The culture of cells in two dimensions does not reproduce the histological characteristics of a tissue for informative or useful study. Growing cells as three-dimensional (3D) models more analogous to their existence in vivo may be more clinically relevant. Discuss the potential of using three dimensional cell cultures for anti-cancer drug screening.

1. GENES AND TISSUE CULTURE
TECHNOLOGY
By
Chan Le Roy (0329157)
Bilal Tahir (0328555)
Aizul Jeffri (0329547)
Lim Ke Wen (0329129)
Siau Yaw Wen (0333548)
Group 3
Potential of using 3-dimensional cell cultures for anti-
cancer drugs screening
SCT60103

2. CONCEPTS
Cell culture
•Studying the behaviour of cells in response to their environment by growing cells in an artificial environment.
•Efficient cell culturing techniques both in vitro and in vivo (Luboya Kombe, Vielle & Casquillas 2019).
2D cell culture
•Cells are grown on flat dishes made of polystyrene plastic (Petri dish) that is very stiff and unnatural.
•Allow cells in vitro to grow in adherent monolayers in xy-plane (Saji Joseph, Tebogo Malindisa & Ntwasa 2019).
3D cell culture
•Biological cells are permitted to grow or interact with their surroundings in all three dimensions.
•Allows cells in vitro to grow in all directions, xyz-plane, similar to how they would in vivo.
•Usually grown in bioreactors, small capsules in which the cells can grow into spheroids, or 3D cell colonies (Luboya
Kombe, Vielle & Casquillas 2019).
Anti-cancer
drug screening
•Long and expensive process
•Micropillar/microwell chip platform, ideally suited for encapsulating primary cancer cells in nanoscale spots of hydrogels
on the chip (Lee et al. 2013)
•The drug discovery and development process for new drugs consists of four phases; drug discovery, preclinical
development, clinical development and regulatory approval.

3. PRINCIPLES
Scaffold
Scaffold-free
Forced floating method
•Use low adhesion polymer-coated well-plates - Prevent their
attachment to the vessel surface by modifying the surface,
resulting in forced-floating of cells.
•Spheroids are generated by filling those well-plates with a cell
suspension after centrifugation (Luboya Kombe, Vielle &
Casquillas 2019).
Agitation-based method
•A cell suspension placed into a rotating bioreactor gradually turn
isolated cells into aggregates that cannot adhere to the container
wall due to the continuous stirring. Broad range of non-uniform
spheroids are eventually generated (Luboya Kombe, Vielle &
Casquillas 2019).
Hanging drop method
•Consists in placing a cell suspension aliquot inside a MicroWell
MiniTray (Nunc).
•By inverting the plates (trays), aliquots become droplets
presenting cell aggregates on it tips, creating compact and
homogeneous spheroids (Luboya Kombe, Vielle & Casquillas
2019).
Matrices and scaffold
•Increases the size and complexity of a 3D model.
•Convenient supports - hydrogels (due to their porosity, scaffolds
facilitate oxygen, nutriment and waste transportation) (Luboya
Kombe, Vielle & Casquillas 2019).
•Cells can proliferate and migrate within the scaffold web to
eventually adhere on it.
Microfluidic cell culture platforms
•Allow accurate micro-environmental parameters control
(microfluidic) (Elveflow 2019).
•Biocompatible microfluidic chips that facilitate tissue manipulation
and study – Creates a long term and controlled 3D cell culture
models.
Custom scaffold: 3D printing
•Uses special printers, 3D printing–like techniques to combine
cells, growth factors, and biomaterials to fabricate biomedical
parts that maximally imitate natural tissue characteristics.
•Utilizes the layer-by-layer method to deposit materials known as
bioinks to create tissue-like structures.

4. 2D VS 3D CELL CULTURE SYSTEMS
(Edmondson et al. 2014)

5. 3D-CELL CULTURING IN ANTI-CANCER
DRUG DEVELOPMENT​
Why in vitro rather
than in vivo?
Reduced costs
More directly assess drug performance
Time-consuming
Offer benefits in terms of
ethical considerations.
(Boussommier 2018; Edmondoson et al. 2014)

6. ADVANTAGES OF 3D-CELL CULTURING IN ANTI-CANCER DRUG
DEVELOPMENT​
Three-dimensional cell
culture systems allow:
 Cells to mimic cells from in-
vivo conditions as cells evolve
in three dimensions in their
natural state.
 Cells to adopt a morphology
and migration mode similar to
that found in vivo.
 Increased degree of
interaction with immediate
surroundings.
 Research and drug discovery
to be carried out in-vitro
rather than in-vivo.
(Boussommier 2018; Edmondoson et al. 2014)

