**3. Three-dimensional cell culture system**

Three-dimensional cell culture was developed to improve the structure of cells and physiological equivalence of *in vitro* experiments performed. It refers to the culture of living cells inside micro assembled devices with a 3D structure mimicking tissue and organ specific microarchitecture [12]. In 3D cell culturing, growth of cells in their 3D physical shape allows better cellto-cell contact and intercellular signaling networks [13]. The 3D environment also facilitates developmental processes allowing cells to differentiate into more complex structures [14].

#### **3.1. Three-dimensional cell culture techniques**

Three-dimensional cell culture techniques are classified as Scaffold-based or non-scaffoldbased techniques. Researchers are required to select the most appropriate model for their cell-based assay.

#### *3.1.1. Scaffold-based cell culture*

potential to damage the cellular structure and possibly kill cells. Once detached, pre-warmed medium is added to stop the activity of trypsin-EDTA or to dilute the cell suspension. Varying amounts of the cell suspension are then transferred into fresh culture vessels and the appropriated amount of pre-warmed medium added and further incubated in 37°C incubator with

Many types of *in vitro* assays are performed in Drug Discovery and Development Research (DDDR), however, use of cell cultures receives extensive use. For example, determination of drug absorption, distribution, metabolism, excretion and toxicity (ADMETox) or drug pharmacokinetics is initially assessed in *in vitro* experiments involving cell cultures. Various cell lines in 2D cultures are used to determine different aspects of ADMETox. For instance, the Human colon carcinoma cells (Caco-2) are commonly used to determine absorption of drug candidates. Cultured Caco-2 cells form tight junctions in a monolayer and mimic intestinal epithelium. Additionally, Caco-2 cells express proteins that are involved in drug transport making them a good model for testing drug absorption [8]. Another cell line commonly used to test absorption is the Madin-Darby canine kidney (MDCK-MDR1) cell line, which mimics efflux activity of P-glycoprotein and allows faster performance of transport assays [9]. Hepatic metabolism plays a critical role in the removal of xenobiotics. Hepatocytes are usually the best model to study drug metabolism [10]. Although immortalized hepatocyte cell lines such as HepG2 and HepaRG are used to test drug metabolism and excretion, freshly isolated hepatocytes are the best model as they exhibit complete expression of metabolic enzymes [10, 11]. Although 2D cell cultures are used widely in DDDR and play a big role in preclinical drug testing, data generated from their use often do not translate to what occurs *in vivo*. Nowadays, 3D cell cultures and co-cultures receive more attention as they exhibit protein expression patterns and intracellular junctions that are similar to *in vivo* states compared to classic monolayer cultures.

Three-dimensional cell culture was developed to improve the structure of cells and physiological equivalence of *in vitro* experiments performed. It refers to the culture of living cells inside micro assembled devices with a 3D structure mimicking tissue and organ specific microarchitecture [12]. In 3D cell culturing, growth of cells in their 3D physical shape allows better cellto-cell contact and intercellular signaling networks [13]. The 3D environment also facilitates developmental processes allowing cells to differentiate into more complex structures [14].

Three-dimensional cell culture techniques are classified as Scaffold-based or non-scaffoldbased techniques. Researchers are required to select the most appropriate model for their

.

*2.1.2. Two-dimensional cell cultures in drug discovery and development*

**3. Three-dimensional cell culture system**

**3.1. Three-dimensional cell culture techniques**

cell-based assay.

humidified atmosphere of 5% CO<sup>2</sup>

24 Cell Culture

Scaffold-based culture technologies give physical support to basic mechanical structures to extra-cellular matrix (ECM)-like matrices, on which cells can aggregate, proliferate and migrate [15]. In scaffold-based techniques, cells are implanted into the matrix and the chemical and physical properties of the scaffold material mold the characteristics of cell. The ultimate aim of a scaffold is to produce characteristics for the native cell function within the ECM. The 3D scaffold is usually biocompatible and it characterizes the shape and function of the assimilated cell structure [16]. The design of scaffold is based on the tissue of interest and the bigger or complex the scaffold is; the more difficult or harder the extraction of cells for analysis becomes [17]. Regardless of the tissue type, there are important factors to consider when designing the scaffold as described in **Table 2**.

