or intra-cerebrally into the hemisphere oblique to the lesion the cells were grafted. A 4.7-T Bruker spectrometer was used to check on rats with grafted stem cells once per week from three to seven weeks after transplantation. On MR scans, the lesion appeared as a hyper-intense signal. Its intensity matched GFP labelling or Prussian blue staining. One week following a transverse spinal cord lesion, MSCs tagged with Endorem were also delivered intravenously into the femoral vein. The lesion cavity appeared as inhomogeneous tissue with a significant hyper-intensive signal on MR images of longitudinal spinal cord slices from animals without spinal grafts but with a lesion. Dark hypo-intense patches were seen as lesions in transplanted animals.

Histological analysis revealed that transplanted mice had a substantial iron positive while lesioned control animals had only a few iron-containing cells. The lesion in grafted animals was significantly smaller than in control rats, indicating that the grafted MSCs had a beneficial impact on lesion repair. There are numerous successful uses of MR tracking in various organs, including the heart, liver, kidney and pancreatic islets. Fluorescent MNPs (FMNPs) can combine with the BRCAA1 antibody to create BRCAA1 antibody-labeled FMMNP probes, BRCAA1 protein showed signs of overexpression in ES CCE cells [27].

#### **5. Application of nanotechnology in stem cell tracking**

Conventional methods to track implanted stem cell fate predicted *in vitro* cell labelling for cell transplantation, subsequent follow-up of cell engraftment and existence concluded by the analysis of histological sections of sacrificed animals or tissue biopsies, by this invasive technique did not permit long-term and continuous experimentation. Recent advances in stem cell therapy need more precise and non-invasive methods for qualitatively and quantitatively tracking transplanted cells inside the host to facilitate the understanding of the prognosis of neurodegenerative treatment and eventually improve patients' health.

The traditional techniques have been developed by the improvement of specific contrast agents, such as endogenous biomolecules with intrinsic fluorescence, exogenous fluorescent proteins or non-fluorescent organic dyes, which have been used

#### *Nanotechnology-Based Stem Cell Therapy: Current Status and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.109275*

for fundamentally two labelling modalities. Which are direct labelling and indirect labelling. (a) direct labelling is the cell incubated with specific intracellular probes; (b) indirect labelling is the cell tracked through the expression of the indicator by a reporter gene inserted in the genome of the cells. Direct methods are simple to apply and less expensive, although potential limitations include fast signal decay due to cell proliferation and subsequent insufficient marker distribution between daughter cells. Alternatively, an indirect technique is much more stable but needs genetic manipulation of cells and has not been suitable for clinical applications. Generally, the active contrast agents frequently present disadvantages like photo-bleaching over time, interference derived from tissue auto-fluorescence, chemical and metabolic degradation *in vivo* and even low transfection efficiency in primary cells and thus are not considered suitable for *in vivo* imaging. Several engineered nanoparticles with unique magnetic and optical properties have been established and employed in biomedicine due to their capability to offer real-time monitoring of tracking intracellular processes at a biomolecular level [42]. The transplanted stem cells labeled by these nanoparticles can be detected by multiple imaging methods, such as magnetic resonance imaging (MRI), nuclear imaging, single-photon emission computed tomography imaging (SPECT), positron emission tomography-computed tomography (PETCT) and photoacoustic imaging [43–46].

Stem cells are tracked by the functional modification of nanoparticles such as Gd-based nanoparticles are the most extensively used T1-contrast agent for labelling and tracking stem cells [47]. The GD-based nanoparticles composed of spherical europium-doped gadolinium oxysulfide (Gd2O2S: Eu3+) have been fabricated and observed by MRI, X-ray imaging and photoluminescence imaging. The number of MSCs labeled by Gd2O2S: Eu3+ have feasible cell tracking in animal models [48].

Au-based nanoparticles are potential contrast agents of photoacoustic imaging developing as a modern method for tracking cells *in vivo*. More significantly, MSCs can be directly labeled by Au-based nanoparticles, so their differentiation after transplantation *in vivo* has been noticed using photoacoustic imaging *in vivo* [49, 50]. Huang et al. [51] synthesized Au-based nanoparticles (AA@ICG@PLL) with dualmodal imaging (CT and near-infrared fluorescence) for labelling and tracking MSCs of mice. AA@ICG@PLL exhibited excellent cellular uptake by MSCs and biocompatibility due to the modification of indocyanine green (ICG) and poly-L-lysine (PLL).

Hsieh et al. [52] described a QD-based NP for labelling human MSCs, in which CdSe was used as the core, and the shell was encapsulated by ZnS. Chen et al. [53] stated an AgS2 QD-based NP for tracking human MSCs transplanted in the mouse by employing fluorescence imaging. Li et al. [54] developed QDs-based nanoparticles (RGD-β-CDQDs) to label and track human MSCs, which were fabricated of QDs, β-cyclodextrin (β-CD) and Cys-Lys-Lys-ArgGly-Asp (CKKRGD) peptide. The QDs altered by b-CD had greater cellular uptake and eased the differentiation of MSCs, due to the small molecule dexamethasone and siRNA carried by b-CD. Further significantly, the labeled MSCs have been identified for one month.

Super-paramagnetic iron oxide nanoparticles (SPIO NPs) are synthesized for labelling MSCs. The labeled MSCs have been tracked in an animal model by MRI [55–58]. Furthermore, these labeled MSCs still upheld differentiation potential. Lee et al. [59] synthesized SPIO NPs with the modification of poly lactic-co-glycolic acid (PLGA) and then utilized fluorescent dye Cy5.5 to functionalize the synthesized nanoparticles for labelling and tracking MSCs to explore the interactions between PLGA-SPIO NPs and MSCs.

Ma et al. [60] stated up-conversion-based nanoparticles, which were fabricated by NaYF4:Yb3+, Er3+ NPs, poly (acrylic acid) (PAA) and poly (allylamine hydrochloride)

(PAH), as a fluorescence maker for tracking bone marrow MSCs *in vitro*. Kang et al. [61] developed a UC-based NP with NIR-controllable properties to label MSCs. In a remote-controllable way, stem cell differentiation was regulated. Moreover, Ren et al. [62] produced conversion-based nanoparticles NaYF4:Yb/Erusedligand free labelling and tracking mouse bone MSCs.

Huang et al. [63] synthesized mesoporous silica nanoparticles altered by fluorescein isothiocyanate, and the labeled MSCs have been identified by imaging to track their viability *in vivo*. Due to clathrin-mediated endocytosis, the nanoparticles have been internalized into MSCs and showed greater cellular uptake. Additionally, Chen and Jokerst [64] used silica nanoparticles to label MSCs and then track the MSCs by ultrasound imaging. The results exhibited that silica nanoparticles have expressively increased the ultrasound signal of MSCs *in vivo*. Yao et al. [65] described unique core-shell nanoparticles in which the core is composed of cobalt protoporphyrin IX (CoPP)-loaded mesoporous silica nanoparticles, and the shell is a 125I-conjugated/ spermine-modified dextran polymer, to label and guide the transplantation of MSCs by PA imaging and SPCT nuclear imaging. Chen et al. [66] developed three sizes of silicon carbide nanoparticles to label MSCs and showed dual-modality imaging of photoluminescence and photoacoustic imaging. Cyanine dye-doped silica nanoparticles have been used to label hMSC without affecting stemness surface marker expression, proliferation, viability and differentiation capability into osteocytes [67].

Lim et al. [68] fabricated bicyclononyne (BCN)-conjugated glycol chitosan nanoparticles (BCN-NPs) as dual-modal stem cell imaging probes for the cellular imaging system. Yin et al. [69] described an organic semiconducting polymer nanoparticle (OSPNC) as a contrast agent for tracking MSCs. The developed cationic nanoparticles revealed the intensive tissue imaging due to the meaningfully higher signal-to-noise (SNR) and improved the cellular uptake for human MSCs because of their biocompatibility, appropriate size and optimized surface property.

#### **6. Nano patterns drive the fate of stem cells into a specific cell lineage**

The instructional and tissue-specific niches of stem cells play a variety of activities, including migration, adhesion and proliferation. Extracellular matrix (ECM) components of nanoscale feature-sized fibrillary collagens, elastin and glycosaminoglycans are particularly prone to affect SCs. The topography and part structure of the ECM can compel stem cells to develop into particular cell lineages.

The crucial step in stem cell-based therapies is to direct SCs with accurate fabrication in a defined direction. Nanotechnologists have created several synthetic nanoplatforms that mimic the topological characteristics of the natural SCs niche to stimulate stem cell activation. The attachment of SCs surface proteins to topography is a fundamental aspect of the mechanism via which stem cells interpret and respond to nanotopographical signals. Focused adhesion, a form of integrin-mediated cell attachment to ECM components, is essential for stem cell regulation. Gene and protein levels will vary with mechanical stimulation and regulating focal adhesions will affect the stem cell differentiation pathway. Integrin-mediated adhesion signaling and other factors that impact the state of the SCs include cytoskeleton (CSK) stress, SC structure and nuclear dynamics. Arginine-glycine-aspartate is a crucial peptide episode in ECM proteins that regulates cell adherence (RGD). Recent studies have concentrated on the effect of RGD-containing nanopatterns on stem cell activity.

#### *Nanotechnology-Based Stem Cell Therapy: Current Status and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.109275*

Cao et al. [70] planned the synthesis of several charged or neutral oligopeptide motifs connected with RGD using quartz substrates as a model and were employed for surface modification. They showed that, in the presence of RGD, positively charged oligopeptide patterns hinder osteogenic development, but negatively charged and neutral oligopeptide patterns may promote it.

Wang et al. [71] investigated the effects of RGD nanospacings ranging from 37 to 124 nm on the conduct of MSC. RGD nanopatterns were developed on PEG hydrogels. Cells were exposed to these nanopatterns at the highest serum level for eight days. They differentiated SCs into adipogenic and osteogenic lineages with large and small nanospacings. Stem cell activity is influenced by the symmetry, size, and regularity of surface nano-topographic features, which have been shown to have a substantial impact. Park et al. [32] showed that MSC activity, which includes differentiation, development and spreading, is significantly dependent on the diameter (d) of self-assembled layers (SAL) of TiO2 nanotubes. They showed that osteogenic differentiation of MSC can be significantly reduced by increasing the diameter of the tube to 50 nm or higher after separating SCs into osteogenic cells through a tube having a 15 nm diameter. Researchers have looked into how different-pitch nanogrooves affect the ability of SCs to self-renew, differentiate and proliferate.

Currently, an important area of research involves the merger of SC nanotechnology (SC-NTech) and tissue engineering ideas. Nano-engineered 3-D scaffolds are frequently used to make it possible for SCs to differentiate into specific cell lineages. These three-dimensional scaffolds might be biodegradable, allowing cells to produce their own ECM as the synthetic scaffold degrades. For example, using nanofibrous scaffolds in bone tissue engineering intensely increased the differentiation of SCs into osteogenic cells compared to controls.

#### **7. Application of nanotechnology in stem-cell-based tissue engineering**

The principle of tissue engineering combined with stem cells enables the development of a stem cell-based therapeutic strategy for human diseases. Stem cell and progenitor cell steering differentiation is presently one hotspot, the differentiation of stem cells that conjugate 3D materials is deliberated as the most perspective tissue engineering. Recently, the developments of several micro/nanofabrication technologies have been used to stimulate stem cells to develop into 3D biodegradable scaffolds. Nanostructured scaffolds are fabricated to initiate stem cells to turn into specific cell types compromising the tissues and organs in the body. Inside these scaffolds, cells secrete their matrix, and as the scaffold degrades, they form a 3D tissue structure that mimics the body's natural tissues. Gelain et al. [72] described that they had established a 3D cell culture system using an exclusive peptide nanofiber scaffold with mouse adult neural stem cells. They prepared 18 different peptides, which directly integrate various functional motifs to stimulate cell adhesion, differentiation and bone marrow homing and engraftment activities. These functionalized peptides are self-assembled into nanofiber scaffolds where cells have been completely entrenched by the scaffold in 3D. Without the addition of neurotrophic factors and soluble growth factors, two of these scaffolds functionalized with bone marrow homing motifs significantly enhanced the survival of the neural stem cells and also encouraged differentiation towards cells expressing neuronal and glial markers.

Carbon nanotube patterns have been used to improve the growth and alignment of MSCs. The MSCs revealed in CNT growth patterns, and the cell culture results

showed that the CNT designs have no harmful consequence on the MSCs [73]. The outcomes demonstrated that CNT patterns have enormous potential as a new platform for basic research and applications expanding stem cells.

Stem cell differentiation is diligently related to their microenvironment. The regulation of stem cells is contingent on their dealings with a highly specialized microenvironment or niche. Secreted factors, stem cell-neighboring cell interactions, extracellular matrix (ECM) and mechanical properties collectively made the stem cell microenvironment. The stem cell niche secretes suitable chemicals to direct the differentiation and development of stem cells. Mineral components are essential to stem cell localization; matrix components are vital to the restraint of stem cells, and bone-forming osteoblasts are also important to the maintenance and proliferation of stem cells, the calcium-sensing receptor located on the surface of HSCs, and other cells are critical to stem cells finding their niche.

Nanotechnology has been employed to create artificial *in vivo* conditions like stem cell microenvironments to discover the fundamental mechanisms of the conversion into differentiated cells. A better solution is presently under exploration: growing the stem cells on a so-called 'lab-on-a-chip'. They synthesize a silicon chip with a thousand nanoreservoir cavities, which surface contains about a thousand reservoir cavities, with each reservoir only about 500 nm across. A reservoir that holds liquid chemicals similar to the stem cells has been exposed to the niche. Each reservoir is covered with a lipid bilayer model resembling a cell membrane. These reservoir bilayers also hold the same voltage-gated channels found in cells. A small charge of electricity has been applied to any individual reservoir to open the channels and allow the chemicals to spill out, delivering them to develop any particular stem cell. The nanoreservoir chip technology also allows the opportunity of growing cells layer by layer, making compound tissues, which are otherwise challenging to produce.