7. POTENTIAL ON 3D CELL CULTURE FOR ANTI-
CANCER DRUGS SCREENING
• 3D cell cultures more physiologically relevant and predictive, showing higher
degree of structural complexity.More relevant cell models
• 3D tissue systems able to model cell interactions due to complex systems
linked together by microfluidics.Interaction between different types of cells
• Cells respond to cellular flow through differentiation and metabolic
adaptationIntegration of flow
• 3D cell culture able to represent tissue barriers which are important in the
bodyEstablishment of barrier tissues
• Microfluidics provide conditions that mimics realistic conditions in cellular
and organ growth
Better simulation of conditions in a living
organism
• It is more reliable to screen drugs against human organs grown on chips
compared to animal modelsReduces use of animal models
• 3D cell culture able to simulate diseased tissues and exhibit realistic growth
and treatment patterns.
More realistic way to grow and treat tumor
cells
(Mimetas 2019)

8. CURRENT DEVELOPMENTS: ORGAN/HUMAN-
ON-A-CHIP​
 Chips containing cell cultures to mimic processes in
humans
 Tumour cells can be added to simulate cancerous
organs
 Drug added to see effects as it passes through the
“organ”
 Potential to be used in personalized medicine
(Souppouris 2012)
(Mauriac, Casquillas & Pannetier 2017)
(Metzger, Cavallasca & Casquillas 2019)

9. EVATAR (MacDonald 2012; National Institutes of Health 2018; Xiao et al. 2017)
 Developed by Northwestern University scientists in 2017
 Chip of female reproductive tract
 Includes ovary, fallopian tube, uterus, cervix, liver
 Can be used to study cancers in female reproductive system and effects of
drug candidates

10. CHALLENGES
• Solution: Improvement of morphometric characterization of multicellular
spheroids and avoiding generalization among different types of spheroid
produced, Using the same concentration of cell.
Different 3d culturing techniques produces
spheroids with different size, shape,
density hence leading to influence testing of drug
efficacy and cytotoxicity.
• Solution: Reduces drug trials periods while making them more precise or and
targeted.
Expensive as compared to 2D monolayer.
• Solution: Confocal imagining helps to some extent, but throughput is limited.
Improvement in analysis tool.
Assays using 3d cell culture models are less
developed for imaging, quantification and
automation due to their complexity. (Adcock,
2015)
• Solution: Co culture combing tumor cells with cancer associated fibroblast .
3d culture lacks vascular components. (Verjans et
al, 2017)
• Solution: Spinner flask.
3d scaffold-based mediums are stationary hence
exchange of nutrients and waste of disposal is
issue . Hence cells cannot be grown for a
longer period of time.

11. CONCLUSION
2D cell culture allow cells to grow in adherent monolayers while 3D cell culture allow cells
to grow in all directions, like in vivo.
3D cell culture has 2 main classes; scaffold-free and scaffold. It also has 6 methods which
all cultured cells can be used in various or specific scenarios/research.
3D cell culture has many advantages such as reliable and realistic endpoints and many
applications such as drug discovery and anti-cancer drug screening
There are huge potentials that 3D cell culture techniques can bring in order to further
advance anti-cancer drug research
Advancements of technology and collaborations between different industries have
produced breakthroughs which allow research to be done more easily and efficiently.
Even with drawbacks 3D cell culturing offers promising results, hence more understanding
of the process is required to achieve significant outcomes related to in vivo studies.

12. REFERENCES
1. Adcock, A. (2015). Three-Dimensional (3D) Cell Cultures in Cell-based Assays for in-vitro Evaluation of Anticancer Drugs. Journal of Analytical & Bioanalytical
Techniques, [online] 06(03). Available at: https://www.omicsonline.org/open-access/threedimensional-3d-cell-cultures-in-cellbased-assays-for-invitro-evaluation-of-
anticancer-drugs-2155-9872-1000249.php?aid=54848 [Accessed 23 Apr. 2019].
2. Boussommier, A. (2018). 3D cell culture: market and industrial needs. [online] Elveflow. Available at: https://www.elveflow.com/organs-on-chip/3d-cell-culture-market-
industrial-needs/ [Accessed 26 Apr. 2019].
3. 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://pdfs.semanticscholar.org/b776/327dc8212170baef1e24100eaa4c60246ddb.pdf [Accessed 20 Apr. 2019].
4. Elveflow. (2019). Microfluidic cell culture: Medium change - Elveflow. [online] Available at: https://www.elveflow.com/microfluidic-tutorials/cell-biology-imaging-
reviews-and-tutorials/live-cell-perfusion/methods-and-techniques/microfluidic-cell-culture-medium-change/ [Accessed 28 Apr. 2019].
5. Howes, A., Richardson, R., Finlay, D. and Vuori, K. (2014). 3-Dimensional Culture Systems for Anti-Cancer Compound Profiling and High-Throughput Screening Reveal
Increases in EGFR Inhibitor-Mediated Cytotoxicity Compared to Monolayer Culture Systems. PLoS ONE, [online] 9(9), p.e108283. Available
at: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0108283 [Accessed 25 Apr. 2019].
6. Langhans, S. (2018). Three-Dimensional in Vitro Cell Culture Models in Drug Discovery and Drug Repositioning. Frontiers in Pharmacology, [online] 9. Available at:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5787088/ [Accessed 28 Apr.il 2019].
7. Lee, D., Choi, Y., Seo, Y., Lee, M., Jeon, S., Ku, B., Kim, S., Yi, S. and Nam, D. (2013). High-Throughput Screening (HTS) of Anticancer Drug Efficacy on a
Micropillar/Microwell Chip Platform. Analytical Chemistry, [online] 86(1), pp.535-542. Available at: https://www.ncbi.nlm.nih.gov/pubmed/24199994 [Accessed 28 Apr.
2019].

