**3. Applications of 3D cultures** *in vitro*

Spurred by the recent advent in cell culture technologies, three-dimensional (3D) cell culture is paving the way in promoting tissue organization and cell differentiation by triggering tissue-based diseased microenvironment. An ideal 3D cell culture system generally composed of tightly bound tissues that involve cell–cell fluent interaction almost mimicking the extracellular matrix (ECM) that is highly dynamic and includes scaffolds of cells in a fluid that enhances them to differentiate (**Table 1**). The key parameter of a 3D culture environment is the ability to organize the spatial arrangement of cells with other surrounding cells along with physical constraints [8]. This significant approach has gardened great focus on understanding complex cellular biology and their responses by validating mammalian tissue studies via linking the gap between *in vitro* and *in vivo* environments. The two-dimensional (2D) cell cultures lack several features that 3D cultures impart such as tissue-specific architecture and complex cellular interactions that make them poor models for complex diseases. Based on the process of preparation, 3D techniques are categorized into (i) scaffold-based, (ii) scaffold-free culture systems. Scaffold-based technique efficiently is more responsive towards cell-to-ECM connection because of their potentiality of mimicking ECM whilst scaffold-free technique persuades physiological and cellular gradients. Scaffold or matrix is designed according to the tissue of interest, higher is the complexity of the scaffold, and the higher is the difficulty to extract the cells for analysis [9]. Majorly, the scaffolds manufactured are polymeric hard material-based support, microfluidic-based assembly, hydrophilic glass fiber, and organoids. In contrast, scaffold-free systems form cellular aggregates called spheroids that have evolved with improvement in their techniques such as magnetic levitation, liquid overlay (low adhesion plates), hanging drop microplates, and spheroid microplates yet the demand for 3D scaffolds preferentially increased due to their immense property of modulating the behavior of cultured cells according to the matrix in which they are cultured.

#### **3.1 3D Spheroids**

3Dspheroids or multi-cellular aggregates are spherical micro-sized cellular constructs that are produced from numerous gamuts of cell types, originally from scaffold-free systems. The most characteristic features of 3D spheroids are the ability to recapitulate a cell's typical physiological behavior, cellular heterogeneity, gene expression, cell–cell signaling, and structural architecture with respect to cell–cell contact [10]. Various types of 3Dspheroids include embryonic bodies, tumor spheroids (spheres of different tumorcells), hepatospheres (spheres of hepatic cells), neurospheres (spheres of different cell types of the central nervous system (CNS)) and mammospheres (spheres of mammary glands) [38]. An ideal 3D spheroid constitute ECM components such as proteoglycans, laminin, collagen, fibronectin, tenascin, and glycosaminoglycans [39] which tightens the spheroid density with close ECM-cell and cell–cell anchors eventually increase interstitial fluid pressure (IFP). Depending on the primary amount of cells seeded, the size of spheroid increases with an elevation in cell number, oxygen, and nutrient gradients equivalent to the tissue of interest [5]. Alongside, the different techniques enabling spheroid cultures are illustrated further.

Hanging drop technique is a non-scaffold method wherein a drop of media containing cells are suspended inversely on the lid of the culture dish (bottom-less and open) such that there is no surface provided for the cells and tend to hang. This attempt forms a cluster called spheroid at the tip of the droplet when cultured for a

*Two-Dimensional and Three-Dimensional Cell Culture and Their Applications DOI: http://dx.doi.org/10.5772/intechopen.100382*

longer period [40]. Spheroids formed through hanging drop cultures have fetched considerable stance in cell culture technology with 100% reproducibility owing to ubiquitous applications in cancer research [41], toxicity testing in hepatocytes [42], and constructing cardiac spheroids [43]. Another method involves the use of a liquid overlay that eases the formation of aggregates and commercially produced as low adhesion plates. These spheroid microplates contain either hydrophilic or hydrophobic coating with V-shaped bottom and allow mild attachment to the surface such that the cells tend to self-aggregate and form spheroid. Unlike the hanging drop technique, low adhesion plates generate one spheroid per plate that signifies its importance for multicellular culture. This ensures a medium-throughput screening that requires no modification in spheroid formation [44]. Spheroids can also be cultured with the use of magnetic nanoparticles with the application of the magnetic field. The process is called magnetic cell levitation that is highly applied to produce spheroids of mesenchymal stem cells and tissue engineering [45, 46]. An *in vivo* study showed that human glioblastoma cells levitated by a magnetic approach closely mimicked the protein expression of human glioblastoma tumor xenografts [47].

### **3.2 Organoids**

Organoids refer to the primary cultures derived from cell aggregates through *in vitro* process that is grown in 3D gels containing ECM to produce organ-like buds with the application of either physical support (cell adherence) or biochemical cues (signaling pathway modulation). Various types of cells such as embryonic, adult, primary, and stem cells are utilized for the development of organoids. Based on organ-like structures formed, organoids are classified into tissue and stem cell organoids. The application of organoids has helped in producing numerous *in vitro* organoids such as rectal [48], gastric [49], lung [50], liver [51], pancreas [52], retina [53], thyroid [54], kidney [55] and intestine [56] that had successfully recapitulated the structural and functional motif of real organs. 3D organoids are extensively used by researchers to decipher the toxicity analysis, examine the genetic pathologies and investigate the local immune responses to infections. In addition to this, current reports have suggested the promiscuous application of organoids in platforms like transcriptomics and proteomics technologies. One such example illustrates the interaction study between Zika virus and Toll-like receptor 3 is performed by the generation of cerebral organoids from embryonic stem cells [57]. They have been also used as models for distinct genetic diseases. For instance, a study applied the rectal organoid model of cystic fibrosis for the investigation of the potency of transmembrane regulator-modulating compounds [48]. Besides, the tubular organoids model of polycystic kidney disease was also used to unravel the cause for cyst formation [55]. Apart from this, organoids have been an excellent source of models to understand the depth of neurodegenerative diseases viz.; Alzheimer, Parkinson's, HIV, diabetes, or cancer.