Substrate topography impacts a wide range of stem cell behaviors in a manner discrete from surface chemistry. One physical difference in the topography of divergent basement membranes is the size of pores and ridges. *In vivo* cell never see flat surfaces: on the nanoscale, no basement membrane or extracellular matrix is flat. The great majority of features in the extracellular environment are in the submicron to the nanoscale range, confirming that an individual cell interacts with numerous topographic features. Nanofibrous structures have favorably modulated osteoblast, osteoclast and fibroblast activities towards the implant or scaffold materials. Nanofibrous matrices are presented as scaffolds that have improved structural similarity to target tissues than their bulk counterparts because leading mechanisms in tissues are nanoscale structures, and cells seem to adhere and proliferate enhanced on nanoscale structures than on bulk materials. The synthesis of natural polymer-based nanofibers is advantageous because of their proven biocompatibility and biodegradation. Strategic aspects of natural polymers include less immune reaction, nontoxicity, hydrophilicity, enhanced cell adhesion and proliferation. The electrospinning method was adapted to fabricate natural polymer nanofibers. Chitosan and alginate, abundant natural polymers have been widely used in tissue engineering, but none had been fabricated into nanostructured matrices until recent years. Uzieliene et al. [74] described that they effectively used chitosan and alginatebased nanofibrous matrices to mimic the extracellular matrix of articular cartilage that mainly contains type II collagen and proteoglycans (glycosaminoglycan, GAG). A nanopit template was created with a conglomeration surface less than 100 nm in diameter. The flat culture surface and nutrient medium of nanopit align ordered the stem cell has been not differentiated. The stem cell could grow to the calcified

ossature cell in the nutrient medium concurrent with well-ordered and unordered aligned nanopit. The surface of the transplanted tissue is the nanoengineering surface that has induced the stem cell to propagate into the ossature. Surface character plays a significant role in stem cell proliferation.

#### **8. Nanoparticle-mediated gene delivery systems for stem cells**

Recent research has previously revealed the therapeutic uses of embryonic stem cells (ESCs), and the generation of progenitor cells with *in vivo* reconstitution properties has also been described for the treatment of severe hereditary, excruciating and degenerative illnesses [75]. A fundamental barrier to the therapeutic uses of these pluripotent cells is the lack of non-invasive and live cell imaging of grafted cells to manage biodistribution (*in-vivo* tracking). Additionally, reproducible methods for the effective intracellular distribution of biomolecules such as RNA, DNA, peptides and proteins are required to control ES cell development should be developed.

Fluorescent multi-walled nanotubes of carbon (dMNTs-C) functionalized with polyamidoamine are very successful at penetrating the CCE embryonic stem cell line in mice [76]. As they are easier to use and can be produced in large quantities than viral vectors, which are riskier for therapeutic use, dendrimers could be a viable non-viral transmission vector. It has been found that dendrimer-modified polyamidoamine (PAMAM) MNPs significantly boost the efficacy of gene delivery [77, 78]. The dMNTs will be a modern method of gene transfer for ESCs and will be used in ES research. Nanoparticles such as MNPs [79] and QDs can penetrate human MSC cells and sustain themselves in ES cells for a long time. Previous studies have shown that SiO2-coated CdTe nanoparticles can bind to and support inside of induceddifferentiated neurons, hematopoietic cells and endothelial cells while exhibiting minimal cytotoxicity at the applied dose. It is simple to show that these transplanted stem cells with MNPs formed teratomas made up of tissues from all three germ layers [41]. Recently, a biological delivery technique that uses nanoneedles and atomic force microscopy (AFM) to transport genes into living cells was created [80].

El-Kharrag et al. [81] examined polymer-based nanoparticles (NPs) for the delivery of mRNA and nucleases to human granulocyte colony-stimulating factor (GCSF)-mobilized CD34+ cells, which might also be employed for *in vivo* administration. The effectiveness of NP-mediated *ex vivo* administration was closely associated with the charge of the nanoparticles and exhibited minimal toxicity. When compared directly to electroporation, NP-mediated gene editing allowed for a 3-fold decrease in reagent usage while maintaining comparable efficiency. Furthermore, employing nanoparticles showed increasing human HSC engraftment capacity in the NSG mice xenograft model. Finally, successfully stored mRNA- and nuclease-loaded nanoparticles were lyophilized, preserving their transfection capacity following rehydration.

#### **9. Nanoparticles as macromolecular delivery systems for stem cells**

Stem cells are unique cells found in the body and are rightly called internal repair systems. The unique properties of stem cells are the ability to proliferate extensively and differentiate into specific cells that are used in therapeutic procedures against dreadful diseases. The biggest challenge ahead of using this cell is to find an effective way to maintain the division and differentiation of stem cells under tightly regulated patterns. It is found that several macromolecules, such as DNA, RNA, proteins and peptides, regulate these pathways effectively. These macromolecules can be introduced into the stem cells at the right time to make it possible. Conventional methods will not work out because of the complexities associated with the cells and macromolecules. Although physical methods such as electroporation and nucleofection could bring out promising results simultaneously, it causes irreversible damage to the cells under some circumstances. Research could also bring in viral vectors concurrently, causing drawbacks, such as toxicity and mutagenesis, and has not been forward to accomplish the transfer. Nanoparticles are found to be very effective after surface modification. They considered several parameters such as size, shape and design of nanoparticles made by Zhu et al. [82] to deliver the plasmid into mouse embryonic fibroblast cells to reprogram it into pluripotent cells. A plasmid carrying OSKM (arginine terminated polyamidoamine) nanoparticle was used to carry out the shipment of macromolecule into the target. In another experiment, Sohn et al. [83] used acid-senitive polyketal-based nanoparticles to activate pluripotency in bone marrow mononuclear cells. The outcome that was fertile in polyketal-based nanoparticles would produce multiple reprogrammed cells. Besides, mesoporous silica nanoparticles were analyzed by Chen et al. [84] for their efficacy against induced pluripotent stem cells. The outcomes of mesoporous silica nanoparticles lead the way by limited cytotoxicity against induced pluripotent stem cells. Positively charged (cationic) nanoparticles were chosen to deliver hepatocyte nuclear 3b factor. It was found that it increased the mRNA concentration in stem cells with liver-specific genes and activated those cells to differentiate into cells resembling hepatocytes with similar functions.

#### **10. Application of nanotechnology in stem-cell-based therapy of neurodegenerative diseases**

Neurodegenerative diseases (ND) can be defined as the gradual degeneration of neurons, which are considered the fundamental unit of the nervous system (**Figure 4**). Neural degeneration could affect the patient, their family members and society. Henceforward, there is no prominent treatment procedure to handle the disease even though the symptoms of ND can be slowed down [85]. A team of doctors, neuroscientists and bioengineers are required to standardize procedures to treat the disease effectively [86]. The neurogenesis cascade of complex mechanism leads to the synthesis of neurons, which makes up the CNS. Stems cells derived from different sources can be subjected to a sequence of processes such as proliferation and differentiation. The differentiated stem cells can be used as ideal drug candidates for cell-based therapy [87].

The key objectives of cell-based therapy are to protect the neurons and to enhance the differentiation and regeneration potential. In recent years, applications of stem cells in cell therapy to treat neurodegenerative diseases have gained more attention among researchers [88]. The neuroprotective effect of stem cells has been scientifically proven [89]. Transplantation of stem cells is positive regulation in Parkinson's disease (PD), spinal muscular dystrophy (SMD) and amyotrophic lateral sclerosis (ALS) [90]. Nanomaterials are considered by the biomedical domain, an effective tool to carry value-added drugs and to deliver those chemicals to the specified target. Since nanoparticles are special and unique properties [91], the nanotechnology domain can be coupled with cell therapy to extend better treatment to people

*Nanotechnology-Based Stem Cell Therapy: Current Status and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.109275*

**Figure 4.**

*Current novel methods to utilize stem cells in cell therapy in treatment of neurodegenerative disease.*

experiencing severe forms of neurodegenerative diseases. The microenvironment of the CNS is called the stem cell niche. When the stem cell niche is administered with pro-neurogenic factors (proteins), it can kindle the proliferation and differentiation of endogenous and exogenous neural stem cells [92]. After successful internalization of the nanoparticles, it can stimulate neurogenesis, which is a much-expected point in the treatment of neurodegenerative diseases. But still, researchers are needed to find out the mystery behind the relationship between stem cells and nanoparticles. If those mysteries might be identified then it may be easy to use the stem cell—nanoparticle complex to treat the neurodegenerative disease effectively.

A high translational potential exists for neural stem cells (NSCs) in transplantation therapy for neural repair. A vital objective of regenerative neurology is to increase the therapeutic potential of these cells through genetic engineering. Major non-viral vectors for the safe bioengineering of NSCs include magnetic nanoparticles (MNPs), which have imperative advantages over viral vectors in terms of safety, scalability and use.

#### **11. Nanomedicine in cancer stem cell therapy**

Conventional therapies such as chemotherapy and radiation therapy remove the tumor but not the cancer stem cell (CSC). Permanent removal of cancer stem cells could result in long-lasting remission of disease, remarkable reduction in metastasis and seems to boost the immune status of the patients. Again, cancer stem cell (CSC) therapies are controlled by nanotechnology and carry them with therapeutic payloads (TPL) [93]. Nanoparticles are engineered in such a way that to attack the cells with over-expressed receptor proteins called CD44. Hyaluronic acid situated on the surface of the B16F10 cells could lead to the bonding with CD44. The study confirms that nanoparticles are remarkably useful for the shipment of CSC suppression antitumour drugs [94].

A novel therapeutic procedure such as nucleus-targeted drug delivery (NTDD) can help researchers to reverse the drug resistance in CSC. Silica nanoparticles are engineered in such a way as to attack the nucleus of the CSC. Surface modulation coupled with thermal sensitive exposure could help to reach the nucleus efficiently. Nucleus-targeted drug delivery facilitates the apoptosis of CSC, which in turn is caused by chemotherapy and thermotherapy [95].

#### **12. Nanoparticles as macromolecular delivery systems for glioblastoma**

Macromolecular drug delivery has taken a bounce in the last 20 years [96]. Due to the robust development in the biotechnology domain, enough novel methods have been developed based on macromolecules such as DNA, RNA, siRNA, proteins and peptides. The U.S Food and Drug Administration (FDA) has classified macromolecular drugs into vaccines, blood and blood components and allergen extracts are used for diagnosis and treatment. In the modern drug delivery system (DDS), an important property called "active targeting" is exploited to make the DDS deliver the drugs selectively to the target without affecting the healthy neighboring cells [97]. Glioblastoma is a deadly form of malignant tumor of the central nervous system (CNS). Current treatment relies on giving radiation therapy followed by a chemotherapeutics regime using a DNA alkylating agent. Life expectancy is also less even after undergoing a series of treatment procedures. The cancer progression leads to the impact of GBM (Glioblastoma) after reaching into the deeper areas of the brain. Hence standard and alternative methods are required to extend drug delivery effectively. In this method, one such effective tool is the solid-lipid nanoparticle developed by Kuo et al. [98]. These nanoparticles are conjugated with metallotransferrin antibodies. Further, the transcytosis property of the nanoparticle across human brain-microvascular endothelial cells was examined and found to be very effective, and at the same time, it inhibits the growth of U87MG cells *in vitro*.

#### **13. Conclusion**

Stem cell nanotechnology begins new avenues for the manufacture, study and potential application of SCs in regenerative medicine. For imaging and labelling, drug or gene administration, tissue engineering scaffolds and stem cell proliferation monitoring, nanomaterials such as fluorescent CNTs, QDs, fluorescent MNPs and fluorescent CNTs, among others, have been used. Differentiation-engineered nanostructures have been employed, and it is anticipated that they would speed up the detection and monitoring of microenvironmental signals. Despite numerous challenges, stem cell nanotechnology offers new opportunities that will considerably improve the identification and tracking of SC-fate and will develop novel stem cell therapies. As a result, stem cell-based therapies would be furnished as an alternative and effective remedy for genetic disorder.

*Nanotechnology-Based Stem Cell Therapy: Current Status and Perspectives DOI: http://dx.doi.org/10.5772/intechopen.109275*

### **Author details**

Ponpandian Samuel1 \*, Shenbagamoorthy Sundarraj2 \* and D.N.P. Sudarmani3

1 Department of Biotechnology, Ayya Nadar Janaki Ammal College (Autonomous), Sivakasi, Tamil Nadu, India

2 Postgraduate and Research Department of Zoology, V.O.Chidambaram College, Tuticorin, Tamil Nadu, India

3 Department of Zoology, Ayya Nadar Janaki Ammal College (Autonomous), Sivakasi, Tamil Nadu, India

\*Address all correspondence to: samuel\_sf432@anjaconline.org and shenvsun@gmail. com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 6**

## 3D Culturing of Stem Cells: An Emerging Technique for Advancing Fundamental Research in Regenerative Medicine

*Sonali Rawat, Yashvi Sharma, Misba Majood and Sujata Mohanty*

#### **Abstract**

Regenerative medicine has been coming into spotlight ever since the realisation that conventional treatments are not enough, and the need for specific therapies has emerged. This, however, has paved way for cell-free therapy using extracellular vesicles. A two-dimensional (2D) cell culture model is widely recognised as the "gold standard" for researching cellular communications ex vivo. Although the 2D culture technique is straightforward and easy to use, it cannot replicate the in vivo ECM interactions & microenvironment. On the contrary, 3D culture culturing technology has emerged which include structures such as spheroids and organoids. Organoids are small replicas of in vivo tissues and organs, which faithfully recreate their structures and functions. These could be used as models to derive stem cells based EVs for manufacturing purposes. The linkages between infection and cancer growth, as well as mutation and carcinogenesis, may be modelled using this bioengineered platform. All in all, 3D culturing derived EVs serves as a novel platform for diagnostics, drug discovery & delivery, and therapy.

**Keywords:** organoid, 2D & 3D cell culture, extracellular matrix, carcinogenesis, bioengineered platform, drug testing

#### **1. Introduction**

Cell culture has become an indispensable tool for elucidating fundamental biophysical and biomolecular mechanisms that govern how cells construct into tissues and organs, how well these tissues function, and how that function is disrupted in disease. Cell culture is now used extensively in biomedical research, biomedical engineering, stem cell therapy, and commercial applications. In regenerative medicine, stem cells are the central players, however the shortcomings and risks associated with cellular therapy are higher as compared to non-cellular therapy thereby EVs are better. Although adherent, two-dimensional (2D) cell culture has

long been the norm, recent research has shifted toward three-dimensional (3D) structures and more feasible biochemical and biomechanical microenvironments. Understanding the *in vivo* processes that leads to the formation and purpose of tissues and organs requires deciphering the mechanisms underlying these behaviours. Laboratory experiments should ideally be carried out using a user-defined threedimensional (3D) model that closely mimics the cell's microenvironment [1–3]. However, challenges in developing such a model include the building of the tissuetissue interface, control of the spatiotemporal distributions of oxygen, carbon dioxide, nutrients or waste, and further followed by the customization of other microenvironmental factors known to regulate activities *in vivo*. It is well understood that cells adapt to their surroundings by reacting to local signals and cues, which has implications for cell proliferation, differentiation, and function [4, 5]. Traditional culture methods for growing mammalian cells *in vitro* are far removed from the complexities that cells encounter in real-life tissues. One of the most noticeable physical differences is the shape and geometry that cells acquire when grown on a flat substrate, such as a cell culture plate or flask. When cells grow on two-dimensional (2D) surfaces, they flatten and remodel their internal cytoskeletons. Lacking the ability to form more natural tissue-like structures, existing *in vitro* 2D cell culture models are frequently a poor substitute when used to study cell growth and various associated aspects [6]. This has a substantial impact on cell performance, as well as, the outcomes of biological assays. Monolayers of cultured cells, for example, are assumed to be more sensitive to therapeutics. Moreover, due to the limited cell interactions, culturing cells on rigid surfaces may increase cell proliferation, but adversely impact cell differentiation. A more adequately engineered cell culture environment might enhance drug discovery predictive accuracy and aid in the interpretation of tissue morphogenesis [4–6].