Scaffolds are manufactured from natural and synthetic materials by a plethora of fabrication techniques. The main natural materials used for scaffold synthesis are different components of the ECM including fibrin, collagen and hyaluronic acid [22–24]. In addition, natural derived materials such as silk and gelatin may also be used [25]. Synthetic materials used for scaffold synthesis include polymers, titanium, bioactive glasses and peptides [26–28]. Polymers have been widely used as biomaterials for the fabrication of scaffolds, due to their unique properties such as high porosity, small pore size, high surface to volume ratio, biodegradation and mechanical properties [29, 30]. Scaffolds are designed to support cell adhesion, cell-biomaterial interactions, adequate transport of gases and nutrients for cell growth and survival and to avoid toxicity [31]. The fabrication technique for scaffold synthesis depends on the size and surface properties of the material and recommended role of the scaffold. The relevant fabrication techniques for a particular target tissue must be identified to facilitate proper cell distribution and guide their growth into 3D space. The various techniques for scaffolds fabrication are given in **Table 3**.


Scaffold-based 3D culture can be broadly divided into two approaches—hydrogels and solidstate scaffolds.

**Table 2.** Scaffold requirements.


*3.1.1.2. Solid state scaffolds*

*3.1.2. Scaffold-free 3D cultures*

described below.

*3.1.2.1. Scaffold-free 3D spheroid cultures*

Culturing cells into a solid scaffold provides 3D space and helps generate natural 3D tissuelike structures. Solid scaffolds for 3D culture can be designed with different materials such as ceramics, metals, glass and polymers. Polymers are mainly used to construct solid scaffolds of different sizes, varying shapes, porosity, stiffness and permeability [63]. The main advantage of solid scaffolds is their ability to create organized positioning of cells *in vitro* in a controllable and reproducible manner [64]. The cell adhesion, growth and behavior in solid scaffold significantly depends on factors such as scale and topography of the internal structure, material used for its construction, the surface chemical properties, permeability and mechanical properties [65]. Solid scaffolds are commercially available, and are distributed sterile and ready to use. One of the main solid scaffolds is described below. An example is the porous scaffold. Porous scaffold creates a 3D microenvironment for cells to enter and maintain their natural 3D structure. It has a homogenous interconnected pore network, allowing cells to interact effectively to create tissue like structures and provides improved nutrient supply to the center of the device [64]. Sponge or foam porous scaffold have been especially used for bone regrowth and organ vascularization. Porous scaffold can be synthesized with specific porosity, pore size, crystallinity and surface area to volume ratio [66]. Synthetic biodegradable polymers such as polylactic-co-glycolic acid (PLGA), polyether ester (PEE ), poly-l-lactic

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acid (PLLA) and PGA are the main materials used for porous scaffolding [67].

Scaffold-free-based 3D systems facilitate the development of multi-cellular aggregates, commonly known as spheroids, and can be generated from wide range of cell types [68]. Common examples of spheroids comprise tumor spheroids, embryonic bodies, mammospheres, neurospheres and hepatospheres. A cellular spheroid 3D model has a variety of properties such as (i) naturally mimicking/imitating various aspects of solid tissues; (ii) establishing geometry and ideal physiological cell-to-cell interactions; (iii) cells form their own ECM components and better cell-ECM interactions; (iv) excellent gradient for efficient diffusion growth factors as well as the (v) removal of metabolic waste [69]. The size of the spheroid can be based on the primary number cells seeded and it can increase in size where until they show oxygen and nutrient gradients similar to target tissue [70]. Spheroids are either self-assembling or are forced to grow as cell clusters [71]. Spheroids can be easily analyzed by imaging using light fluorescence, and confocal microscopy and that is an added advantage of spheroids compared to other 3D models. There are different approaches for facilitating spheroid cultures as

Hanging drop method co-culture used to generate tissue-like cellular aggregates for molecular and biochemical analysis in a physiological suitable model. The hanging drop method was first developed in 1994 and became the basis of the non-scaffold method for the formation of multicellular spheroids. In hanging drop method, cells are cultured in a drop of media suspended on the lid of a cell culture dish, which is carefully inverted and placed on top of the

