*2.1.1. Sub-culturing cells*

As cells reach confluency, they must be sub-cultured or passaged. The first step in sub-culturing adherent cells is to detach them from the cell culture plate or flask. This is done by subjecting them to trypsin-EDTA or by physically scraping them off the plate using a sterile cell scraper. One must take care because some mechanical and chemical methods have the


DMEM, Dulbecco's Modified Eagles Medium; MEM, minimum essential medium; RPMI, Roswell Park Memorial Institute; FBS, fetal bovine serum.

**Table 1.** Common 2D cell culture media recipes.

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

*3.1.1. Scaffold-based cell culture*

when designing the scaffold as described in **Table 2**.

scaffolds fabrication are given in **Table 3**.

state scaffolds.

**Table 2.** Scaffold requirements.

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

Two-Dimensional (2D) and Three-Dimensional (3D) Cell Culturing in Drug Discovery

http://dx.doi.org/10.5772/intechopen.81552

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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

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

**Property Purpose References** Biocompatibility Ability to provide normal cellular function [18] Bioactivity Ability to activate fast tissue attachment to the implant surface [18] Biodegradability Allow cells to produce their own ECM [19]

implantation and must have enough mechanical integrity for the

adequate diffusion of nutrients to cells and mean pore size should large

[20]

[21]

Mechanical response Scaffold should be strong enough to allow surgical handling during

completion of the remodeling process

Scaffold architecture Porous interconnected structure provide cellular penetration and

enough to allow cells to migrate into the structure

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

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.