Some significant aspects of cancer cells, for example, cannot be adequately modelled in 2D cultures. To overcome the limitations, novel 3D cell culture platforms that better mimic in vivo conditions are now being developed, which are sometimes referred to as spheroid or organoid culture. In many cases, these new platforms have shown to be more capable of stimulating *in vivo*-like cell fates for the processes under investigation. 3D research shows that increasing the dimensionality of the extracellular matrix (ECM) surrounding cells from 2D to 3D has a significant impact on cell proliferation, differentiation, mechano-responses, and cell survival [5, 7, 8].

For example, Extracellular vesicles (EVs) are membrane-enclosed structures that are released by almost all cell types. They transport biologically active molecules such as RNAs, lipids, and proteins from the delivering cell to the target cell, allowing for a novel mode of intercellular communication. The use of EVs as diagnostic tools is highly influenced not only by the molecular cargo but also by the quantity of EVs derived from various cell subpopulations in tissues and body fluids. Moreover, overall mechanisms and factors influencing EV release are still unknown [9, 10]. Organoids are obtained from animal or patient samples, cultured in 3D matrices like Matrigel under well-defined conditions, and retain the cellular heterogeneity found in in vivo epithelial tissues. As a result, they depict one of the most cutting-edge technologies for studying human diseases, allowing for the investigation of pathways and factors that influence EV release.

Although these findings suggest that 3D systems should be used anytime feasible, the system of choice is often governed by the specific process of interest, and there is currently no universal 3D platform; additionally, 2D cell culture approaches can still recapitulate in vivo behaviour for many bioactivities, and new advances in substrate

*3D Culturing of Stem Cells: An Emerging Technique for Advancing Fundamental Research… DOI: http://dx.doi.org/10.5772/intechopen.109671*

configuration continue to offer new capabilities for this platform [11]. All in all, 3D platforms are probable to become a more appealing alternative to 2D cell culture as technology advances to enable a broader range of processes. Technological advances have opened up new avenues for cell culture and the formation of 3D tissue-like frameworks. This is primarily due to research activities between cell biology and biophysical sciences, which has introduced new materials and manufacturing techniques to produce technologies tailored to support 3D cell growth in vitro [12]. The culture of cells in 3D is rapidly progressing, as evidenced by the increasing number of publications in the scientific literature. The adoption, validation, and implementation of these novel strategies will guarantee the effectiveness of this technology. This will most likely take time as the scientific community recognises the limitations of traditional 2D cell culture and recognises the value of new methods to reliably culture cells in 3D.

#### **2. Technologies for 2D and 3D cell culture**

#### **2.1 2D cell culture techniques**

Traditional 2D cell culture relies on the cells adhering to a flat surface, typically a glass or polystyrene petri dish, to provide mechanical support. Culturing in 2D monolayers allows the cell access to a significant amount of growth factors and nutrients in the form of media, resulting in homogeneous proliferation and expansion [13, 14]. However, a majority of these 2D approaches do not allow for regulation of cell shape, which influences biophysical cues that affect cell biological properties *in vivo*. Micropatterned surfaces, such as cell-adhesive islands, microwells, and micropillars, have indeed been developed to control cell shape in 2D culture and aid in the investigation of the effects of cell shape on bioactive components [15]. This induced polarity may alternate cell functions such as expansion and migration for perceiving soluble components and other microenvironmental signals. One of the strategies to completely eradicate apical-basal directionality is by the sandwich culture procedure, which adds a layer of ECM atop the cells and provides a mimic of 3D ECM that can be used to mitigate the effect of cell polarisation in 2D cell culture (**Figure 1**). The sandwich culture method, which involves placing cells between two layers of ECM, polyacrylamide, collagen or any type of suitable ECM, has long been shown to produce cell cultures with morphology and function that more closely resemble the *in vivo* behaviour. This is especially important in drug discovery, in which scientists aim to understand pharmacokinetic profile in relation to the organs [15]. The sandwich method was discovered to reduce oxygen diffusion sufficiently, resulting in an 80% survival rate over 5 days, compared to a 32% success rate over 2 days in a mixing process population culture. Many researchers have been able to examine the consequences of pharmacokinetics, which is crucial to consider when modelling physiological and pathological events, thanks to the sandwich culture [16, 17]. Another strategy could including micropatterning, which is a designed 2D surface that allows imprinting and alteration to create a 2D microenvironment for cell culture that contains distinct physiochemical factors, topography, stiffness, and mechanical load. In a typical 2D cell culture, cells are subjected to a homogeneous surface free of defects that could interfere with their development [18, 19]. Moreover, cells cultured on patterned and un-patterned PLLA surfaces differentiated at a slower rate than cells cultured on tissue culture-treated polystyrene (PS), the experiment's control. In terms of lipid production, it was discovered that a later time points, shaped

**Figure 1.**

*The representative diagram showing different kinds of cell culture techniques and their impact on cells fate.*

PLLA surfaces produced the most lipids, followed by PS, and then non-patterned PLLA [20, 21]. The findings revealed that the micro-patterning and surface type both influenced the rate of cell differentiation. Overall, since the early 1900s, two-dimensional (2D) cell culture has been the technique used to culture cells, which plays an important role in research but has many drawbacks due to 2D models imprecisely representing tissue cells *in vitro* [22–24].

### **2.2 3D cell culture techniques**

#### *2.2.1 Aggregate cultures and the formation of spheroids*

A 3D culture model is supposed to provide a tissue-like microenvironment in which cells can proliferate, aggregate, and differentiate. This could have an application in predicting the effect of a drug on cells. For several reasons, cells cultured in 3D respond differently to drugs than cells cultured in 2D [25, 26]. Changes in physical and biological features between 2D and 3D cultures make 2D cultured cells more susceptible to drug effects than 3D cultured cells because 2D cultured cells cannot maintain constant morphology like 3D cells. Since 3D cultured cells have greater depth than 2D cells, the variation in shape between 2D and 3D cultured cells creates a change in local pH levels within the cells. Lower intracellular pH levels have been shown to reduce drug efficacy, giving back to drug resistance. Further, Microfluidics, microchips, embryoid bodies (EBs), collagen gels (GELs), and hanging-drop culture are all methods for spheroid cultures [26, 27]. Various studies have established the experiments in two different 3D culture methods based on the differentiation and

#### *3D Culturing of Stem Cells: An Emerging Technique for Advancing Fundamental Research… DOI: http://dx.doi.org/10.5772/intechopen.109671*

proliferation of embryonic stem cells (ESCs). To promote adherence, cells were cultured as embryoid bodies (EBs) in either a collagen type I gel (GEL) or in non-tissue culture-treated dishes. GEL and unattached EB cultures produced cluster morphology that was similar, with defined boundaries and the occasional cavity. The existence of enlarged masses along the edges of the gel form presented a marked difference in the morphology of GEL cultures. Over the same 12-day period, free EBs had more continuous change in the genotypic expression profiles of cytoskeletal genes than GEL cultures [28, 29]. These findings suggest that, despite having similar morphologies, the gene expression of each kind of 3D culture is unique in its adaptation to its microenvironment. Similarly, hanging drop method is another technique to establish spheroids but because of the difficulty in maintenance measures, like changing of media, the traditional hanging-drop method does not allow for extended cell culture.

All such 3D scaffolds and associated cell-encapsulation techniques provide valuable tools for understanding how the ECM influences cell fate. During the last decade, major advances have been made in the techniques for encapsulating cells in 3D using tissue engineering scaffolds with customised biochemical and biophysical components. Majorly, biopolymers derived from animal tissues are especially popular because they contain similar biochemical components to those found in cells' native tissue and may encourage tissue regeneration. However, one of the most pressing issues is the inability to individually control the key elements required in modulating cell bioactivities, such as biochemical properties, matrix elasticity, and macro-porosity [26, 28, 29]. Therefore, a prefabricated scaffolds has the advantage of a configurable biochemical composition, matrix elasticity, and micro-architectures. These scaffolds can be made using polymer phase separation, 3D printing, lyophilizing, gas foaming, stereo-lithography and porogen leaching with soluble templates to form pores or channels. However, current methods for creating prefabricated scaffolds frequently involve procedures that create circumstances which are too severe for cells to survive, such as extreme pressure, non-physiological osmotic pressure, and the use of organic solvents [28, 29]. As a result, cell diffusion is primarily used to deliver cells into scaffolds, and this method is frequently associated with low cell penetration rates and poor scaffold cellularization. Hydrogels made up of various types of biopolymers have been broadly used as scaffolds in contrast to prefabricated scaffolds because of their ease of cell encapsulation. They have tissuelike water content as well as effortlessly controllable biochemical and mechanical characteristics. Most hydrogels, on the other hand, are composed of micron/ nanometre-sized mesh that is frequently too small to facilitate post fabrication cellularization and lack the microtopography required for influencing cell shape and supporting cell mobility, proliferation, and matrix production [30–32]. The main disadvantage of hydrogels in tissue regeneration is that matrix degradation simultaneously changes biochemical elements and matrix elasticity, both of which require careful control. Furthermore, matching the rate of hydrogel degradation with the rate of tissue formation is extremely difficult, which is essential for maintaining the shape and structural stability of tissue engineering.

Making multiple layers of cell sheets is another method for engineering organs and tissues without relying on constructed scaffolds. A plethora of studies have successfully replicated cardiomyocyte pulsatile function and functional dopaminergic neurons in a 3D construct by stacking multiple cell sheets or on the single cell sheet by twitching the mechanical properties of scaffold [29, 31–33].

Apart from this, bioreactors are designed to study cell behaviour during the development of micro-tissues or organs and to generate more cells for clinical use or laboratory research. Large-scale bioreactors involve simple systems like spinner flasks and rotating wall bioreactors, which enable for semi-adherent cell growth, in addition to more complicated systems like gravimetric bioreactors. The impacts of fluid transport on a cell membrane scale have been investigated using bioreactors with cell-sized conduits. Micro-bioreactors have also shown promise in drug screening and controlling the cell microenvironment.

#### *2.2.2 Organoid formation from diseased microenvironment and microfluidic 3D cell culture*

The shift from 2D to 3D culture techniques is a significant step toward more biologically relevant tissue models. However, 3D culture techniques do not yet capture the multicellular intricacies of tissues, they lack vasculature, do not provide precise control over gradients, and exchange medium at discrete time points rather than continuously. Microfluidic techniques enable spatial control of fluids in micrometresized channels, which can be used to investigate the biological significance of 3D culture models (**Figure 1**) [29]. Early examples of spatial patterning of adhesion molecules and hydrogels, which are still employed in microfluidic 3D cell culture, are depicted in **Figure 1**. The three foremost drivers for using microfluidic methodologies in 3D cell culture today are as follows:


An even more functional aspect that can be introduced using microfabrication techniques is mechanobiological aspects such as active stretch and tension. Microfluidic devices can conquer the drawbacks of traditional cell/stem cell culture techniques and tissue engineering approaches by better simulating *in vivo* interplay between ECM and cells thereby enabling high-resolution in situ imaging [25]. The combination of the unique benefits of microfluidics and the breadth of possibilities offered by stem cell technologies can also provide alternatives for the management of neurodegenerative diseases such as Alzheimer's and Parkinson's, as well as other disorders or injuries of the central or peripheral nervous system. This method has even progressed so far as to suggest the development of devices known as "brainon-a-chip" [34, 36]. In neuro-regeneration, for example, these systems enable the development of uniform populations of neuronal and glial cells. The potential to co-culture cells in a 3D arrangement, better controlled signalling, and the ability to combine diffusion and laminar flow are the most significant advantages of microfluidic cell culture. Microfluidics allows researchers to precisely control stem cell/ cell numbers and growth conditions, as well as arrange or design cells in spatially controlled positions and track cell responses to various internal/external mechanical, chemical, and optical stimuli [35–37]. Furthermore, microfluidics techniques enable high-throughput studies of single cells in microenvironments closely mimicking biologically relevant conditions by creating gradients of mechanical forces and different chemical agents. These benefits are classified into four categories: (a) fabrication characteristics, (b) Biomaterial ingredients, (c) Biochemical properties, and (d) bio-physico-mechanical (**Table 1**) [37–39].

*3D Culturing of Stem Cells: An Emerging Technique for Advancing Fundamental Research… DOI: http://dx.doi.org/10.5772/intechopen.109671*


#### **Table 1.**

*Benefits of different aspects of 3D cell culture.*

Overall, the fabrication of an artificial human organ (even going as far as a brain) is beginning to be considered possible due to the multidisciplinary overlap of biology and engineering combined with emerging new trends such as microfluidics, stem cells, and nanotechnology; this has been imagined as a "Organ-on-a-chip." Organs on a chip will provide much better mimicking of real human physiology and will be beneficial for tissue engineering, disease modelling, and drug screening; however, much more well-designed research in this field is still required [37–39, 49].