**Table 3.** The different scaffold fabrication techniques and their advantages.

#### *3.1.1.1. Hydrogel scaffolds*

Hydrogels are water swollen polymeric materials formed by chemical reactions of monomers that generate main-chain free radicals that make cross-link junctions or by hydrogen bonding [46]. Hydrogels are one of the most used scaffolds because they mimic the ECM to a certain extent [17]. Hydrogels are highly hydrated hydrophilic polymer networks with pores and void space between the polymers [47]. The hydrophilic structure facilitates absorption and retention of large quantities of water. It is regarded as a powerful method when applied for biomedical purposes [48]. Because hydrogels have properties such as soft and rubbery consistence, low surface tension and high water content, they are more suitable substitutes for natural tissues [49]. Sources of hydrogels can be natural, synthetic or a mixture of both (hybrid) materials, offering a broad spectrum of chemical and mechanical properties. The natural materials used for hydrogels are collagen, gelatin, alginate, fibrin, hyaluronic acid, agarose, chitosan and laminin [50–53]. Natural hydrogels confer l adhesive properties, high cell viability, controlled proliferation and differentiation. Collagen is the most widely used natural polymer for hydrogel preparation and it is the main component of tissues such as ligament, bone, cartilage skin and tendon [54, 55].

Synthetic hydrogels can mimic biological properties of ECM and are ideal material to use for 3D scaffolds. They have well defined chemical, physical and mechanical properties to achieve stiffness and porosity [56]. The main synthetic materials used to formulate hydrogels are polyacrylic acid, polyethylene glycol (PEG), polyvinyl alcohol, polyglycolic acid (PGA) and poly (2-hydroxy ethyl methacrylate [57–60]. Synthetic hydrogels are the most used hydrogels because of their longer service life, high gel strength and water absorption capacity [61]. PEG and its derivatives are used mainly for synthetic hydrogels [62].

#### *3.1.1.2. Solid state scaffolds*

Culturing cells into a solid scaffold provides 3D space and helps generate natural 3D tissuelike structures. Solid scaffolds for 3D culture can be designed with different materials such as ceramics, metals, glass and polymers. Polymers are mainly used to construct solid scaffolds of different sizes, varying shapes, porosity, stiffness and permeability [63]. The main advantage of solid scaffolds is their ability to create organized positioning of cells *in vitro* in a controllable and reproducible manner [64]. The cell adhesion, growth and behavior in solid scaffold significantly depends on factors such as scale and topography of the internal structure, material used for its construction, the surface chemical properties, permeability and mechanical properties [65]. Solid scaffolds are commercially available, and are distributed sterile and ready to use. One of the main solid scaffolds is described below. An example is the porous scaffold. Porous scaffold creates a 3D microenvironment for cells to enter and maintain their natural 3D structure. It has a homogenous interconnected pore network, allowing cells to interact effectively to create tissue like structures and provides improved nutrient supply to the center of the device [64]. Sponge or foam porous scaffold have been especially used for bone regrowth and organ vascularization. Porous scaffold can be synthesized with specific porosity, pore size, crystallinity and surface area to volume ratio [66]. Synthetic biodegradable polymers such as polylactic-co-glycolic acid (PLGA), polyether ester (PEE ), poly-l-lactic acid (PLLA) and PGA are the main materials used for porous scaffolding [67].

#### *3.1.2. Scaffold-free 3D cultures*

*3.1.1.1. Hydrogel scaffolds*

Solvent casting/particulate

leaching

26 Cell Culture

Hydrogels are water swollen polymeric materials formed by chemical reactions of monomers that generate main-chain free radicals that make cross-link junctions or by hydrogen bonding [46]. Hydrogels are one of the most used scaffolds because they mimic the ECM to a certain extent [17]. Hydrogels are highly hydrated hydrophilic polymer networks with pores and void space between the polymers [47]. The hydrophilic structure facilitates absorption and retention of large quantities of water. It is regarded as a powerful method when applied for biomedical purposes [48]. Because hydrogels have properties such as soft and rubbery consistence, low surface tension and high water content, they are more suitable substitutes for natural tissues [49]. Sources of hydrogels can be natural, synthetic or a mixture of both (hybrid) materials, offering a broad spectrum of chemical and mechanical properties. The natural materials used for hydrogels are collagen, gelatin, alginate, fibrin, hyaluronic acid, agarose, chitosan and laminin [50–53]. Natural hydrogels confer l adhesive properties, high cell viability, controlled proliferation and differentiation. Collagen is the most widely used natural polymer for hydrogel preparation and it is the main component of tissues such as ligament, bone, cartilage skin and tendon [54, 55].

