Common 2D cell culture media recipes.
Cell culture is an indispensable in vitro tool used to improve our perception and understanding of cell biology, the development of tissue engineering, tissue morphology, mechanisms of diseases and drug action. Efficient cell culturing techniques both in vitro and in vivo allow researchers to design and develop new drugs in preclinical studies. Two-dimensional (2D) cell cultures have been used since 1900s and are still a dominant method in many biological studies. However, 2D cell cultures poorly imitate the conditions in vivo. Recently three-dimensional (3D) cell cultures have received remarkable attention in studies such as drug discovery and development. Optimization of cell culture conditions is very critical in ensuring powerful experimental reproducibility, which may help to find new therapies for cancer and other diseases. In this chapter, we discuss the 2D and 3D cell culture technologies and their role in drug discovery.
- 2D cell culture
- 3D cell culture
- drug discovery
- cell-based assays
The discovery and development of new drugs is a very lengthy and costly process. The cost of developing a new drug and bringing it to the market is between $800 million and $2 billion, and can take up to 15 years. In part, termination of the development process is due to failure at late preclinical stages of development at great expenditure . The drug discovery and development process for new drugs consists of four phases; drug discovery, preclinical development, clinical development and regulatory approval. Most drugs fail at phase II and phase III clinical stages due to poor efficacy and safety issues . The high attenuation rates in drug discovery suggest that the main reasons for drug failure are inappropriate preclinical testing methods and
Cell-based assays are crucial in the drug discovery and development process. Mammalian cell culture provides a defined platform for investigating cell and tissue physiology and pathophysiology outside of the organism. For over a century, traditional 2D cell culture was used in drug discovery. In 2D cell culture, cells are grown on flat dishes optimized for cell attachment and growth (Figure 1). Nowadays, 2D cell culture models are still used to test cellular drug responses to drug candidates. Although 2D cell culture is generally accepted and has increased understanding of drug mechanisms of action, there are limitations associated with it. The main limitation is that the cells grown as a monolayer on flat petri plates or flasks. This is a stiff platform, offering unnatural growth kinetics and cell attachments. Therefore, natural microenvironments of the cells are not fully represented . Recently, significant work by researchers produced improvements in the form of better
While traditional monolayer cultures still are predominant in cellular assays used for high-throughput screening (HTS), 3D cell cultures techniques for applications in drug discovery are making rapid progress [6, 7]. In this chapter, we provide an overview of 2D and 3D cell culture techniques, and their role in the discovery of new drugs.
2. Cell culture system
Cell culture involves the dispersal of cells in an artificial environment that is composed of an appropriate surface, nutrient supply, and optimal conditions of humidity, temperature and gaseous atmosphere . Usually cells are grown for days or weeks in a sterile 37°C humidified incubator with 5% CO2 until a sufficient number of cells are reached. This system allows the study of cellular response to different environmental cues such as physiological stimulants or agonists/antagonists, potential drugs or pathogens.
2.1. Two-dimensional (2D) cell culture system
Two-dimensional culture conditions vary widely for each cell type. Appropriate cell culture medium suitable for the growth of particular cells has to be used. Various laboratories use different recipes of cell culture media prepared in the laboratory or commercially produced. The commercially produced cell culture medium is obtained sterile and ready to use in liquid or powder form and is usually dissolved in sterile water. Most laboratories obtain commercial components, which are mixed in the lab to make a complete culture medium for optimal cell growth. In addition, the culture media are usually supplemented with antibiotics and/or fungicides to inhibit contamination (Table 1).
|Adherent cells||Non-adherent cell lines|
|Cancer cell lines||Non-cancerous cell lines|
|Cell culture medium||89% DMEM or MEM with high glucose, l-glutamine + 10% FBS + 1% penicillin/streptomycin||89% DMEM or MEM with medium/low glucose, l-glutamine + 10% FBS + 1% penicillin/streptomycin||89% RPMI-1640 + 10% FBS + 1% penicillin/streptomycin|
Many continuous mammalian cell lines can be maintained on a relatively simple medium such as MEM supplemented with serum and antibiotics. However, most laboratories use DMEM as mammalian cells can be easily grown in DMEM supplemented with serum as well as antibiotics. When working with specialized cell types, a specialized cell culture medium may be required to maintain the growth of cells such as RPMI-1640 medium that is mostly used to grow cells in suspension such as HL-60 (promyelocytic leukemia) with varying serum amounts.