#### **3. Impact of culture strategy on the secretion and content of extracellular vesicles**

Extracellular vesicles are a novel modality in the scope of diagnosis, drug delivery & regenerative medicine. These are nanoscale vesicles which can be sub divided based upon their mode of synthesis, size and content. The sub category among them which has recently gained popularity is the small vesicles identified in the range

of 30–150 nm & found to be synthesised via the endosomal route, also commonly known as exosomes. These vesicles are naturally found to be involved in mediating inter-cellular communication. They carry a diverse compass of functional molecules including DNA, RNA, miRNA, Proteins, Enzymes & many other are yet under discovery. The release & content of these vesicles are largely influenced by the cell microenvironment & the extracellular cues which are received in a mechanosensitive manner [9]. These vesicles happen to package the content from inside the cell & deliver them to the cell-in-need. These are primarily known to fuse with the cells & release their content in order to facilitate the functioning, however, at certain instances they might as well be phagocytosed. Recent gain of limelight have brought up diverse and divergent functions & applications of these small vesicles, for e.g. Tian et al., suggested the role of these vesicles in the detection of breast cancer. They suggested 8 EV proteins that could serve as a biomarker for diagnosing and differentiation between non metastatic and metastatic breast cancer [50]. Being membrane bound structures, these circulating EVs succeed in preserving their content & can be easily captured by any kind of normal cells. When released from the cancer cells, EVs have been evidenced to enhance the malignancy by causing malignant transformations in the recipient normal cells. Tumorigenicity and cancer spread are highly attributable to the intercellular communication in the tumour microenvironment & in the blood stream via the release of EVs. It was stated by Bebelman et al., that cancer cells are found to exhibit higher EV secretion as compared to the non-cancerous cells [51]. This could be attributed to the fact that cancer cells hold a diminish property of contact inhibition, therefore forming a tightly packed 3D layered & inculcated mass of cells which advances into a tumour. In order to study the biology of this deadly near-pandemic disease, it is essential that the conditions subjected to the tumour in vivo be replicated in in vitro set ups so that the exact nature & habits of a tumour could be elucidated. This has led to invoking the question regarding the culture conditions in which cancer cells & their fate are studied. The most popular choice of cell culture is based upon a 2D sub culturing method wherein the cells are allowed to form a sheet like structure and their propensities are studied [25, 27]. A 2D culture set up is popular due to its ease of handling and other properties as discussed in the previous section, however it does not necessarily confirm that the results being obtained from such a set up are actually a simulation of the in vivo scenario. On that account, 3D culture is the recent technique of choice of researchers as it is expected & anticipated to model the tumour microenvironment in vivo. Pertaining the same, the EVs which are released from the 3D kind of culture have also been found to mimic the in vivo secretions in a more identical manner. Upon comparison of EVs from 2D and 3D culture of the same cell types, it was also found that there were differences in both EV secretion and EV content.

#### **3.1 Effect of 3D culture on EV secretion**

Extracellular vesicles have been the modality of interest for diagnostic & therapeutic purposes. However, their less yield hinders their successful commercialization. Thereby in order to enhance the yield of EVs for commercial purposes, many strategies have been explored. One of these strategies is the culturing of cells in a 3D manner [46]. Kim et al., compared the secretion of mesenchymal stem cell derived EVs in a 2D monolayer culture format vs. a 3D culture format via spheroid formation using the hanging drop method & the poly-HEMA coating. From this study, they found that exosome secretion was significantly enhances upon culturing in a 3D format. This

#### *3D Culturing of Stem Cells: An Emerging Technique for Advancing Fundamental Research… DOI: http://dx.doi.org/10.5772/intechopen.109671*

led them to sought the cause of increase in EV production, which was found to be the creation of hypoxic niche in a 3D culture format, along with the increased cell density and circular cellular morphology [52]. This finding has also been evidenced by many other studies, for e.g. Yan et al., cultured umbilical cord derived MSCs in a hollow fibre bioreactor & found that the EV secretion was increased by 7.5 folds as compared to the EV release in monolayer culturing [53]. Similar finding was also reported by Haraszti et al., wherein 3D culturing of MSCs resulted in 20 fold increase in exosomes concentration when combining 3D culturing of MSCs along with isolation via differential ultracentrifugation. They also established another technique wherein they combined 3D culturing with Tangential flow filtration which thereby enhanced the yield up to 27 folds. Patel et al., developed a culture system by combining a tubular perfusion bioreactor system & a 3D printed scaffold, wherein they found a 100-fold increase in EV production by endothelial cells [54]. Not just in primary cells, but these results are also observed in cancer cells based 3D Culturing [55]. Hwang et al. suggested that the EV release was increased upon 3D culturing in colorectal cancer [56].

#### **3.2 Effect of 3D culture on EV content**

The effect of 3D culturing of cells on EV content is exceptionally significant. 3D culturing has proven time and again that it is a better model to study the cell-ECM & cell-cell interactions as the dynamics of 2D and 3D culturing are poles apart. As a means of cell-cell communication, EVs regulate vibrant interplay mechanisms, and thereby 3D culturing leads to modifications in the cargo of EVs as the stimuli perceived by the cells is varied as compared to 2D culture. It was concluded in one of the studies that 3D culturing leads to an overall depression of protein expression while upregulation of miRNA cargo in EVs due to the downregulation of ARF6 pathway influencing the cell arrangement & secretion profile thereof [57]. Many studies have presented varied views on this matter. It was found that culturing of HeLa cell line in a 3D manner results in the secretion of EVs which were up to 96% similar in their RNA profile with the circulating EVs collected from the plasma of a cervical cancer patient, however the genetic profile of EVs i.e. DNA was unaltered [58]. It is also speculated that EV release in 3D culture systems is aided by the higher expression of transporters [59]. Due to the mechano-sensing based activities in cells upon culturing in the 3D microenvironment, the gene and protein expression of the same cells are differential. For e.g., Eguchi et al. observed that upon 3D culturing, the neuroendocrine adenocarcinoma cells formed large organoids in a steady growing pattern which further expressed numerous stem cell specific markers, neuroendocrine markers and intercellular adhesion molecules. While in case of 2D culturing, it was found that cells had a faster growth rate, while intercellular adhesion molecules were decreased and mesenchymal transition was increased. It was deduced thereby that the 3D culturing of cells leads to the formation of more realistic tumoroids in terms of morphology & gene expression [60]. Furthermore due to enhanced intercellular communication in 3D culturing, EVs which are involved in transcellular transport are more in number when compared to the 2D culture system. Tu et al., realised 3D culturing as a better model for tumour progression, as they estimated the miRNA content of exosomes and protein expression of GPC-1, and found that the trend observed in EVs derived from spheroids presented higher relevance to the progression of pancreatic cancer [61]. Not just in cancer, but 3D culturing has also been tested for primary cells like Mesenchymal Stem Cells (MSCs). It was found the culturing of MSCs in a 3D manner leads to multi-fold increase in the exosome concentration & enrichment of cargo such

that they were more efficient in their uptake capabilities and improved the viability of the recipient cells [62]. Furthermore, it was deduced that the cargo content of MSCs-EVs was vividly distinct when derived from a 3D culture microenvironment. The results of a microarray suggested that expression of 193 miRs were varied wherein 68 miRs were up regulated and 125 miRs were downregulated [63]. Yu et al., also explored the EV dynamics in 3D vs. 2D culture of mesenchymal stem cells & observed that there was a 2.5 fold increase in exosome production upon 3D culturing along with 2.9 fold increase in the enrichment of proteins. Furthermore, they also suggested that exosomes derived from 3D culturing of MSCs had heightened expression of osteo-inductive genes and proteins, which could be attributed to the upregulation of YAP pathway [64]. 3D culture system derived exosomes were also proven to possess extended anti-inflammatory effects and were able to restore the homeostatic balance of Th17 and Treg cells in a model of periodontal inflammation. This was suggested to be happening as a result of enhanced expression of miR1246 in the 3D derived exosomes, thereby affecting the Nfat5 expression which plays a role in Th17 polarity [65]. However, there have also been studies that suggest that EVs isolated from MSCs cultured in a 3D manner did not sufficiently execute the properties which are a characteristic of their parent cells like immunomodulation & anti-fibrotic activity. It was observed that the level of IDO was significantly downregulated when the cells were cultured in 3D & there was a rise in pro-inflammatory capacity of macrophages upon culturing of EVs derived from 3D culture as compared to 2D culture. This could be due to the extensive networking and interactions between the cells itself during 3D culturing & the decline in cell volume thereby affecting the packaging of EVs so released [66].

3D culturing for EV derivation is a budding area of research, and so there is yet nothing conclusive about the possible effects of 3D culturing on exosome release & cargo profile. There have been many contrasting views that support or reject the hypothesis of culturing cells in a 3D manner. It can be accepted that 3D culture model might best be able to replicate the cancer biology due to its ability to replicate tumour like interactions & features in vitro, and concurrent release of in vivo like EVs. Such a culture model could aid the advancements in identification of cancer biomarkers which may be specifically analysed in a simulated manner. While 3D culturing is being preferred in carcinoma-based studies, culturing of primary cells in a 3D microenvironment is still a topic of debate. There have been divergent perspectives of researchers regarding the derivation of EVs from 3D culture of primary cells however, it still needs to be developed further to enhance its benefits, more than its shortcomings. EVs are a recent popular candidate for therapeutics, drug loading & delivery, and 3D culture has shown tremendous potential in enhancing their yield, therefore it could be an interesting application & strategy to exploit these modalities for commercial manufacturing of the EVs.

#### **4. Future prospects of using organoid as drug screening and EVs as biomarker analysis**

Research in cancer has relied heavily for a considerable amount of time on cancer cell lines as a model system. Recent research has made use of high-throughput screening of broad panels of cancer cell lines to detect patterns of drug sensitivity and to correlate drug sensitivity to genetic changes [67]. These high-throughput cell-line-based research paint a picture of a complex network of biological variables

#### *3D Culturing of Stem Cells: An Emerging Technique for Advancing Fundamental Research… DOI: http://dx.doi.org/10.5772/intechopen.109671*

that affect sensitivity to most cancer medicines. It is possible, for example, that there is no direct connection between sensitivity to a particular medicine and individual genetic changes. Instead, the outcome of drug sensitivity may be determined by the complicated interactions that occur between several genetic changes, which are difficult to find. Therefore, despite the new information that has recently become available, it is still difficult to develop algorithms that can accurately predict the drug sensitivity of a patient's tumour based on the spectrum of genomic alterations that are present, in the context of the individual's specific genetic background [68]. Even though there is a great deal of information accessible on the biology of cancer, there are still a great deal of questions regarding this international health issue [69]. There is a clear and pressing requirement to keep researching and developing improved therapies for cancer patients. Incomplete or inaccurate modelling of cancer is one of the primary roadblocks in the way of the development of additional treatment regimens. This is because, at times, cancer models can only poorly recapitulate clinical conditions. Because of this, a significant number of medications that produce encouraging outcomes in cancer models fail when tested in humans. Therefore, despite the fact that animal models appear to provide useful insights into the fundamentals of cancer biology, it is vital to keep in mind that these models frequently fail to faithfully recreate the pathogenic processes that take place in patients [70]. As a result, the field of oncology requires the development of new methodologies and approaches to create fresh targeted medicines and to continue lowering the number of fatalities caused by cancer.

An important advance in scientific methodology over the course of time has led to the development of three-dimensional (3D) organoid culture as well as 3D printed scaffolds, both of which are able to simulate human biology as well as diseases more accurately. In 1946, Smith and Cochrae were the first people to use the term "organoid," which means "resembling an organ," to describe a case of cystic teratoma. This term was used to describe the growth of a cystic teratoma [71]. However, the term "organoid" now has a more restricted definition. This definition states that organoids are self-assembled in vitro 3D structures, which are primarily generated from primary tissues or stem cells such as adult stem cells, induced pluripotent stem cells (iPSCs), and embryonic stem cells (ESCs). The production of organoids is dependent on the self-assembly and differentiation of cells, as well as the signalling signals from the extracellular matrix (ECM) and the conditioned medium. This is true regardless of the circumstances. When the three-dimensional constructions are complete, they are able to replicate the intricate features of their real-life organ equivalents, as well as undergo genetic engineering, long-term expansion, and cryopreservation [72]. Organoids and 3D cultures have emerged because of several attempts to replicate the biology of human organs, such as stem cell development in 2D cultures with or without a 3D matrix, cell culture on a microfluidic device (organon-a-chip), and bio-printing of cells. Opportunities for medication discovery and human disease study have been expanded because of these modelling initiatives [73]. The term "organoids" refers to three-dimensional structures that can self-organise through the processes of self-renewal and tenogenic differentiation. These structures are formed from pluripotent stem cells that have been cultivated from organ-specific tissues. Organoids have a distinct organisation that places them in the category of micro physiological systems. This is because they are capable of both self-renewal and self-organisation, and, more importantly, that they display organ functionalities that are analogous to those of the tissue(s) from which they originated. Therefore, it is essential to establish cultures of functional tissues, but these cultures should be

devoid of the mesenchymal, stromal, immune, and neuronal cells that interspace tissues in vivo. This will allow for the development and maintenance of optimal conditions for organoid design. In fact, this process is dependent on the construction of artificial extracellular matrices in order to allow organoid self-organisation into structures that are analogous to the architecture of the native tissue [69, 74]. To this day, organoids have been successfully created from the intestine, liver, pancreas, colon, and prostate of murine animals, as well as from the small intestine, colon, stomach, and prostate of human beings. The fact that these organoids can be grown for an extended period and, according to whole-genome sequencing, match the patient tissue from whence they originated suggests that their phenotypic and genetic traits are consistent [75].

Patient derived organoids (PDOs) have recently proven valuable in translational research because these models can be maintained for an extended period and cryopreserved. In addition, PDOs are genetically stable, which makes them a perfect choice for modelling diseases. In addition, PDO models are helpful because they enable the expansion of normal cells as well as tumour cells in parallel, which contributes to the formation of a living tumour organoid biobank. PDO models, on the other hand, solely represent the epithelial tissues of organs; they do not include the stroma, nerves, or vasculature that are seen in real organs, which is an essential distinction to make. When organoids are generated from different types of tissues, different types of growth components are required (**Figure 2**) [74].

The use of murine and human embryonic stem cell lines and induced pluripotent stem cell lines to generate organoids gets around the limited availability of high-quality human primary material. However, in order to perform directed differentiation, in-depth knowledge of the factors involved in germ layer formation and subsequent lineage specification is required. In contrast to the employment of ESCs, the utilisation of iPSC lines necessitates the performance of an additional step. Specifically, the expression of OCT4, KLF4, SOX2, and MYC29 is required in order to transform somatic cells into iPSCs. Following this step, embryonic stem cells (ESCs) and


**Figure 2.** *Culture additives/growth factors used for generation of different organoid models.*

#### *3D Culturing of Stem Cells: An Emerging Technique for Advancing Fundamental Research… DOI: http://dx.doi.org/10.5772/intechopen.109671*

induced pluripotent stem cells (iPSCs) are subjected to germ layer and tissue-specific patterning factors. Next, the cells are embedded in Matrigel in order to facilitate the development of 3D architecture. Finally, the cells are treated with differentiation factors in order to produce the desired organoids.

The first successful mesoderm-derived organoids were reported not too long ago. Renal organoids were formed by manipulating GSK3 and FGF signalling pathways in human iPSCs. This was done while the cells were in an intermediate mesodermal state. The architecture and segmentation of human foetal nephrons into ducts, tubules, and glomeruli is replicated in these organoids, which have the same name [76]. The human renal organoids provide a 3D model to study human renal development and disease under well-defined conditions, thus overcoming various limitations of previous models such as 2D monolayers, short-term 3D aggregates, and co-cultures with mouse fibroblasts [77, 78].

