Gene and Tissue Culture Technology Assignment Presentation by Group 4

1. Development of cancer therapeutics is often carried out in 2D
cultures prior to testing on animal models. In comparison to
2D cultures, discuss the potential of using 3D in vitro models
for drug efficiency testing.
SCT 60103: Genes and Tissue Culture
ALEX LEE WEI YAN 0331003
WONG HEY WEI 0331364
LEE HAKHYUN 0331561
YEE YONG SHENG 0327783
HARIZ ABU HASSAN 0330318

2. What is 2D culture?
● For over a century, two-dimensional (2D) cell cultures have been used as in
vitro models to study cellular responses to stimulations from biophysical and
biochemical cues (Duval et al., 2017).
● 2D culture is also used to conduct drug screening and testing (Gupta et al.,
2016).
● This culture technique is culturing cells into a monolayer and this monolayer
system allows cell growth over a polyester or glass flat surface with
presenting a medium that feeds the growing cell population (Kombe, Vielle
and Casquillas, 2018).
● It is commonly used to understand of cell behavior, growing evidence and cell
bio activities in vivo response (Edmondson et al., 2014).

3. Why must cancer therapeutics testing carried out in 3D culture rather than
2D culture (Edmondson et al., 2014) ?
● The behavior of 3D-cultured cells is more reflective of in vivo cellular
responses
● Able to observe cells in the 3D culture environment differ
morphologically and physiologically from cells in the 2D culture
environment
● 3D culture systems provide a model that better mimics cell–cell
interactions and cell–ECM interactions compared to the traditional 2D
monolayer
● However, the data gathered with 2D cell culture methods could be
misleading and non-predictive for in vivo applications (Duval et al., 2017).
● This is because it grown cell under simplified and unrealistic conditions
thus, it does not fully reflect the essential physiology of cells, such as cell
signaling, tissue-specific architecture, mechanical/biochemical signals, and
cell-to-cell communication (Kombe, Vielle and Casquillas, 2018).

4. WHY cancer therapeutics is still carried out in 2D
cultures prior to testing on animal models?
Why 2D culture is used currently in researches? (Oortweg, 2018)
1. It is simple and inexpensive
2. It is well established and recognised by scientists, has comparative literature
3. Easier cell observation and measurement
Limitations of 2D culture (Kapalczynska et al., 2016)
1. The culture does not represent the real cell environment
2. The morphology or phenotypes of cells can be differ
3. Cells in monolayer has unlimited access to the ingredients of medium
4. It changes gene expression and splicing, topology and biochemistry of cells
5. It allows for the study of only one cell type

5. What is 3D culture ?
● 3D cell culture simulates physiological in vivo conditions, of a heterogeneous microenvironment, it can contribute to
the understanding of tumor cell growth and survival, therapy resistance and identification of novel cancer targets.
There were a few models of models of 3D culture (Gangadhara et al., 2016).
● 3D cell culture can be classified as scaffold based and non- scaffold based (Larson, 2015).
Scaffold Based
● Polymeric Hard Scaffolds
● Biologic Scaffolds
● Micropatterned Surface Microplates
Non-Scaffold Based
● Hanging Drop Microplates
● Spheroid Microplates containing Ultra-Low Attachment (ULA) coating
● Microfluidic 3D Cell Culture

6. What is 3D culture ?(Larson, 2015)
Scaffold based
● Polymeric Hard Scaffold: Culturing cells on pre-fabricated scaffolds, or matrices, designed to mimic the in vivo ECM.
Cells attach, migrate, and fill the interstices within the scaffold to form 3D cultures.
● Biological Scaffold: Culturing cells on natural or biological scaffolds, such as protein, which can be found easily in
vivo ECM. They provide the correct microenvironment of soluble growth factors, hormones and other molecules that
can alter gene and protein expression.
● Micropatterned Surface Microplates: Micrometer sized compartments regularly arranged on the bottom of each well
with various shapes (e.g. square, round..etc). Different configuration optimized for spheroid or cell networking
formation depends of cell types.
Non-Scaffold based
● Hanging Drop Microplates: Cells, in the absence of surface to attach, self assemble into 3D spheroid structure. Each
plate conforms to SBS standards with well bottoms containing opening.
● Spheroid Microplates containing Ultra-Low Attachment coating: Create same round multi-cell tissue or tumour
models using hanging drop plates. It has typical well shape and depth.
● Microfluidic 3D Cell Culture: Create similar heterogenous model and introduce perfusive flow aspect to cellular
environment

7. Advantages and disadvantages of 3D culture
Advantages (Brajša, 2016)
- Cells grown in 3D culture support their natural 3D physical shape which facilitates cell-cell regulatory mechanism and
signalling networks.
- It can be prepared 3D co-culture with other cells and cellular components in their microenvironment which supports
the growth, proliferation and migration of cell through a network of signal propagated by interactions.
- Can explore complex interactions not possible on monolayer. (Ravi et al., 2015)
- Drugs may have a different effect on a 3D structure with many layers than a monolayer. (Ravi et al., 2015)
Disadvantages (Antoni et al., 2015)
- Potentially more expensive, as certain techniques may require expensive specialized equipment. (Breslin and
Driscoll, 2012)
- 3D culture is not optimised due to lack of a quantifiable entity of biomarkers of three dimensionality.
- Unequal exposure of cells in well to drug activity.
- Time consuming. (Joshi and Lee, 2015)
- Control of culture conditions.
- Limited of capacity to scale up or down a single 3D format and handling of post culturing processing.