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 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% CO2.
2.1.2. Two-dimensional cell cultures in drug discovery and development
Many types of
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
3. Three-dimensional cell culture system
Three-dimensional cell culture was developed to improve the structure of cells and physiological equivalence of
3.1. Three-dimensional cell culture techniques
Three-dimensional cell culture techniques are classified as Scaffold-based or non-scaffold-based techniques. Researchers are required to select the most appropriate model for their cell-based assay.
3.1.1. Scaffold-based 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 . 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 . 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 . Regardless of the tissue type, there are important factors to consider when designing the scaffold as described in Table 2.
|Biocompatibility||Ability to provide normal cellular function|||
|Bioactivity||Ability to activate fast tissue attachment to the implant surface|||
|Biodegradability||Allow cells to produce their own ECM|||
|Mechanical response||Scaffold should be strong enough to allow surgical handling during implantation and must have enough mechanical integrity for the completion of the remodeling process|||
|Scaffold architecture||Porous interconnected structure provide cellular penetration and adequate diffusion of nutrients to cells and mean pore size should large enough to allow cells to migrate into the structure|||
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, 23, 24]. In addition, natural derived materials such as silk and gelatin may also be used . Synthetic materials used for scaffold synthesis include polymers, titanium, bioactive glasses and peptides [26, 27, 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 . 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 fabrication techniques||Advantages||References|
|Solvent casting/particulate leaching||Easy method, pore size can be controlled, desired crystallinity, highly porous structure|||
|Melt molding||Able to construct scaffolds of any shape by changing the mold geometry, free of organic solvents, controlled pore size and porosity|||
|Gas foaming||Controlled porosity and pore size, free of strong organic solvents||[34, 35]|
|Fiber bonding||Large surface area for cell attachment, interconnected fiber structure and high porosity|||
|Freeze drying||High porosity and interconnectivity, controlled pore size, leaching step not required, work at low temperature||[37, 38]|
|Electrospinning||Controlled over porosity and pore size, produces ultra-thin fibers with special orientation and large surface area||[39, 40]|
|Fiber mesh||Variable pore size, large surface area for cell attachment||[41, 42]|
|Porogen leaching||High porosity, controlled pore size and geometry, bigger pore size and increased pore interconnectivity||[43, 44]|
|Micro molding||It is biologically degradable, mechanical and physical complexity|||
Scaffold-based 3D culture can be broadly divided into two approaches—hydrogels and solid-state scaffolds.
18.104.22.168. 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 . Hydrogels are one of the most used scaffolds because they mimic the ECM to a certain extent . Hydrogels are highly hydrated hydrophilic polymer networks with pores and void space between the polymers . The hydrophilic structure facilitates absorption and retention of large quantities of water. It is regarded as a powerful method when applied for biomedical purposes . 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 . 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, 51, 52, 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 . 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, 58, 59, 60]. Synthetic hydrogels are the most used hydrogels because of their longer service life, high gel strength and water absorption capacity . PEG and its derivatives are used mainly for synthetic hydrogels .
22.214.171.124. Solid state scaffolds
Culturing cells into a solid scaffold provides 3D space and helps generate natural 3D tissue-like 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 . The main advantage of solid scaffolds is their ability to create organized positioning of cells
3.1.2. Scaffold-free 3D cultures
126.96.36.199. 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 . 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 . 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 . Spheroids are either self-assembling or are forced to grow as cell clusters . 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 . 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).
The use of low adhesion plates helps to promote self-aggregation of cells into spheroids . 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 . 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, as well as creating defined geometry suitable for multicellular culture . 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 . The dynamic conditions are generated by stirring or rotating using spinner flask or NASA (National Aeronautics and Space Administration) rotating wall vessel, respectively . The rotating wall vessel produces larger sized spheroids than spinner flask . Bioreactors provide greater spheroid production control and reproducibility . However, production of spheroids through this method requires expensive instruments and high quality cell culture medium.