The existence of cell types other than those intended for lineage in ESCs and iPSCs is one of the most distinguishing characteristics of organoids created from primary tissue as opposed to those generated from ESCs and iPSCs. This is due to the fact that the factors that are used for the directed differentiation of ESCs and iPSCs are not completely efficient in driving all of the cells toward the lineage of choice. As a result, many ectodermal and endodermal organoids, such as those of the intestine, stomach, and kidney, have reported the limited presence of mesenchymal cell types [79, 80]. Despite these developments, some tissues remain resistant to organoid culture but have been successfully cultivated in 3D as whole-tissue explants or organotypic/ mechanically supported cultures (for example, skin or ovary) [81]. Understanding the endogenous stem cell microenvironment and signalling pathways driving lineage specification in organoid cultures is crucial. Our limited knowledge in these areas for certain tissues prevents us from logically designing niche parameters for organoid formation. Identifying stem cells is not necessary for growing primary tissue units but understanding the stem cell niche is essential for long-term culture sustainability [82]. Small-molecule modulators of critical signalling pathways and organ-specific hormones could facilitate organoid growth from organs like the ovaries. A tight dependence on growth factor/signalling gradients for stem cell renewal and lineage specification may also complicate organoid formation from tissues. Microfluidics could be utilised to create in-vivo-like concentration gradients [83]. In vivo stem cell behaviour and differentiation are also highly impacted by local biomechanical factors, such as interactions with the extracellular matrix49. In order to create more robust organoid culture models for a larger spectrum of tissues, researchers are screening for substrates and ECM factors that influence cell behaviour in vitro [84].

In the field of cancer research, the improvement of culture techniques has been applied to the study of EVs, which models the environment and physiological conditions that are present in the area surrounding tumours. The role of EVs in tumour physiology is not limited to cell-to-cell communication; rather, they are also a promising source of biomarkers, a tool to deliver drugs, and a mechanism to induce antitumor activity [10, 85]. Extracellular vesicles, also known as EVs, were discovered in the 1980s and were initially thought to be carriers of waste products that were produced inside of cells [86]. After it was discovered that RNA could be found enclosed within the lipid membrane of EVs, approximately 20 years later, EVs began to be seen in a different light; specifically, as crucial mediators in the process of intercellular communication [87]. The nucleic acids contained in EV RNAs were found to be distinct from those found in the cell from which they originated, displaying distinct sequences and even concentration profiles. In the course of time, research has demonstrated that EVs

are responsible for transporting nucleic acids, such as RNA and DNA, as well as a wide variety of biomolecules, which includes proteins and lipids, into and out of cells [88]. For instance, in cancer, the EV content of the tumour is tumour-like, and a class of EVs known as exosomes help the progression of the tumour by signalling to the tumour cells that they should establish the pre-metastatic niche. In another scenario, EVs that are released from cells that have been infected with a virus such as HIV can contain fragments of viral RNA as well as viral proteins; consequently, the function of EVs in HIV is unclear at the present time. In addition, extracellular vesicles released by breast cancer cells have been shown to contribute to the spread of the disease to the brain and to have triggered the breakdown of the barrier that separates the blood and the brain. In general, EVs can break through natural barriers such as the blood-brain barrier and others. These mechanisms can be exploited to deliver therapeutic agents to parts of the body that are difficult to reach. EVs have also been shown to play a role in reproductive biology, the differentiation of stem cells, angiogenesis, and a variety of other biological processes [89]. Various clinical trials are ongoing and have been completed where they have used EVs for therapy of various cancer types [90]. EVs are critically important for tumour communication with their intended target cells. Therefore, the study and modification of EVs have opened so many doors for diagnosis and therapy. It is wellestablished that elevated levels of circulating EVs are linked to the development of most cancers. Blood EV concentration has also been shown to correlate with tumour volume in several tumour types. These EVs have become the substrate for biomarker mining in a variety of cancers, including prostate cancer, due to the valuable information they transport about the tumour (**Table 2**) [91–93].

Three-dimensional (3D) culture enables cell growth in a physiological topology, and organoids and spheroids continue to release EVs, which are essential for tumour communication with targeted cells, and the released EVs are functional (**Figure 3**). The extracellular vesicles that are released by pancreatic cancer organoids have the


#### **Table 2.**

*Human clinical trials using EVs for therapeutic purposes.*

#### *3D Culturing of Stem Cells: An Emerging Technique for Advancing Fundamental Research… DOI: http://dx.doi.org/10.5772/intechopen.109671*

ability to activate p38 MAPK and increase the expression of F-box protein 32 and UBR2 in myotubes. In the case of colorectal cancer stem cells, 3D cultures exhibit a higher level of EVs release in comparison to 2D conformations. The presence of APC mutations in colon cancer organoids that activated the WNT pathway resulted in an increase in the amount of EVs released in cultures based on Matrigel. This release was presumably likely helped along by the presence of collagen, which is a component of the extracellular matrix and is present in this sort of gel [61]. Collagen is a component of the gel. Additionally, a further potential hypothesis is that the greater expression of transporters in 3D cultures may be partially responsible for the release of EVs [94–96]. It was shown that tumoroids of colon cancer cells with improved stemness had significant levels of expression of the ATP-binding cassette transporter G1, which is a cholesterol lipid efflux pump. Similarly, inhibiting this transporter prevents the release of EVs and leads to an increase in the number of vesicles found inside the cell [97].

Research has been done to investigate the spontaneous effect of EVs derived from normal cells in order to use them as natural antitumor agents. For instance, extracellular vesicles produced from glia have been shown to have an anticancer effect in spheroids composed of glioma cells. This effect was demonstrated by a gradual reduction in the tumour potential to invade surrounding tissue. Another example is the EVs that are produced by mesenchymal stem cells (MSCs). These EVs could initiate angiogenesis and preserve vascular homeostasis in activated endothelial cells [98, 99]. On the other hand, most of the publications centre their attention on the prospect of loading EVs with anticancer medicines and biomolecules such as amino acids, lipoproteins, or nucleic acids. In a microfluidic system that contained a variety of cell types, an anticancer effect of EVs that were loaded with a particular miRNA (miR-497) was evaluated [100]. These kinds of devices are helpful when used in conjunction with an extracellular matrix because doing so makes it possible to investigate migration in response to a factor that is controlled via microfluidic channels. In this experiment, the non-small cell lung cancer cell (NSCLC) line A549 was

#### **Figure 3.**

*The schematic diagram showing the establishment of patient derived organoid cell culture depicting the organoid microenvironment and respective EV distribution.*

**Figure 4.** *The schematic diagram shows the isolation of EVs and modification of EVs for targeted delivery.*

cultured alongside human umbilical vein endothelial cells (HUVEC). Both cell types were grown in a dish (HUVEC). When the experiment was run under these conditions, the production of tubes by endothelial cells was prevented, and the amount of tumour migration was significantly reduced in comparison to the control. Both types of cells were separated in the microfluidic devices using the matrigel component. This was done so that the limitations of cocultures that are related with cell separation after analysis could be avoided [101, 102]. This is a fascinating illustration of how 3D culture may be used to recreate the physiological intricacy of tumours (**Figure 3**).

One of the most fascinating uses of 3D cultures is the large-scale and standardizable production of EVs. This is one of the most exciting applications of 3D cultures because till yet there is no established biomanufacturing platform for EVs, which poses restriction for clinical translation (**Figure 4**). The utilisation of bioreactor flasks is a straightforward method that can be utilised because these flasks boost the creation of EVs that are discharged by tumour cells. Utilising cell cultures on microfluidic substrates is a more interesting application of this technology [103, 104]. These automated systems can manufacture therapeutic exosomes, which could also be modified, and harvest them in real-time from the cultures that are performed on the chip. As a device of this kind has been utilised in the process of isolating leukocytes from human blood. An alternative method that has been utilised is a 3D-printed scaffold-perfusion bioreactor system to investigate the impact that dynamic cultures have on the production of EVs from endothelial cells. Because of this method, the cells were able to keep up their level of functionality (i.e., pro-vascularization bioactivity or pro-angiogenic gene expression) [95, 101].

#### **5. Conclusion**

Three-dimensional (3D) cell culture models are more functionally important than two-dimensional (2D) cell cultures and include a broad range of structures such as embryoid bodies, spheroids, patches, and scaffolds. Whereas, organoids and

#### *3D Culturing of Stem Cells: An Emerging Technique for Advancing Fundamental Research… DOI: http://dx.doi.org/10.5772/intechopen.109671*

3D culture systems are becoming being recognised as a tool for advancing medical research without relying on animal models. Improvements to these methods may even result in new methods for creating 3D models. Understanding the limitations of the systems is critical for their improvement and determining the model's suitability for investigating EVs. Overall, cellular architecture influences the concentration and cargo profile of EVs. Many studies, for example, found that 3D in vitro systems secreted more EVs than their 2D culture counterparts. Moreover, the possibility of a necrotic core developing in multicellular cultures poses a unique challenge to isolating EVs from 3D in vitro systems. The necrotic core can generate EVs composed primarily of apoptotic bodies rather than small vesicles or large vesicles. To address the challenges, developing cell viability criteria and measures to normalise the outcomes compared against controls such as 2D cell monolayer cultures are required. With the start of human clinical trials for EV therapeutics, these challenges become even more important. Despite the widespread use of organoids in biology, the technology is still in its infancy for certain disorders. Most neurodevelopmental or neuropsychiatric disorders, such schizophrenia, Parkinson, or autism, are examined using animal models. Autism spectrum diseases or Parkinson's have clinical heterogeneity (epilepsy, sleep disruptions, motor difficulties), making organoid culture challenging to use. Researchers have been developing techniques to create more mature and complicated brain organoids. Organoids can be utilised to explore developmental brain injuries and disorders (DBD) Stem cells are linked to several disorders. Scientists do not know how stem cells develop abnormalities or which type of specialised cell to generate. The organoids method can answer questions about stem cells in diseases like emphysema, when lung stem cells fail to heal damage. Further, scientists have suggested utilising organoids to screen medications that can produce specialised cell types for hereditary illnesses like cystic fibrosis, where ciliated cells that remove mucus from the lung do not work properly. Generate organoids from cystic fibrosis patient tissues, then design a medication to make ciliated cells operate better in organoid cultivation. Since scientists can co-culture organoids with immune cells, the approach can be used to investigate autoimmune disease mechanisms and screen medications.

#### **Acknowledgements**

The authors would like to acknowledge the Biorender software for figures.

#### **Conflict of interest**

The authors declare that they have no competing interest.

#### **Abbreviations**



### **Author details**

Sonali Rawat, Yashvi Sharma, Misba Majood and Sujata Mohanty\* Stem Cell Facility, DBT-Centre of Excellence for Stem Cell Research, All India Institute of Medical Sciences, India

\*Address all correspondence to: drmohantysujata@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*3D Culturing of Stem Cells: An Emerging Technique for Advancing Fundamental Research… DOI: http://dx.doi.org/10.5772/intechopen.109671*

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#### **Chapter 7**

## Female Germline Stem Cells: A Source for Applications in Reproductive and Regenerative Medicine

*Hong-Thuy Bui, Nhat-Thinh Nguyen, Truc Phuong Lam Do, Anh My Le Ba and Nguyen Van Thuan*

#### **Abstract**

One of the most significant findings in stem cell biology is the establishment of female germline stem cells (FGSCs) in the early 21st century. Besides the massive contribution of FGSCs to support ovarian function and fertility of females, the ability to create transgenic animals from FGSCs have high efficiency. Whether FGSCs can differentiate into mature oocytes for fertilization and complete embryonic development is a significant question for scientists. FGSCs were shown to produce oocytes, and the fertilized oocytes could generate offspring in mice and rats. This discovery has opened a new direction in human FGSCs research. Recently, cryopreservation of ovarian cortical tissue was already developed for women with cancer. Thus, isolation and expansion of FGSCs from this tissue before or after cryopreservation may be helpful for clinical fertility therapies. Scientists have suggested that the ability to produce transgenic animals using FGSCs would be a great tool for biological reproduction. Research on FGSCs opened a new direction in reproductive biotechnology to treat infertility and produce biological drugs supported in pre-menopausal syndrome in women. The applicability of FGSCs is enormous in the basic science of stem cell models for studying the development and maturation of oocytes, especially applications in treating human disease.

**Keywords:** ovarian stem cell, primordial germ cell, female germline stem cell, oocyte-like cell, regenerative medicine

#### **1. Introduction**

It is widely known that mammals begin their lives with a fixed number of oocyte-containing follicles, which do not increase but only degenerate after birth. This explains why fertility decreases with age, and the phenomenon of menopause in women is an indication that reserve resources of oocytes have been depleted over age. This differs from the male that can produce sperm throughout their life due to the presence of spermatogonia stem cells (SSCs), which could allow them to constantly proliferate and differentiate to maintain their persistent spermatogenesis, allowing male mammals to have a long-lasting reproductive age [1]. Since the 1950s, the dogma in reproductive biology has been widely accepted that primordial germ cells (PGCs)-derived oogonia in mammals cease proliferation and differentiate into primary oocytes shortly after birth, which will arrest in prophase of meiosis I until fertilization triggers the completion of meiosis [2]. In other words, shortly after birth, mammalian ovaries can differentiate to produce new oocytes to compensate for the consumption of ovulation. This explanation was well accepted due to the shorter gestational age of females compared to that of males. This theory of a fixed ovarian reserve had been the central principle in the field of reproduction.

The evolutionary and molecular processes of female reproductive aging have been highly debated. In the early 21st century, scientists at Harvard Medical University ignited the debate about the unexpected ability of mouse ovaries to regenerate immature oocytes after destruction [3]. Then, the report raised numerous questions by showing that these proliferative ovarian cells, termed female germline stem cells (FGSCs), could produce immature oocytes. Transplantation of these FGSCs into the ovaries of adult mice, was able to differentiate them into mature eggs that are able to ovulate, fertilize, and produce viable offspring [4]. This study opens a new direction in the study of stem cells in human ovaries. If we succeed in establishing the human FGSCs (hFGSCs), they will play a very significant role in reproductive medicine and the treatment of menopause symptoms in women. This would not only recover fertility in infertile women but also delay early menopause in women and treat pre-menopausal syndrome for women without hormone replacement therapy. Notably, the preservation of hFGSC can restore fertility and endocrine function for patients after cancer treatment. This is a huge challenge for scientists "Do hFGSCs exist in humans as they do in mice or not?". Recent studies from researchers supporting the existence of oogenesis in postnatal mammalian ovaries raised some questions, and opened a new avenue for the investigation of stem cells in human ovarian tissue. Stem cells are thought to have numerous uses in cell therapy. Chemotherapy, radiation, genetic induction, or hormonal stress can all result in ovarian failure [5]. Additionally, premature ovarian failure (POF), which affects 1% of young women, is a common cause of ovarian dysfunction before the age of 40 [6]. Furthermore, young women are also rendered sterile by some diseases causing oocyte loss, such as polycystic ovary syndrome [7]. However, very little progress has been made in solving this problem. With assisted reproductive technologies, the findings that FGSCs play a role in fertility provide the future application for clinical therapies [8]. Cryopreservation of ovarian cortical tissue has already been developed for female patients with cancer. Isolation and expansion of FGSCs from this tissue before or after cryopreservation may be helpful for new fertility applications [9]. Some progress has been made in addressing this issue through assisted reproductive technologies. It has been discovered that FGSCs play a role in fertility, offering the potential for future clinical applications. However, the question arises whether the egg cells are produced from hFGSCs or not. If hFGSCs exist in ovaries, why do women still have the phenomenon of menopause? The remaining challenge is to clearly elucidate the origin, roles, and capabilities of these cells, and to be able to use them for therapeutic applications. To this end, studies in mammalian models other than the mouse need to be done because several mechanisms of biological processes for oocytes in mice are different from those in humans. This review will present recent studies on the existence of germline stem cells (GSCs) in the mammalian ovary and summarize the current understanding of ovarian germline stem cells (OSCs) and FGSCs (**Figure 1**).