**Scaffold fabrication techniques Advantages References**

Gas foaming Controlled porosity and pore size, free of strong organic solvents [34, 35]

step not required, work at low temperature

with special orientation and large surface area

Fiber mesh Variable pore size, large surface area for cell attachment [41, 42]

Micro molding It is biologically degradable, mechanical and physical complexity [45]

highly porous structure

Fiber bonding Large surface area for cell attachment, interconnected fiber structure and high porosity

Freeze drying High porosity and interconnectivity, controlled pore size, leaching

Electrospinning Controlled over porosity and pore size, produces ultra-thin fibers

Porogen leaching High porosity, controlled pore size and geometry, bigger pore size and increased pore interconnectivity

porosity

**Table 3.** The different scaffold fabrication techniques and their advantages.

Melt molding Able to construct scaffolds of any shape by changing the mold

Easy method, pore size can be controlled, desired crystallinity,

geometry, free of organic solvents, controlled pore size and

[32]

[33]

[36]

[37, 38]

[39, 40]

[43, 44]

Synthetic hydrogels can mimic biological properties of ECM and are ideal material to use for 3D scaffolds. They have well defined chemical, physical and mechanical properties to achieve stiffness and porosity [56]. The main synthetic materials used to formulate hydrogels are polyacrylic acid, polyethylene glycol (PEG), polyvinyl alcohol, polyglycolic acid (PGA) and poly (2-hydroxy ethyl methacrylate [57–60]. Synthetic hydrogels are the most used hydrogels because of their longer service life, high gel strength and water absorption capacity [61]. PEG

and its derivatives are used mainly for synthetic hydrogels [62].

#### *3.1.2.1. Scaffold-free 3D spheroid cultures*

Scaffold-free-based 3D systems facilitate the development of multi-cellular aggregates, commonly known as spheroids, and can be generated from wide range of cell types [68]. Common examples of spheroids comprise tumor spheroids, embryonic bodies, mammospheres, neurospheres and hepatospheres. A cellular spheroid 3D model has a variety of properties such as (i) naturally mimicking/imitating various aspects of solid tissues; (ii) establishing geometry and ideal physiological cell-to-cell interactions; (iii) cells form their own ECM components and better cell-ECM interactions; (iv) excellent gradient for efficient diffusion growth factors as well as the (v) removal of metabolic waste [69]. The size of the spheroid can be based on the primary number cells seeded and it can increase in size where until they show oxygen and nutrient gradients similar to target tissue [70]. Spheroids are either self-assembling or are forced to grow as cell clusters [71]. Spheroids can be easily analyzed by imaging using light fluorescence, and confocal microscopy and that is an added advantage of spheroids compared to other 3D models. There are different approaches for facilitating spheroid cultures as described below.

Hanging drop method co-culture used to generate tissue-like cellular aggregates for molecular and biochemical analysis in a physiological suitable model. The hanging drop method was first developed in 1994 and became the basis of the non-scaffold method for the formation of multicellular spheroids. In hanging drop method, cells are cultured in a drop of media suspended on the lid of a cell culture dish, which is carefully inverted and placed on top of the dish containing media to maintain a humid atmosphere. Suspended cells then come together and form 3D spheroids at the apex of the droplet of media [72, 73]. This method has many advantages such as cost effectiveness, controlled spheroid size, and various cell types can be co-cultured and produced into spheroids [74, 75]. Moreover, it has been reported that 3D cell culture generated with hanging drop method have 100% reproducibility [69]. Due to limited volume of droplets generated with this technique, it is difficult to maintain spheroids and change the medium. Presently, there are many commercial devices for hanging drop culture (**Figure 2**).