188.8.131.52. Scaffold-free organoid cultures
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
Spheroid 3D cell cultures have been used for modeling the microenvironments, signaling, invasion and immune characteristics of cancer, also for studying cancer stem cells . Studies have shown that cancer cell line spheroids have been used to analyze different characteristics of the cancer invasion process such as endothelial cell to tumor cell contact  and invasion of cells in a spheroid into the nearby 3D ECM structure . Additionally, organoid cell cultures have been used to model number of diseases infectious diseases, neurodevelopmental and neuronal degeneration disorders . For example, intestinal organoids were used to investigate genetically reconstituted tumorigenesis , gastrointestinal infection with rotavirus ,
Gene expression patterns seen in 3D systems are more similar to
Three-dimensional cell culture models have been shown to be more accurate in assessing drug screening, selection and efficacy than 2D models of the diseases [115, 124]. For instance, spheroids obtained from patients were used to identify an effective therapy for 120 patients with HER2-negative breast cancer of all stages. The results indicated that spheroid 3D culture models display present guideline treatment recommendation for breast cancer . In addition, 3D cell culture models are very powerful in analyzing drug induced toxicity. Organ buds of heart, liver, brain and kidney can be used to identify drug toxicity . For instance, liver cell spheroid 3D culture used for investigating drug induced liver injury, function and diseases. Spheroids generated from human primary hepatocyte found to be phenotypically stable and retained morphology and viability for almost 5 weeks, providing toxicity analysis of drug molecules . Liver spheroids and organoids also have been used to understand the metabolism of drug molecules.
However, many challenges remain in 3D cell culture technologies in the drug discovery process. Three-dimensional culture are different in terms of size, morphology, complexity and protocol for assaying compared to 2D cell culture, which can lead to challenges in systematic assessment, culture and assay protocol standardization. It also has complexity of identifying specific phenotypes for drug screening . Moreover, some 3D models have limited permeability, which can impact cell viability and functions thus making it difficult to have accurate automated system for HTS. A summary of the differences between 2D and 3D cell cultures is given in Table 5.
|Characteristics||2D cell culture||3D cell culture||References|
|Morphology||Cells grow on a flat surface and have flat or stretched shape||Cells grow naturally into 3D aggregates/spheroids in a 3D environment and natural shape retained|||
|Cell shape||Single layer||Multiple layers|||
|Cell to cell contact||Limited cell to cell contact, only on edges||Physiologic cell to cell contact similar to |||
|Distribution of medium||Cells receive an equal amount of nutrients and growth factors from the medium during growth.||Cells do not receive an equal medium during growth. The core cell receive less growth factors and nutrients from the medium and tend to be in a hypoxic state, which is very similar to ||[115, 127]|
|Cell proliferation||Generally, cells proliferate at a fast rate than ||Cells proliferate faster or slower depending on the type of cell or 3D system used||[128, 129, 130]|
|Protein/gene expression||Protein and gene expression profiles differ compared with ||Protein and gene expression profiles more similar to |||
|Cell differentiation||Moderately differentiated||Properly differentiated|||
|Response to stimuli||Poor response to mechanical stimuli of cells||Good response to mechanical stimuli of cells|||
|Viability||Sensitive to cytotoxin||Greater viability and less susceptible to external factors|||
|Drug sensitivity||Cells are more sensitive to drugs and drug show high efficacy||Cells are more resistant to drugs and drug show low potency|||
|Cell Stiffness||High stiffness||Low stiffness|||
|Sub-culturing time||Allows cell to be grown in culture for up to 1 week||Allows cells to be grown in culture for almost 4 weeks|||
Two-dimensional and 3D cell culture models have been widely used for improving the productivity of pharmaceutical research and development. It is evident that 3D culture systems hold great potential as a tool for drug discovery compared to 2D cell culture. This is due to the improved cell-cell and cell-ECM interactions, cell populations and structures that similar to
List of abbreviations
|ADMETox||absorption, distribution, metabolism, excretion and toxicity|
|CaCo-2||human colon carcinoma|
|DDDR||drug discovery and development research|
|DMEM||Dulbecco’s Modified Eagle Medium|
|HEP-G2||liver hepatocellular carcinoma|
|HER-2||human epidermal growth factor receptor 2|
|MDCK-MDR1||Madin-Darby canine kidney cells|
|MEM||minimum essential medium|
|RMPI||Roswell Park Memorial Institute medium|