#### **3.3 Cancer co-culture models**

Cancer cell lines have emerged as an eminent tool for comprehending complex physiology of cancers. The cell cultures have eased the outlook in preclinical research to understand the process of disease, morphological changes occurring in tissue, gene function, cell biology and tissue engineering [58]. They have evolved with immense features of offering homogenous samples without any sort of modification and

variations. However, a big leap was noted when monolayer cell cultures (2D) obtained from solid tumors were incapable of mimicking the structural elements of tumor microenvironment. Thusly, 3D cancer cell culture models have placed an enduring platform recently whereby ECM in 3D construct is same as that of original cell culture and imparted knowledge of predicting tumor response to treatment [59]. The application of 3D cell culture models of tumors have ought to manifest typical properties of tumor microenvironment such as gene and protein expressions, morphology, angiogenesis, malignancy and invasiveness. From this standpoint, 3D tumor cell culture models scintillate anticancer therapeutics and cancer drug discovery. To date, a vast content of literature owes the significance of these 3D co-cultures models in varying applications. In a study, tumor-associated macrophages (TAM) or cancer-associated fibroblasts (CAF) and gelatin hydrogel microspheres (GM) have been applied to produce cancer co-culture models from different cancer cells including HepG2 (liver), MCF-7 (breast) and WA-hT (lung) in order to inspect sustained release of drugs. They induced metastatic proteins involved in epithelial-mesenchymal transition (EMT) with transforming growth factor-β1 (TGF-β1) and reported elevation in N-cadherin and Vimentin proteins with deceleration in E-cadherin protein [58]. Recently, cancer co-culture models evinced interest in numerous approaches such as 3D breast cancer co-culture models obtained from MCF-7, MRC-5 and MDA-MB-231 tumor cells were used in investigating radiation-induced fibrosis [59], tumor-associated fibroblast differentiation [60] and development of immunotherapies [61], 3D lung cancer co-culture models derived lung squamous carcinoma and Non-Small Cell Lung Cancer Cells (NSCLC)fromTUM622, A549 and Colo699 tumor cells were utilized to explore tumor-stroma interactions [62, 63], 3D renal cancer co-culture models formed from Caki 1 (skin metastasis derived) and ACHN (pleural effusion derived) were sought for determining the efficacy of produced 3D models in stem cell physiology research and drug toxicity screening [64]. 3D colon cancer co-culture models acquired from LS 174 T, HCT 116, Colo205, MCF7, SW480, SW620, CCD-18Co, Caco-2, HT-29, and H446 have also been used to explore tumor-stroma interactions [65].

#### **3.4 Tissue co-culture models**

*In vitro* tissue models with the use of co-culture cells have emphasized greater applications to represent varying mechanisms of human body which is a daunting task. These models have served a vital role over several animal models that once were used to examine human physiology and pathophysiology. The major limitation of animal models was the failure of mimicking true human facets and their ethical constraints. This led to fetch insights into development of tissue models as a research tool from co-cultures such that the created models would possibly recapitulate the natural microenvironment of cells and examine the pathophysiological bases of diseases. Distinct *in vitro* 3D tissue models have been achieved with the approach of tissue engineering comprising human characteristics with increased complexity as compared to the 2D monolayers. Some of the examples of 3D models constructed from tissue co-cultures include kidney [66], neuro-glia [67], lung [68], liver [69], ovary [70] and intestine [71] that have potentially predicted and represented physiological responses of the original culture of cells. Most probably, primary cells are used as they possess feasible *in vivo* features of not being immortal, incapable of getting transformed and consist of limited survival time in culture. Pertaining to these advantages, they have been applied to develop models that would combat various disease and physiological studies. Reportedly, tissue models have been revolutionized in terms of investigating

### *Two-Dimensional and Three-Dimensional Cell Culture and Their Applications DOI: http://dx.doi.org/10.5772/intechopen.100382*

multiple changes in real-time processes. A pulmonary endothelium model was constructed to investigate massive inflammation in patients with acute respiratory disease syndrome (ARDS). The authors performed this study using lavage samples of the patients for determining the etiology of ARDS that took place during the process of disease [72]. In addition, endothelial cells have also played a key role in constructing 3D tissue models with their fascinating physiological roles. For instance, *in vitro* gastrointestinal epithelial cell cultures derived from adult murine colon allowed the authors to analyze epithelial cell–cell interactions, microbiological infections and cellular signaling [73]. Another instance showed the potential of hepatocyte tissue cultures in maintaining the cancer cell hierarchy in human hepatocellular carcinoma [74].