as well as creating defined geometry suitable for multicellular culture [78]. These plates have initial higher volume capacity than hanging droplets and there is no need to manipulate the

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Spheroids can also be cultured by using bioreactors under specific dynamic conditions [79]. The dynamic conditions are generated by stirring or rotating using spinner flask or NASA (National Aeronautics and Space Administration) rotating wall vessel, respectively [80]. The rotating wall vessel produces larger sized spheroids than spinner flask [81]. Bioreactors provide greater spheroid production control and reproducibility [82]. However, production of spheroids through this method requires expensive instruments and high quality cell culture

Organoids are *in vitro* derived 3D cell aggregates that are capable of self-renewal, self-organization, and exhibit organ functionality [83]. Organoids are produced either from stem cells or primary tissues by providing suitable physical (support for cell attachment and survival) and biochemical (modulate signaling pathways) cues [84]. Organoids are classified into tissue organoids and stem cell organoids, based on how the organ buds are created [85]. Distinctive examples of tissue organoids culture are intestine, prostate, mammary and salivary glands. Stem cell organoids are created from either embryonic stem cells or primary stem cells (neonatal tissue) or induced pluripotent cells. Presently, different *in vitro* organoids have been set to simulate numerous tissues such as functional organoids for pancreas [86], liver [85], intestine [87], kidney [88], lung [89], retina [90], stomach [91] and thyroid [92]. Organoids mimic some of the structure and function of real organs [83]. Several approaches have been used to obtain organoids. The first approach is to culture cells as a monolayer on an ECM coated surface; organoids are then produced after the cells differentiate. The second is a mechanically supported cell culture to provide further differentiation of primary tissues. The third approach is to produce embryoid bodies through hang drop culture or on the low adhesion plates [93]. The main disadvantages of organoids are the lack of vasculature, lack of key cell types found *in vivo* and some organoids only replicate early stages of organ development [83].

**3.2. Three-dimensional cell culture in drug discovery and development**

Cell-based assays are the major tool used to evaluate the potency of a new compound in drug discovery. Three dimensional cell culture technologies have been used in different stages of drug discovery including diseases modeling, target identification and validation, screening, target selection, potency profiling and toxicity assessment. **Table 4** indicates the 3D models used in different stages of drug discovery. Three-dimensional culture models behave similarly to the cells *in vivo*, and are therefore used in the early stage of the drug discovery process, especially in cytotoxicity tests [94] such as MTT, Flow Cytometry and so on. The most effective cell-based assays with 3D cultures are cell viability, proliferation, signaling and migration [95]. It is now broadly accepted that cells act differently in 3D environments compared to 2D ones, especially when it comes to drug discovery—many prospective cancer therapeutics look favorable in the 2D cell culture dish, but fall painfully later on in clinical development.

spheroids.

medium.

*3.1.2.2. Scaffold-free organoid cultures*

The use of low adhesion plates helps to promote self-aggregation of cells into spheroids [76]. Low adhesion plates have been developed as the commercial product of the liquid overlay technique, which is a low cost highly reproducible culture method that easily promotes 3D aggregates or spheroids [77]. Low adhesion plates are spheroid microplates with round, V-shaped bottoms and very low attachment surfaces to generate self-aggregation and spheroid formation. Plates are designed with hydrophilic or hydrophobic coating, which reduces cell from attaching to the surface. The main advantage of low adhesion plates is the potential to produce one spheroid per well making it appropriate for medium-throughput screening,

**Figure 2.** (a) A schematic of the hanging drop plate and (b) Schematic of spheroid formation techniques for hanging drop spheroids.

as well as creating defined geometry suitable for multicellular culture [78]. These plates have initial higher volume capacity than hanging droplets and there is no need to manipulate the spheroids.

Spheroids can also be cultured by using bioreactors under specific dynamic conditions [79]. The dynamic conditions are generated by stirring or rotating using spinner flask or NASA (National Aeronautics and Space Administration) rotating wall vessel, respectively [80]. The rotating wall vessel produces larger sized spheroids than spinner flask [81]. Bioreactors provide greater spheroid production control and reproducibility [82]. However, production of spheroids through this method requires expensive instruments and high quality cell culture medium.