*Female Germline Stem Cells: A Source for Applications in Reproductive and Regenerative… DOI: http://dx.doi.org/10.5772/intechopen.110438*

**Figure 1.**

*The timeline of major discoveries in the history of female germline stem cells research.*

#### **2. The existence of germline stem cells in adult ovary**

For several decades, it has been believed that females are born with a limited pool of oocytes and lose their capacity for oocyte renewal under perturbed conditions. The presence of mammalian FGSCshas been a highly debated area of reproductive technology since 2004, the Harvard University group suggested a source of cells for oogenesis during the reproductive period [3]. In this study, they discovered that the atresia follicle formation rate in mice was lower than the consumption rate of non-atretic follicles. Hence, they believed that there is a renewal of follicles in mice. Moreover, immunostaining showed the presence of proliferating germ cells by expression of germ cell markers. In their final set of experiments, wild-type mouse ovaries were grafted to transgenic GFP-positive mouse ovarian bursa to provide additional evidence for ongoing folliculogenesis in postnatal life. They found that GFP was positive after 3–4 weeks in the grafted ovarian tissue, leading to the conclusion that oogenesis continues in the postnatal ovary. This has sparked much debate about whether germline stem cells do exist in the postnatal ovary. In addition, it was reported that both immature and follicles were detected after transplanting the bone marrow-derived cells from the adult mice into the ovary of infertility mice [10]. Both reports opened many arguments on whether FGSCs existed in the postnatal ovary. Kerr's study in 2006, in which the number of ovarian follicles in mice were counted at different ages, revealed that the average number of follicles did not significantly decrease between 7 and 100 days after birth. Since postnatal mice mature sexually in around 8 weeks (56 days) and ovulation consumes a portion of the follicular pool, it is suggested that postnatal mice have an oocyte replacement mechanism [11]. The culture of cells attained from scrapings of the human ovarian surface epithelium (OSE) resulted in the formation of large oocyte-like cells (OLCs) expressing zona pellucida proteins [12], leading the authors to suggest that putative germ cells within the OSE of the postnatal ovary differentiate from mesenchymal progenitors in the ovarian tunica albuginea. In line with this possibility, small round (2–4 μm diameter) c-kit/stage-specific embryonic antigen (SSEA)-positive cells were isolated from human OSE cells. These cells expressed early PGC markers, including

OCT4 (POU5F1), NANOG, and SOX2 [13]. In 2009, the first successful result in isolating and purifying the FGSCs from the ovary of neonatal and adult mice was reported, by using a magnetic bead sorting technique against Vasa protein, a germline-specific marker [4]. In order to dissipate the doubts about the existence of putative FGSCs in postnatal mammalian ovaries, this group utilized another germline cell-specific protein, Fragilis, to isolate and purify putative FGSCs in postnatal mice. They successfully purified the cells using magnetic sorting techniques, which showed the same characteristics as FGSCs isolated by Vasa protein. Then, FGSCs were transplanted with different genes and subsequently implanted into the ovaries of infertile female mice to create transgenic animals after mating with normal male mice [14]. This research provided significant evidence to support the existence of germline stem cells in postnatal female mammals and opened a new direction in the study of stem cells in the human ovary. Suppose the FGSCs can be successfully isolated from humans, the benefit of this type of cells will play a key role in reproductive studying, medicine, and treatment for menopausal syndrome in women, and other relevant clinical applications. Infertile women can have the ability to give birth again. Besides, further investigation on FGSCs can open a new method for delaying early menopause in women and treating pre-menopause syndrome for women without using hormone replacement therapy.

In 2012, remarkably, FGSCs were able to be isolated from the cortex of adult human ovaries and differentiated into oocyte-like structure cells *in vitro*. Moreover, xenotransplantation of hFGSCs modified to express GFP into immunodeficient female mice resulted in the development of follicles harboring GFP-positive oocytes in the human ovarian cortex after 1–2 weeks [15]. Further, these isolated FGSCs formed into follicles containing oocytes when transplanted into the immunodeficiency mice and could be expanded for months and spontaneously generate 35–50 μm oocytes [16]. This discovery has opened a new direction in research on human FGSCs. Therefore, FGSCs can have an important role in the treatment of diseases caused by infertility females or in extending the period of menopause, as well as the application of stem cell therapy.

We have successfully established pig FGSC from ovarian tissue *in vitro* culture, and porcine PGCs-like Putative Stem Cells (PSCs) continue to maintain their germ stem cell identity *in vitro* and can differentiate into OLCs under appropriate culture conditions. Moreover, experimental evidence showed that PGCs-like PSCs are probably generated from Vasa-positive stem cells *in vitro*. Finally, we demonstrated the critical role of ovarian cell-derived regulatory factors and the proximal stem cell niche in the establishment of porcine PSCs [17]. In addition, many studies provided evidence available to support the existence and potential of putative germline stem cells in the adult mammalian ovary, such as bovine, monkey, and human [18–21]. A finding demonstrated the presence of stem-like cells with ovarian germline properties within the otherwise exhausted oocyte reserve of menopausal human ovaries. Using immunomagnetic enrichment based on membrane DDX4 expression followed by single-cell sorting under a dielectric field, large culture-derived OLCs expressing markers of mature and haploid oocytes were obtained from fertile women as well as menopausal women [22].

Besides, other groups failed to observe replenishment of the follicle pool by donor bone marrow-derived cells [23] or after chemical depletion [24]. They also failed to observe the generation of new follicles even after depletion with busulphan toxin, thus putting into question a need to explain the regeneration of follicle numbers, a finding further supported by mathematical modeling [25]. Lei and Spradling traced the numbers of follicles over time using tamoxifen-induced random labeling of cells. They argued that the follicle pool is highly stable with a half-life of 10–11 months, which would make the follicle pool at birth large enough to support ~500 ovulations required during

#### *Female Germline Stem Cells: A Source for Applications in Reproductive and Regenerative… DOI: http://dx.doi.org/10.5772/intechopen.110438*

the life of a mouse [26]. In a study to confirm the existence of FGSCs in postnatal mouse ovaries, transplantation of premeiotic female PGCs and companion pre-follicular cells into the ovaries of adult mice has been shown to be capable of supporting the formation of new follicles. However, the transplanted PGCs could only form follicles with their prefollicular cells and vice versa [27]. Although the authors concluded that neo-oogenesis does not normally occur in the ovaries of adult mice, the results nonetheless provide an answer to the important question of whether adult ovaries can support neo-oogenesis from transplanted PGCs. Taken together, we suggest that germline stem cells themselves may not persist in postnatal and adult mammalian ovaries but that progenitor cells/small PSCs in the ovary may instead differentiate into germline stem cells under appropriate conditions [17]. Therefore, although experimental evidence supports the existence of cells with germline progenitor/stem cell characteristics in ovaries of various species, including humans, the existence of GSCs in postnatal ovaries remains ambiguous.

#### **3. Location of female germline stem cells (FGSCs) in the ovary**

In female mammalian species, during the embryonic stage, a subset of blastula cells can form PGCs by germ cell determination under some signal induction. Millions of germ cells are formed during embryogenesis. However, most of the germ cells degenerate after embryonic development. Most oogonia die in this period, while the remaining enter the first meiotic division. These latter cells, called primary oocytes, proceed to the first meiotic prophase after replicating their genomes. These primary oocytes are then arrested at this stage of development until the first menstrual cycle. Only a few numbers of oocytes periodically resume meiosis after puberty. Millions of germ cells are produced during embryonic development, but only hundreds of oocytes mature during a female's lifetime [28]. In a menstrual cycle, when the primary oocyte enters metaphase I, its nucleus (germinal vesicle) breaks down, and the metaphase spindle migrates to the periphery of the cells. In telophase, the chromosomes are evenly divided, but one of the two daughter cells retains almost all of its cytoplasmic components, while the other cell has almost no cytoplasm. The smaller and larger cells are called the first polar body and the secondary oocyte, respectively. Moreover, the same phenomenon takes place during the second division of meiosis. Nearly all the cytoplasm is retained by the mature egg (the ovum), and a second polar body receives little more than a haploid nucleus. Thus, the purpose of oogenic meiosis is to conserve the volume of cytoplasm in a single oocyte. Ovulation begins shortly thereafter, in which the follicle ruptures and the secondary oocyte is released into the uterine tube, yet the second meiotic division has not occurred yet. Meiosis of a secondary oocyte is completed only if a sperm succeeds in penetrating its barriers. Meiosis II then resumes, producing one haploid ovum that, at the instant of fertilization by a (haploid) sperm, becomes the first diploid cell of the new offspring (a zygote) [29, 30].

The general idea in reproductive biology is that FGSCsdifferentiate into primordial oocytes through fetal development and that oogenesis begins with a pool of primordial follicles, which is the case in the majority of animals. Several studies have reported the presence of FGSCs in the ovary. Parte and colleagues in 2011 reported the discovery of very small pluripotent stem-like cells deposited in the OSE of adult rabbits, sheep, monkeys, and menopausal humans [18]. Two different populations of putative stem cells (PSCs) of varying sizes were found in scraped OSE. While the larger 4–7 μm cells with cytoplasmic localization of Oct-4 and little expression of SSEA-4 were likely the tissue-committed progenitor stem cells, the smaller

1–3 μm very small embryonic-like PSCs were pluripotent in nature. To demonstrate characteristics of these cells derived from OSE, the PSCs underwent spontaneous differentiation, c-Kit, DAZL, GDF-9, VASA, and ZP4 germ cell markers were used to immunolocalize in oocyte-like structures. Mammalian ovaries include a unique population of extremely small embryonic-like PSCs and tissue-committed progenitor stem cells that have the ability to develop into oocyte-like structures *in vitro*, contradicting the conventional belief that OSE is a bipotent source of oocytes and granulosa cells.

In 2014, we indicated that the ovary contains a considerable number of undifferentiated cells with stem cell characteristics. These might remain in the adult ovary and cannot proliferate normally, but they can undergo proliferation and differentiate into OLCs under appropriate conditions. PSCs were found to comprise a heterogeneous population based on c-kit expression, cell size, and expressed stem and germ cell markers. Analysis of PSCs molecular progression during establishment showed that these cells undergo cytoplasmic-to-nuclear translocation of Oct4 in a manner reminiscent of gonadal PGCs. Flow cytometry analysis revealed abundant PSCs proliferation after isolation and culture for 1 week. Of these, 4.65% of the cells were positive for the germ cell marker Vasa, and some were also positive for additional germ and stem cell markers, such as Fragilis, Thy-1, SSEA4, and c-kit. At this time, two populations of PSCs were observed: one with a cell diameter of 5–7 μm and one with a cell diameter of 10–12 μm. The cells became identical in size after 2 weeks in culture, at 10–12 μm, with an increasing percentage of cells positive for germ and stem cell markers [17]. About 2.8% of all mouse testicular cells were c-kit positive [31] and had the capacity to become multipotent germline stem cells, whereas c-kit-negative cells go on to become SSCs [32]. We similarly observed two distinct subsets of cells (c-kit positive versus c-kit negative) within the PSCs population. This finding was strengthened by immunofluorescence analysis showing that, after 1 month in culture, most of the PSCs expressed high levels of the reprogramming factor Oct4. In contrast, only 22% of the PSCs expressed high levels of c-kit [17].

#### **4. FGSCs aging and stem cell niche in the ovary**

While much evidence support the existence of OSCs, it raises the question that ovarian have follicle reserve. Why do they not appear to contribute to postnatal follicle formation, and why does the phenomenon of menopause occur only in females? The researchers believe that the FGSCs aging directly determines ovarian aging.

The stem cell niche is the key to elucidating the entire mechanism of stem cell senescence; Schofield proposed the hypothesis in 1978 that the components surrounding stem cells act as a microenvironment that promotes their growth and protects them from external damage [33]. Other than FGSCs, stem cell niche can be found in almost any stem cell, such as intestinal, myocardial, neural, and hematopoietic stem cells. Stem cell niche can support the growth of stem cell, and disturbance of these niches can cause stem cell damage and eventually leads to certain diseases. Ovarian stem cell niche aid FGSCs to continually proliferate to differentiate into postnatal follicles and oocytes, by regulating to divide into new stem cells and differentiate into germ cells. FGSCs niche is extensively studied in Drosophila Melanogaster. Although the structure and the function of FGSCs niche in mammals have not been fully understood, the component of the niche is believed to be similar to that of drosophila, thus predicting that it may at least be composed of follicular membrane-stromal cells, granulosa cells, extracellular matrix, blood vessels, immune system-related cells, and cytokines. It was suggested that damage to the stem cell niche is a major cause of the ovarian recession and is more

#### *Female Germline Stem Cells: A Source for Applications in Reproductive and Regenerative… DOI: http://dx.doi.org/10.5772/intechopen.110438*

closely related to aging in the ovary than the stem cells themselves. Thus, factors that damage the stem cell niche may have a critical impact on ovarian regression.

Factors that lead to stem cell decay are nutritional and energy deficiencies in the stem cell niche. Insufficiency of energy is caused by mitochondrial depletion associated with aging women, thus only supplies limited amounts of ATP. As a result, the risk of birth defects and infertility is increased due to the reduced energy of FGSCs provided by stem cell niche. The immune system has a critical role in the maintenance of OSC niche to support FGSCs. The perivascular compartment of the stem cell niche forms a bridge to connect the niche and both cellular and humoral aspects of immunity. Cellular immunity that provides support in stem cell niche are monocytes, macrophages, and T cells. Cytokines and immunoglobulins are the humoral aspects of immunity that help stem cell niche maintenance. These are essential for the derivation of OSCs into new germline cells. Weakened immunity due to aging is causing difficulty in the maintenance of the stem cell niche and resulting in ovarian recession.

