**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.


Gene expression patterns seen in 3D systems are more similar to *in vivo* conditions compared to 2D cell culture systems [123]. For instance, analysis of gene expression in mesothelioma cell lines cultured in spheroids shows the basic cause of chemoresistance in malignant mesothelioma [108]. In addition, cancer cell lines grown in 2D and 3D models show different gene expression levels of various genes responsible for proliferation, chemo sensitivity, angiogenesis and invasion [63]. Ovarian cells grown in 3D system shown higher level of gene expression of the cell receptors integrins compared to 2D cell culture [99]. Moreover, 3D cell cultures are cost effective and time saving for drug screening because they decrease drug trail time whilst generating accurate representation of *in vivo* conditions [6]. Screening using cell-based assays has been the initial point for identifying the potential compounds in the early stage of drug discovery. Most 3D cell culture models, together with HTS and HCS (high-content screening)

**Characteristics 2D cell culture 3D cell culture References**

Cell shape Single layer Multiple layers [6]

Cell differentiation Moderately differentiated Properly differentiated [132]

factors

Cell Stiffness High stiffness Low stiffness [105]

4 weeks

low potency

Viability Sensitive to cytotoxin Greater viability and less susceptible to external

shape retained

Cells grow naturally into 3D aggregates/ spheroids in a 3D environment and natural

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Physiologic cell to cell contact similar to *in vivo* [127]

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 *in* 

Cells proliferate faster or slower depending on

Good response to mechanical stimuli of cells [133]

Cells are more resistant to drugs and drug show

Allows cells to be grown in culture for almost

Protein and gene expression profiles more

*vivo* tissues, especially in tumors

the type of cell or 3D system used

similar to *in vivo* models

[126]

[115, 127]

[128–130]

[131]

[134]

[135]

[136]

processes shows promise in identifying clinically relevant compounds.

Morphology Cells grow on a flat surface

shape

Cell to cell contact Limited cell to cell contact,

Cell proliferation Generally, cells proliferate

models

Drug sensitivity Cells are more sensitive to

Sub-culturing time Allows cell to be grown in

efficacy

Response to stimuli Poor response to

Distribution of medium

Protein/gene expression

only on edges

Cells receive an equal amount of nutrients and growth factors from the medium during growth.

at a fast rate than *in vivo*

mechanical stimuli of cells

drugs and drug show high

culture for up to 1 week

**Table 5.** Characteristics of 3D cell culture versus 2D cell culture.

Protein and gene expression profiles differ compared with *in vivo*

and have flat or stretched

**Table 4.** Three-dimensional culture techniques used in different stages of drug discovery.

Three-dimensional cell cultures promise to bridge the gap between traditional 2D cell culture and *in vivo* animal models. Studies have shown that cellular response to drug treatment in 3D cell culture are more similar to what occurs *in vivo* compared to 2D cell culture [96–98]. In addition, a number of studies show that cells cultured in 3D models are more resistant to anticancer drugs than those in 2D cultures [99, 100]. For example, the cell viability of ovarian cancer cells in 3D spheroid cell cultures after paclitaxel treatment was reduced by 40%, while the same treatment led to 80% reduced cell viability in 2D cell cultures [101]. The stronger drug resistance in 3D culture can be attributed to different factors including, phenotype and genotype changes [100], signals from cellular interactions between cells and ECM [102], activation of genes involved in cell survival and drug sensitivity due to limited diffusion through the spheroid [103].

Spheroid 3D cell cultures have been used for modeling the microenvironments, signaling, invasion and immune characteristics of cancer, also for studying cancer stem cells [104]. 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 [116] and invasion of cells in a spheroid into the nearby 3D ECM structure [117]. Additionally, organoid cell cultures have been used to model number of diseases infectious diseases, neurodevelopmental and neuronal degeneration disorders [83]. For example, intestinal organoids were used to investigate genetically reconstituted tumorigenesis [118], gastrointestinal infection with rotavirus [119], *Cryptosporidium parvum* infection [106], and colon cancer stem cell biology [107]. A large number of genetic disorders that have not been possible to model in animals can be modeled using organoid 3D cultures. For example, intestinal organoids derived from patient biopsies have been used to understand onset and progression of genetic disorders [120, 121]. Organoid 3D culture model is also a powerful tool for modeling neurodevelopmental disorders such as microencephaly, caused by Zika virus infection at early stages of brain development. Moreover, brain organoid model of neural stem cells was used to understand implications of Zika virus infection during neurogenesis [122]. These are some examples of uses of 3D cell cultures as models to study disease.