8. Table shows the comparison of 2D cultures vs 3D cultures (Edmondson, 2014)

9. Application
● The cell line that used in the research is A2780 cell line which is a
ovarian cancer cell line.
● The A2780 cells were seeded in 3D culture and incubated for 4 days
in standard growth media. (RPMI with GlutaMAX, 10% FBS) On day
4, the samples were treated with the cancer therapeutic drug with
the 25 μg/ml. Cell viability was assessed using the CTG assays on
the final days.
● A2780 cells growing in 3D culture is the most effective model to
investigate the efficacy of cancer therapeutic drugs.
A research was carried out by using Automation To Optimize 3D Bloom Microtissue
Conditions (3D Culture) And Drug Efficacy Screening In an Ovarian Cancer Cell
Model.(Bailey and Langhoff, 2018)

10. Results
The curves show the efficacy of a drug
as the concentration is increased. Cell
viability decreased drastically as the
concentration increased for both Drug
A and Drug B. Both drugs have a
stronger dose-response than the
market competitor.
GI50 is used in the research to find out the
concentration of the drug for the 50%
inhibition of cell proliferation.

11. Result
The figure above shows the Immunostaining and
confocal imaging to visualize drug-cell interactions.
Red = drug; Blue = cell nuclei; Green =actin
cytoskeleton.
Figure above shows the Bright field
imaging to visualize compound
efficacy by using drug from market
competitor, Drug A and Drug B

12. References
1. Antoni, D., Burckel, H., Josset, E. and Noel, G. (2015). Three-Dimensional Cell Culture: A Breakthrough in Vivo. International Journal of Molecular Sciences, 16(12), pp.5517-5527.
2. Bailey, J. and Langhoff, J. (2018). [online] Cellspring.co. Available at: http://www.cellspring.co/assets/an_screening_drug_efficacy_in_3d.pdf [Accessed 26 Sep. 2018].
3. Brajša, K. (2016). Three-dimensional cell cultures as a new tool in drug discovery. Periodicum Biologorum, 118(1), pp.59-65.
4. Breslin, S. T. and Driscoll, L. O. (2012) ‘Three-dimensional cell culture: the missing link in drug discovery.’ Drug Discov Today, 18 pp. 240-249 [Online] DOI: 10.1016/j.drudis.2012.10.003
5. Duval, K., Grover, H., Han, L., Mou, Y., Pegoraro, A., Fredberg, J. and Chen, Z.(2017). Modeling Physiological Events in 2D vs. 3D Cell Culture. Physiology, [online] 32(4), pp.266-277.
Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5545611/ [Accessed 26 Sep. 2018].
6. Edmondson, R., Broglie, J., Adcock, A. and Yang, L. (2014). Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors. ASSAY and Drug
Development Technologies, [online] 12(4), pp.207-218. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4026212/ [Accessed 26 Sep. 2018].
7. Gangadhara, S., Smith, C., Barrett-Lee, P. and Hiscox, S. (2016). 3D culture of Her2+ breast cancer cells promotes AKT to MAPK switching and a loss of therapeutic response. BMC Cancer,
[online] 16(1). Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4888214/ [Accessed 26 Sep. 2018].
8. Gupta, N., Liu, J., Patel, B., Solomon, D., Vaidya, B. and Gupta, V. (2016). Microfluidics-based 3D cell culture models: Utility in novel drug discovery and delivery research. Bioengineering &
Translational Medicine, [online] 1(1), pp.63-81. Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5689508/ [Accessed 26 Sep. 2018].
9. Joshi, P. and Lee, M. Y. (2015) “High Content Imaging (HCI) on Miniaturized Three-Dimensional (3D) Cell Cultures”, Biosensors, 5(4) pp. 768-790 [Online] DOI:10.3390/bios5040768
10. Kapalczynska M., Kolenda T., Przybyla W., Zajaczkowska M., Teresiak A., Filas V., Ibbs M., Blizniak R., Luczewski L. and Lamperska K. (2016), ‘2D and 3D cell cultures - a comparison of
different types of cancer cell cultures’, Archives of Medical Science, 14(4), viewed 25 September 2018. <https://www.researchgate.net/publication/310494093_2D_and_3D_cell_cultures_-
_a_comparison_of_different_types_of_cancer_cell_cultures>.
11. Kombe, H., Vielle, H. and Casquillas, D. (2018). 3D cell culture methods and applications. [online] Elveflow Plug and Play micro fluidics. Available at: https://www.elveflow.com/organs-on-
chip/3d-cell-culture-methods-and-applications-a-short-review/ [Accessed 26 Sep. 2018].
12. Larson, B. (2015). 3D Cell Culture: A Review of Current Techniques. [online] BioTek. Available at: https://www.biotek.com/resources/white-papers/3d-cell-culture-a-review-of-current-techniques/
[Accessed 26 Sep. 2018].
13. Oortweg J.H. (2018), 2D versus 3D Cell Cultures: Advantages and Disadvantages, MIMETAS, The Netherlands, viewed 25 september 2018. <https://mimetas.com/article/2d-versus-3d-cell-
cultures>
14. Ravi, M., Paramesh, V., Kavjya, S. R., Anuradha, E., Solomon, F.D. (2015) ‘3D cell culture systems: advantages and applications.’ J Cell Physiol., 230(1) pp. 16-26 [Online] DOI:
10.1002/jcp.24683