Dysfunctional gonads can be regenerated by transplanting niche cells of germline stem cells (mostly Sertoli cells or mesenchymal cells). Thus, it could be helpful as first-line therapy to permanently restore gonadal function in POF and cancer patients. Thus, it is clinically more important to reestablish the niche of the FGCSs than to inhibit the aging of the FGCSs themselves in order to delay ovarian aging. The study of the FGSC niche is relatively new, and there are still several issues that we still need to know about. To be able to use for the application of the FGSC niche in clinical practice, these should be addressed and clarified. Understanding the mechanism and application of stem cell niche in FGCS can be crucial in regenerative medicine. Although the result of transplanting FGSCs into infertile female ovaries remains controversial, this has the potential to regenerate.

Researchers have put out solutions to answer this question. One of the most widely accepted theories is that stem cell functions would decline with age, which would result in a loss of renewal capacity [34]. They explained that the aging of FGSC is related to the aging of the stem cell niche. The niche is a specialized microenvironment that gives stem cells specialized cues in the form of adhesion molecules, differentiation and self-renewal-regulating signals, spatial organization, and metabolic support to stem cells. As a result, the niche is crucial for controlling stem cells' fundamental processes and protecting them from cell damage and toxins. Changes in the niche may result in to decline in stem cell function. To demonstrate this hypothesis, Bukovsky observed that the niche of FGSCs formed during early embryonic development consists of nonspecific ovarian monocyte-derived cells (MDCs), T cells, and vascular endothelial cells, In contrast to the nests of adult ovarian germinal stem cells, which are made up of primary CD14 + MDCs, activated HLA-DR + MDCs, and T cells [12]. Furthermore, when the ovarian tissues of older mice were transplanted into young mice, the young mice's ovarian tissues were found to have fewer follicles and no mature follicles [35].

Another hypothesis proposed by researchers suggests that ovarian function may decline by systemic aging-related signals despite the presence of oogonial stem cells [36]. For example, progressive loss of ovarian estrogen (E2) production drives reproductive aging and menopause [37]. To demonstrate the hypothesis, they have shown that mouse OSCs express E2 receptor-α (Erα) by RT-PCR and western blot analysis. To test for potential interactions of E2-activated Erα with meiotic regulatory pathways in OSCs, chromatin immunoprecipitation (ChIP)-PCR assays were applied to assess the Stra8 promoter. Results showed that Erα occupied a consensus ER response element (ERE) in the Stra8 promoter. Moreover, E2 treatment increased the number of GFP-positive cells. Thus, OSCs have differentiated in response to E2. In reverse, Era-deficient shows a loss of Stra8 expression and oocyte numbers. This study will

provide more information on how changes in ovarian estradiol production with aging in women are related to age-related ovarian dysfunction and reproductive aging.

Moreover, oxidative stress also has an essential role in the aging of FGSCs. ROS is a chemically reactive oxygen atom or group of atoms produced during cellular metabolism. The development of ovarian granulosa cells was inhibited by ROS, which also damaged mitochondria and lowered the production of the anti-oxidative enzyme. Additionally, it might diminish ovarian function and trigger an inflammatory response, impairing fertility [38]. Resveratrol (RES) is a naturally occurring substance with many pharmacological roles, including antioxidant, anti-inflammatory, immune-regulating, cell-protective, anti-tumor, and anti-apoptotic effects. RES therapy can be employed to enhance ovarian follicle function by lowering TNF-levels, which was validated by lowering LH levels and the ratio of LH/FSH, two markers of ovarian function. The scientists discovered that RES significantly increased body weight, ovarian index, follicle quantity, and decreased follicular atresia in POF mice.

#### **5. Isolation, maintenance, and characterization of FGSCs**

After debating the existence of germline stem cells in ovaries, proponents of their existence continue to question what types of FGSCs exist in ovaries and their characteristics. FGSCs' sizes vary considerably, ranging from 2 to 8 μm. Tilly's group focused on bigger-sized (5–8 μm) OSCs [16], whereas smaller (2–4 μm) pluripotent stem cells, very small embryonic-like stem cells (VSELs) were found in ovary surface epithelium (OSE) [39]. However, OSCs of both sizes express germ-line markers and differentiate into OLCs. Thus, they concluded that there are two different populations of stem cells, the small-sized, pluripotent VSELs, and the bigger OSCs. VSELs are pluripotent stem cells produced from epiblast that are identical to PGCs and persist in small numbers in adult gonads [39]. In contrast, OSCs are tissue-specific progenitors that are bigger and have different gene expressions from pluripotent VSELs. It has been reported that VSELs are the most primitive population of quiescent SCs found in adult tissues compared to OSCs, which quickly divide and produce germ cell nests before differentiating into oocytes. FSH receptor (FSHR) expression was observed on both very small embryonic-like stem cells (VSELs) and ovarian stem cells (OSCs) by immune-localization and immunophenotyping studies. FSH treatment increases germ cell clusters and stimulates stem cells to undergo proliferation and clonal expansion to form germ cell nests. This was further confirmed by the differential expression of OCT-4 in VSELs and NUMB in OSCs. Immunohistochemical expression of OCT-4, proliferation, and FSHR were noted on stem cells located in the OSE of ovarian sections of sheep. Therefore, the establishment of FGSCs is significant for many applications.

Using adult porcine ovaries to isolate, identify, and characterize FGSCs to elucidate their origin and then examine the capability of these cells to proliferate, grow, and differentiate. These cells were heterogeneous, depending on both c-kit expression and cell size, and also expressed stem cell and germline markers. Importantly, we clearly demonstrated that cells with characteristics of early PGCs are present in the adult porcine ovaries. Once FGSCs were established, they could be expanded *in vitro* for months without the loss of identifying markers and proliferative potential. Under appropriate conditions, FGSCs can be differentiated into OLCs. These have the potential to make new oocytes, support ovarian function and fertility, and may support therapeutic strategies in humans [17]. The methods used to isolate, maintain, and characterize FGSCs from each of the research teams studied to date are summarized in **Figure 2**.

*Female Germline Stem Cells: A Source for Applications in Reproductive and Regenerative… DOI: http://dx.doi.org/10.5772/intechopen.110438*

#### **Figure 2.**

*The female germline stem cells (FGSCs) were successfully isolated from porcine ovarian tissue by enzymatic digestion and different adhesive selection, then cultured in vitro, in DMEM/F-12 and N21 free-serum supplemented. Two populations of FGSCs, one with a cell diameter of 5-7μm and the other with a cell diameter of 10-12μm, were observed with spherical morphology and expressed specific germline characteristics (Vasa, Stella, Oct4, c-kit). The cells became identical at 10-12 μm after ten days in culture, Oct4 expression was reduced in the cytoplasm and augmented in the nucleus, forming groups of cells clustered around theca cell colonies. The large clumps of ovarian cells will be removed during cell passages by the filter of 40μm, and FGSCs were maintained for one month. After one month, following the gradual death of the ovarian cells, the FGSCs were transferred onto mitomycin C-treated MEF feeder layers for long-term culture with the addition of stem cell factor.*

The formation of germ cell nests has provided evidence in support of neo-oogenesis from the stem cells [40]. VSELs may endure radiotherapy and chemotherapy and preserve lifelong homeostasis. Due to the loss of function brought on by disturbing ecology, its impairment results in host aging, and the existence of overlapping pluripotency markers raises the possibility that it may also be linked to epithelial ovarian cancer. Many methods for isolation and culture of FGSC have been studied in recent years. Briefly, FGSCs can be isolated by enzymatic digestions (collagenase and trypsin) and purified with either MVH/Fragilis-magnetic bead sorting or/with Flow Cytometerbased SSEA-4, FRAGILIS, DDX4 (VASA) or long-term passage methods to eliminate ovarian somatic cells. Isolated FGSCs are able to be cultured in both absence or presence of feeder cell layers, MEF, STO, or granulose cells. The gelatin-coated surface is typically used in the culture of non-sorting cells. The concentration of FBS varies from 5 to 15% and is used in maintenance and differentiation medium. Besides that, growth factors GDNF, EGF, and bFGF are important for the expansion of FGSCs; LIF is used for FGSCs maintenance, while bone morphogenetic protein and retinoic acid can significantly induce germ cell differentiation [41, 42]. In addition, SCF was also suggested to increase the number of Fragilis- and MVH-positive cells and enhance colony formation efficiency [17, 43]. Other supplements such as antibiotic and antimycotic, pyruvate, non-essential amino acids, antioxidants, insulin, transferrin, and selenium are added to increase the viability and proliferation of ovarian cells. Cultured cells normally express general pluripotent, germ cell, and oocyte markers. While Oct4 is always strongly expressed in cultured OSCs, others, such as SOX2 and Nanog, are still in conflict. VASA,


**Table 1.**

*Summary of isolation and culture of FGSCs in mammalian.*

#### *Possibilities and Limitations in Current Translational Stem Cell Research*

**166**

*Female Germline Stem Cells: A Source for Applications in Reproductive and Regenerative… DOI: http://dx.doi.org/10.5772/intechopen.110438*

PRDM1, FRAGILIS, and DAZL are considered germline-associated genes. Oocytespecific markers, including ZP1–3, GDF9, NOBOX, and SCP3, are rarely detected or weakly expressed [15, 44]. A summary of these methods is shown in **Table 1**.

#### **6. FGSCs share characteristics with epiblast-derived PGCs**

We have successfully established FGSCs from porcine ovaries and demonstrated that these FGSCs derived from PGCs have been retained and become inactivated. These PGCs were thought to exist only during the fetal period, and all transformed into oocytes before the individual was born [17]. In addition, a study has also shown similar patterns of gene expression profiles between FGSCs and PGCs [51].

In the current model, PGCs are derived from a small number of epiblast cells and are identified before differentiation into different germ layers begins. PGCs then undergo a complex migration process, passing through the abdominal cavity, along the developing hindgut, and finally into the dorsal mesentery, where gonads develop. Despite the apparent importance of these early developmental processes for future fertility, little is understood. It was suggested that PGCs temporarily reside in an "Allantoic Core Domain" (ACD), which they propose has similar functions to the Spemann organizer, consisting of a stem cell pool that extends the body axis in a posterior direction—contributing not only to the germ cell lineage but also the three germ layers. This creates a solid interface between the future umbilical cord and the developing embryo [52]. The stem cells in the ACD express Oct4, Blimp1, Stella, and Fragilis—markers thought to be specific for PGCs but appear to contribute also to other tissues [53]. These observations and the fact that hematopoietic stem cells also migrate from the proximal epiblast to the embryonic aorta-gonad-mesonephros during the same development period. Implies that it is theoretically possible that there may be "intermixing" or that there is a common precursor pool for PGCs and a subpopulation of bone marrow stem cells. Cell lineage tree analysis based on somatic mutations accumulated in microsatellites has shown that oocytes form a completely distinct cluster from other cell populations, suggesting no mixing of germline progenitor pools with other cell types but the bone marrow stem cells [54]. It is conceivable that very rare subpopulations of these cells would be missed in this type of analysis. Their result also shows that aging and unilateral ovariectomy increase the number of mitotic divisions of oocytes. This may be explained by the recruitment of oocytes in the order in which they first differentiate during development. However, it is also consistent with the idea that oocytes are continuously produced from circulating stem cells. Many researchers have suggested that if GSCs exist, they are most likely derived from normal developmental precursors of oocytes, that is, PGCs or oogonia (which have not yet differentiated into oocytes and can undergo mitosis) [55, 56]. The close relationship of PGCs to pluripotent cells is evidenced by the fact that they can be returned to a pluripotent phenotype called embryonic germ cells *in vitro* without genetic manipulation after isolation from the embryo [57, 58].

In 2014, we investigated the developmental origin of porcine PSCs. In normal development, c-kit, SSEA1, and SSEA4 are expressed by the majority of pre-gonadal PGCs and are progressively downregulated when PGCs enter meiosis in the embryonic ovary [59]. In contrast, Vasa protein is detectable only when PGCs enter the gonadal ridges and remains elevated in human fetal and postnatal oocytes [60]. VASA (DDX4) negative VSEL stem cells (2–4 μm) isolated from the human OSE express genes typical of ESCs, such as NANOG and SOX2, thereby indicating their undifferentiated status. After culture for 3 weeks under differentiation conditions, VASA-negative cells are

transformed into OLCs expressing VASA and ZP2, a marker for oocytes. In the present study, small Vasa-positive porcine PSCs (5–7 μm in diameter) began to reduce their expression of Nanog, Sox2, and Rex1 after 1 week in culture, indicating their transformation to a differentiating status. Previous investigations showed that Vasa-positive VSEL stem cells isolated from adult organs express several characteristic markers of early PGCs, including fetal-type alkaline phosphatase, Oct4, SSEA-1, CXCR4, Stella, Fragilis, Nobox, and Hdac6. Since the porcine PSCs described herein similarly express a number of typical, early PGC markers, these findings might indicate a close association of PSCs with Vasa-positive VSELs and epiblast-derived PGCs [17].

#### **7. Self-renewal capacity of FGSC**

The studies have focused on the development of FGSCs into oocytes both *in vivo* and *in vitro*. Transplantation of the GFP-FGSCs back into ovaries leads to the generation of fertilization-competent eggs that produce embryos and offspring [4]. Furthermore, GFP\_FGSCs have generated GFP-positive OLCs enclosed in host somatic cells, as characterized by morphology and expression of oocyte-specific markers after injection into adult human ovarian cortical tissue and transplantation into an immune-deficient mouse [61].

Maintaining and extending FGSCs *in vitro* is a crucial step to obtaining fully active germ cells. The effects of various supplements on FGSCs proliferation have been evaluated to optimize conditions for *in vitro* culture of FGSCs. Ovarian tissue plays an essential role in maintaining the properties of FGSCs *in vitro* culture [62]. Follicle-stimulating hormone (FSH) and basic fibroblast growth factor (bFGF) were considered to induce the proliferation of FGSCs and retain their potential for spontaneous differentiation into oocyte-like structures in extended cultures [39]. Preliminary studies by our group have successfully established porcine FGSCs and maintained them for more than 6 months without loss of proliferative potential. Expression of identified germline markers was also maintained. The estimated cell doubling time was 48–72 hours. Subsequently, long-term culture increased the number of differentiated cells among FGSCs, but many FGSCs that were positive for both BrdU and Oct4 or Vasa retained high proliferative potential [17].