Gene expression patterns seen in 3D systems are more similar to *in vivo* conditions compared to 2D cell culture systems [123]. For instance, analysis of gene expression in mesothelioma cell lines cultured in spheroids shows the basic cause of chemoresistance in malignant mesothelioma [108]. In addition, cancer cell lines grown in 2D and 3D models show different gene expression levels of various genes responsible for proliferation, chemo sensitivity, angiogenesis and invasion [63]. Ovarian cells grown in 3D system shown higher level of gene expression of the cell receptors integrins compared to 2D cell culture [99]. Moreover, 3D cell cultures are cost effective and time saving for drug screening because they decrease drug trail time whilst generating accurate representation of *in vivo* conditions [6]. Screening using cell-based assays has been the initial point for identifying the potential compounds in the early stage of drug discovery. Most 3D cell culture models, together with HTS and HCS (high-content screening) processes shows promise in identifying clinically relevant compounds.


**Table 5.** Characteristics of 3D cell culture versus 2D cell culture.

Three-dimensional cell cultures promise to bridge the gap between traditional 2D cell culture and *in vivo* animal models. Studies have shown that cellular response to drug treatment in 3D cell culture are more similar to what occurs *in vivo* compared to 2D cell culture [96–98]. In addition, a number of studies show that cells cultured in 3D models are more resistant to anticancer drugs than those in 2D cultures [99, 100]. For example, the cell viability of ovarian cancer cells in 3D spheroid cell cultures after paclitaxel treatment was reduced by 40%, while the same treatment led to 80% reduced cell viability in 2D cell cultures [101]. The stronger drug resistance in 3D culture can be attributed to different factors including, phenotype and genotype changes [100], signals from cellular interactions between cells and ECM [102], activation of genes involved in cell survival and drug sensitivity due to limited diffusion through

Organoids [106, 107]

Organoids [113, 114]

[108]

**Drug discovery stages 3D model References** Disease modeling Spheroids [104, 105]

Organoids

Screening Spheroids [109–111] Efficacy profiling Spheroids [112] Toxicity profiling Spheroids [114, 115]

**Table 4.** Three-dimensional culture techniques used in different stages of drug discovery.

Target identification Spheroids

Spheroid 3D cell cultures have been used for modeling the microenvironments, signaling, invasion and immune characteristics of cancer, also for studying cancer stem cells [104]. 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 [116] and invasion of cells in a spheroid into the nearby 3D ECM structure [117]. Additionally, organoid cell cultures have been used to model number of diseases infectious diseases, neurodevelopmental and neuronal degeneration disorders [83]. For example, intestinal organoids were used to investigate genetically reconstituted tumorigenesis [118], gastrointestinal infection with rotavirus [119], *Cryptosporidium parvum* infection [106], and colon cancer stem cell biology [107]. A large number of genetic disorders that have not been possible to model in animals can be modeled using organoid 3D cultures. For example, intestinal organoids derived from patient biopsies have been used to understand onset and progression of genetic disorders [120, 121]. Organoid 3D culture model is also a powerful tool for modeling neurodevelopmental disorders such as microencephaly, caused by Zika virus infection at early stages of brain development. Moreover, brain organoid model of neural stem cells was used to understand implications of Zika virus infection during neurogenesis [122]. These are some examples of uses of 3D cell cultures as models to study

the spheroid [103].

30 Cell Culture

disease.

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 [113]. 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 [83]. 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 [115]. Liver spheroids and organoids also have been used to understand the metabolism of drug molecules.

DMEM Dulbecco's Modified Eagle Medium

EDTA ethylenediaminetetraacetic acid

HEP-G2 liver hepatocellular carcinoma

HTS high-throughput screening

MEM minimum essential medium

PEE polyether ester

PEG polyethylene glycol

PLGA polylactic-co-glycolic acid

RMPI Roswell Park Memorial Institute medium

Jitcy Saji Joseph, Sibusiso Tebogo Malindisa and Monde Ntwasa\*

Department of Life and Consumer Sciences, College of Agriculture and Environmental

[1] Kola I, Landis J. Can the pharmaceutical industry reduce attrition rates? Nature Reviews

[2] Arrowsmith J, Miller P. Trial watch: Phase II and phase III attrition rates 2011-2012.

\*Address all correspondence to: ntwasmm@unisa.ac.za

Nature Reviews. Drug Discovery. 2013;**12**:569

Sciences, University of South Africa, South Africa

Drug Discovery. 2004;**3**:711-715

PGA polyglycolic acid

PLLA poly-l-lactic acid

2D two dimensional 3D three dimensional

**Author details**

**References**

MDCK-MDR1 Madin-Darby canine kidney cells

HER-2 human epidermal growth factor receptor 2

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

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ECM extracellular matrix

HCS high-content screening

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 [125]. 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**.