GSK3 inhibitors are involved in this process by promoting the proliferation of FGSCs by activating both β-CATENIN and E-CADHERIN [63]. FGSCs that exhibit pluripotency are highly capable of self-renewal, which involves a number of genes and signaling pathways. CADHERIN-22 (CDH22), a member of the cadherin superfamily, functions in FGSC maintenance and self-renewal through its interaction with JAK–STAT and β-CATENIN. The knockdown of CDH22 strongly affected FGSC proliferation by its inhibition and triggering apoptosis, decreased phosphorylation levels of p-JAK2 and p-STAT3, and led to the downregulation of β-catenin [64, 65]. CDH22 also interacts with PI3K to phosphorylate AKT3 and increase the expression of N-myc and cyclin family of FGSCs to promote self-renewal [65].

A study in 2012 evaluated the effects of leukemia inhibitory factor (LIF) and other growth factors, epidermal growth factor (EGF), beg, and glia-derived neurotrophic factor (GDNF), on the proliferation and colony formation of FGSCs. Results showed that these growth factors promote FGSCs proliferation through activation and upregulation of β-CATENIN and E-CADHERIN, and LIF has a significant positive effect on cell colony number [63]. Activation of the GDNF signaling pathway is mediated by GFRα1 (GDNF receptor), which is related to circGFRα1 by leading to the expression of GFRα1 [66]. In addition, YAP1, an effector of the Hippo signaling pathway,

*Female Germline Stem Cells: A Source for Applications in Reproductive and Regenerative… DOI: http://dx.doi.org/10.5772/intechopen.110438*

regulates FGSCs proliferation and differentiation *in vitro* and ovarian function [67]. The Hedgehog (Hh) signaling pathway plays a vital role in the fertility of FGSCs. Blocking the Hh pathway by GANT61 depletes ovarian germ cells and FGSCs [68].

Furthermore, gene expression analysis inferred increased expression of proliferation-related genes c-Myc and Cyclin A in the OSE and cortical cells, while expression of the differentiation marker Zp3 was significantly decreased. Rapamycin inhibits the activation of primordial follicles, promotes FGSCs proliferation, and inhibits their differentiation, thus providing a new prospect for delaying ovarian senescence. Furthermore, the novel administration of Daidzein to FGSCs promoted the survival and proliferation of FGSCs by activating the Akt signaling pathway through Type C lectin domain family 11 member a, which functions as a growth factor [66].

#### **8. Differentiation of FGSCs**

Stem cell differentiation is critically dependent on the ability to differentiate stem cells into a specific cell type with a highly efficient and scalable system. Recent evidence has demonstrated that the differentiation signals are strongly modified by adhesive and mechanical factors. Furthermore, an environment that mimics the microenvironment in tissues is desired to stimulate stem cell potential and differentiation. Mimicking the cellular microenvironment *in vitro* is increasingly influential in guiding stem cell proliferation and differentiation [69]. Studies claim that cells from both menopausal and non-menopausal women may produce *in vitro* oocyte-like cells (OLCs-large spherical cells). This suggests that neo-oogenesis may take place during ovarian senescence. Tilly and colleagues discovered that OSCs developed *in vitro* to produce OLCs with gradual expansion up to 30–50 μm in diameter. These cells have expressed terminal markers such as zona pellucida (ZP) glycoproteins, GDF–9, NOBOX, YBX2, and SYCP3. In addition, they have examined that OLCs have the haploid karyotype [15]. OLCs generated by regulatory factors or spontaneous development lose expression of developmental pluripotency-associated genes, resulting in strong expression of oocyte makers and maintenance of germline markers. However, although many groups have been studied for the establishment of FGSCs, the differentiation potential of FGSCs in mammalian ovaries remains a controversial issue among germline biologists and stem cell researchers. To date, no mammals other than mice and rats have successfully produced offspring from FGSCs [14, 48]. Whether FGSCs can undergo growth, maturation, and fertilization to become functional oocytes is one of the crucial questions for us.

While most researchers have focused on the role of media (cytokines and growth factors) in regulating FGSCs differentiation into OLCs, the ideal culture system condition has yet to be established. We have investigated the effect of culture medium on FGSCs and further studied the effect of different culture systems (gelatin-coated dish, MEF feeder layer, co-culture with granulosa cells, and co-culture with MEF cells) on isolated FGSCs differentiation into OLCs. Co-culture of stem cells with a somatic cell population has been investigated as an alternative growth factor to induce stem cell differentiation [70]. This system provides growth factors and overcomes the requirement for exogenous growth factors to promote stem cell differentiation [71]. Somatic cells have been demonstrated to support oocyte development through cell-to-cell communication. This plays a vital role in oocyte growth and functioning via the transport of metabolites [72, 73]. Granulosa cells are one of the most important cell types which support oocyte development in the follicle. It was also proved that granulosa cells enhance oocyte development competence for *in vitro* culture [50, 74]. Another important cell type, MEF feeder cells,

can produce and secrete growth factors and cytokines to provide an environment for stem cell migration, differentiation, and proliferation [75]. Hence, co-culture systems between FGSCs and somatic cells should be studied to improve the quality of OLCs.

2D culture system (monolayer) has been used as a conventional method for stem cell culture and differentiation. Recently, a new advanced 3D low attachment system has been developed. In this system, the primary cells exhibit a higher level of a specific function for a more extended period *in vitro* in 3D culture compared to monolayer culture. Furthermore, the 3D low attachment culture enhances the differentiation and stabilizes the functions of stem cells. [76]. During oocyte culture and development, it has been proved that improvement in ovarian culture systems increased the survival and growth of preantral follicles after the long-term culture period [77]. The majority of work on *in vitro* oocyte culture was undertaken using a conventional 2D culture system [72]. In recent years, further technical advancement has emerged form of 3D culture, which improved oocyte quality and development competence [78].

Our group also has successfully isolated FGSCs from adult pig ovaries and differentiated them into OLCs [50]. FGSCs were passed to the differentiation medium. Firstly, the old culture medium was gently removed from the tissue culture plate. Cells were then washed three times with PBS solution. After removing the PBS solution, 0.25% Trypsin-EDTA was added to the dish for 3–5 minutes. The dish was shaken slightly to separate FGSCs from the bottom of the surface. 10% FBS was added to stop trypsin action. The cell suspension was then divided into new tubes and centrifuged at 1000 rpm for 5 minutes twice using DMEM solution (Sigma). The supernatant was taken out, and

#### **Figure 3.**

*The female germline stem cells (FGSCs) were isolated from porcine ovarian tissue and cultured in vitro, in DMEM/F-12, and N21 free-serum supplemented. These cells possessed spherical morphology and expressed specific germline characteristics (Vasa, Stella, Oct4, c-kit). For in vitro differentiation induction, using FGSCs 1-week after isolation, where ovarian somatic cells remained in the culture, or using 1-month cultured cells with less or no ovarian somatic cells. Co-culturing the isolated FGSCs with MEF cells under three-dimensional (3D) cell cultures supplemented with follicle fluid. After 1-month in differentiation culture, OLCs could reach about 70 μm in diameter, with a large number of surrounding somatic cells. OLCs expressed germ cell-specific markers (Vasa, Blimp1, Fragilis, C-kit) and oocyte-specific markers (ZP, ZPC, SCP3, GDF9b), contained large nuclei, about 25-30μm, with filamentous chromatin, similar to the oocyte.*

*Female Germline Stem Cells: A Source for Applications in Reproductive and Regenerative… DOI: http://dx.doi.org/10.5772/intechopen.110438*

the pellet was resuspended with a differentiation medium. This study evaluated four differentiation conditions to differentiate FGSCs into OLCs *in vitro* (**Figure 3**).

#### **9. Application of FGSCs**

Preservation of fertility is one of the most important qualities of life issues fertility preservation (FP) for young women with threatening premature ovarian insufficiency, especially for young cancer survivors who have not completed their family upon a cancer diagnosis. Among the currently available options for FP, especially in young patients, is cryopreservation of ovarian cortical tissue containing primordial follicles followed by autotransplantation [79]. Although ovarian tissue cryopreservation (OTC) currently represents an experimental approach, it offers not only FP but a restoration of endocrine function for women with cancer prior to undergoing gonadotoxic treatments. Re-implantation of OTC is currently the only option to use stored tissue, but in many cases, this procedure carries the potential risk of reintroducing the malignancy. However, in cancer patients, especially women with leukemia, re-transplantation is not an option due to the presence of malignant cells in the ovaries [80, 81].

FGSCs have the great clinical potential to be one of the options for the treatment in regenerative medicine for restoring declined female reproductive function caused by ovarian aging and perimenopausal-related diseases and preservation of fertility for patients with post-gonadotoxic therapy for ovarian cancer. In the former case, activation of FGSCs may restore ovarian function through their self-renewal and ability for committed differentiation into oocytes. The ovary is one of the most important female organs and reflects physiological signs of aging. Delay of ovarian age may avoid the negative consequences of menopause on one's health and its climacteric symptoms. It can also be applied to the fertility of women with POF to prolong their lives. Regarding the latter, FGSCs can be isolated and preserved for future use by cryopreservation after the biopsy of the ovarian cortex from the patient. Then, it might be able to use for *in vitro* fertilization by starting *in vitro* maturation to mature oocytes or injected back into the patient's ovaries to undergo neo-folliculogenesis. These will offer several advantages. First, the collection of ovarian cortex samples does not create the need to delay life-saving treatment, contrary to ovarian superovulation regimens. Additionally, more new follicles and oocytes may be obtained from FGSCs than from cryopreserved tissue or ovarian stimulation. However, the actual clinical application has not yet been achieved, due to technical and regulatory issues. Thus, research on FGSCs for therapeutic application has become an important topic.

Nowadays, OTCs and re-transplantation is a viable methods to preserve fertility in cancer patients. Therefore, most research focuses on technical aspects of OTCs, including follicle survival from freezing/thawing and fragment size, and duration of ovarian function after re-transplantation [82–84]. Study on *in vitro* culture for human OTCs is limited by the extreme difficulty of this technique and the unavailability of human tissue. Despite the undeniable advancements strengthening the protocols currently used for OTCs in domestic animals and endangered species has been achieved, this technique is still considered to be experimental for livestock such as swine [85], caprine [86], ovine [87], bovine [88], equine [89]. Using OTCs to establish FGSCs and differentiate into functional oocytes will answer the question about the possibility of creating gametes cells from adult mammalian ovaries. Gamete cells are ready for insemination with sperm to form embryos. This study will contribute significantly to the study of biological processes in human eggs in infertility treatment.

Moreover, FGSCs have great applicability in the basic science of stem cell models to study oocyte development and maturation, especially for treating human disease. Besides, FGSC is also very important in the production of transgenic animals. Transgenic animals are the animals with modified genome. A foreign gene is inserted into the genome of the animal to alter its DNA. This method is done to improve the genetic traits of the target animal. Until now, people have created many products of this type, and many products are suitable for use as food or medicine. Zhang and colleagues established FGSCs in mice and transplanted various genes [14]. FGSCs were transferred into the ovaries of infertile female mice. The result was to create transgenic mice after coordination with normal male mice. Scientists have suggested that the ability to produce transgenic animals in this method would be a great tool for biological reproduction in the future. Producing transgenic livestock can significantly improve human health, enhance nutrition, protect the environment, increase animal welfare, and decrease livestock disease. Especially, the creation of transgenic animals with biotechnology-based pharmaceuticals to produce precious protein for human or animal organ replacements, such as miniature pigs, due to the size of their organs being similar to humans.

In order to control these factors, a suitable culture system must be designed and optimized. From the basic research on the ability to generate eggs from FGSCs, applying this method to produce transgenic animals would be carried out efficiently. A study showed that FGSCs were established in mice and transplanted different genes into them. Then these FGSCs were transferred into the mouse ovaries of infertile females. The result was to create transgenic mice after coordination with normal male mice [14]. Recently, scientists have proven that FGSCs were a useful tool for the genetic manipulation of animals by creating transgenic rats [48]. Moreover, a study reported the success to restore ovarian function that suffers from cancer chemotherapy treatment and eventually produces offspring for the first time by transplanting the FGSCs [90]. Therefore, FGSCs played an essential role in the treatment of diseases caused by infertility females or in extending the period of menopause, as well as the application of stem cell therapy. In addition, FGSCs may play a significant role in treating diseases caused by infertile females or in extending the period of menopause, as well as the application of stem cell therapy.

#### **10. Summary**

FGSCs have a vital role in the treatment of diseases caused by infertility of females or in extending the period of menopause, as well as the application of stem cell therapy. Research on FGSCs opened up a new direction in reproductive biotechnology to treat infertility and produce biological drugs supported in pre-menopausal syndrome in women.

Our results support the theory that the ovary contains a small number of undifferentiated cells with stem cell characteristics. They may remain in the postnatal and adult ovary, but they are generally unable to proliferate due to inhibitory factors in the ovary. Under appropriate conditions, however, they can proliferate, differentiate into OLCs, and self-renewal of FGSCs. The presence of such FGSCs in mammalian ovaries and the depletion of ovarian reserve as the female reproductive system ages leads to the hypothesis that such "neo-oogenesis" was present in ancestors and is still present in insects, some fish, and mollusks. Nevertheless, it has been lost in terrestrial vertebrates during evolution. FGSCs are usually unable to proliferate in the ovary due to the presence of inhibitory factors unless placed under appropriate conditions. Although we have successfully established pFGSCs and differentiated them into

*Female Germline Stem Cells: A Source for Applications in Reproductive and Regenerative… DOI: http://dx.doi.org/10.5772/intechopen.110438*

OLCs, it is still inconclusive whether FGSCs become functional oocytes through their growth, maturation, fertilization, and embryonic development in large animals.

In summary, FGSCs appear to exist in ovaries and have been independently isolated by different research groups and from various species (e.g., humans, pigs, mice, rats, etc.). Furthermore, these cells can be manipulated *in vitro* and transplanted to produce offspring. However, only mice and rats have successfully produced offspring from FGSCs. Although the biological significance of these cells remains controversial, their identification and isolation are expected to provide a valuable model for understanding germ cell development and represent a significant step forward in the future for reproductive biotechnology and infertility treatment. Thus, research on the isolation and culture of FGSCs from ovarian tissue before or after cryopreservation may be helpful in the treatment of fertility in women.

Acknowledgments: Our research is funded by Vietnam National University HoChiMinh City (VNU-HCM) under grant number B2022-28-01.

#### **Author details**

Hong-Thuy Bui1,2\*, Nhat-Thinh Nguyen1,2,3, Truc Phuong Lam Do1,2, Anh My Le Ba1,2 and Nguyen Van Thuan1,2

1 Cellular Reprogramming Lab, School of Biotechnology, International University, Ho Chi Minh City, Vietnam

2 Vietnam National University, Ho Chi Minh City, Vietnam

3 School of Medicine-VNU, Ho Chi Minh City, Vietnam

\*Address all correspondence to: bhthuy@hcmiu.edu.vn

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### Section 3