13. 8. Luboya Kombe, H., Vielle, H. and Casquillas, G. (2019). 3D cell culture methods and applications - a short review -Elveflow. [online] Elveflow. Available at:
https://www.elveflow.com/organs-on-chip/3d-cell-culture-methods-and-applications-a-short-review/#_ftn1 [Accessed 28 Apr. 2019].
9. MacDonald, A. (2017). Organs-on-Chips: Applications, Challenges, and the Future [online]. Technology Networks.
Available at: https://www.technologynetworks.com/drug-discovery/articles/organs-on-chips-applications-challenges-and-the-future-288031 [Accessed 21 Apr. 2019].
10. Mauriac, H., Casquillas, G.V., Pannetier, C. (2017). Organs on chip review [online]. Elveflow. Available at: https://www.elveflow.com/organs-on-chip/organs-chip-
review/ [Accessed 22 Apr. 2019].
11. Metzger, M., Cavallasca, J., Casquillas, G.V. (2019). Recent research breakthrough in lung-on-chip
technology [online]. Elveflow. Available at: https://www.elveflow.com/organs-on-chip/organs-chip-review/microfluidic-lung-on-chip/ [Accessed 24 Apr. 2019].
12. Mimetas (2019). 2D Versus 3D Cell Cultures | Mimetas. [online] Available at: https://mimetas.com/article/2d-versus-3d-cell-cultures [Accessed 28 Apr. 2019].
13. National Institution of Health. (2018). Modeling the Female Reproductive Tract in 3-D: The Birth of EVATAR™ [online].
Available at: https://ncats.nih.gov/pubs/features/evatar [Accessed 21 Apr. 2019].
14. Nguyen, H., Nguyen, S. and Van Pham, P. (2016). Concise Review: 3D cell culture systems for anticancer drug screening. Biomedical Research and Therapy, [online]
3(5), pp.625-632. Available at: http://www.bmrat.org/index.php/BMRAT/article/view/96.

14. 15. Saji Joseph, J., Tebogo Malindisa, S. and Ntwasa, M. (2019). Two-Dimensional (2D) and Three-Dimensional (3D) Cell Culturing in Drug Discovery. Cell Culture. [online]
Available at: https://www.intechopen.com/books/cell-culture/two-dimensional-2d-and-three-dimensional-3d-cell-culturing-in-drug-discovery [Accessed 28 Apr. 2019].
16. Shin, Y., Han, S., Jeon, J., Yamamoto, K., Zervantonakis, I., Sudo, R., Kamm, R. and Chung, S. (2012). Microfluidic assay for simultaneous culture of multiple cell types on
surfaces or within hydrogels. Nature Protocols, [online] 7(7), pp.1247-1259. Available at: https://experiments.springernature.com/articles/10.1038/nprot.2012.051 [Accessed
28 Apr. 2019].
17. Singh, D. and Thomas, D. (2019). Advances in medical polymer technology towards the panacea of complex 3D tissue and organ manufacture. The American Journal of
Surgery, [online] 217(4), pp.807-808. Available at: https://www.ncbi.nlm.nih.gov/pubmed/29803500 [Accessed 28 Apr. 2019].
18. Souppouris, A. (2012). Lung-on-a-chip research could give us new ways to fight disease [online]. The Verge.
Available at: https://www.theverge.com/2012/11/8/3617042/lung-on-a-chip-pulmonary-edema-research-wyss-institute-harvard [Accessed 21 Apr. 2019].
19. Verjans, E., Doijen, J., Luyten, W., Landuyt, B. and Schoofs, L. (2017). Three-dimensional cell culture models for anticancer drug screening: Worth the effort?. Journal of
Cellular Physiology, [online] 233(4), pp.2993-3003. Available at: https://www.ncbi.nlm.nih.gov/pubmed/28618001 [Accessed 24 Apr. 2019].
20. Xiao, S., Ceppeta, J.R., Rogers H.B., Isenberg, B.C., Zhu, J., Olalekan, S.A., McKinnon, K.E., Dokic, D., Rashedi, A.S., Haisenleder, D.J., Malpani, S.S., Arnold-Murray, C.A.,
Chen, K., Jiang, M., Bai, L., Nguyen, C.T., Zhang, J., Laronda, M.M., Hope, T.J., Maniar, K.P., Pavone, M.E., Avram, M.J., Sefton, E.C., Getsios, S., Burdette, J.E., Julie Kim, J.,
Borenstein, J.T., Woodruff, T.K. (2017). A microfluidic culture model of the human reproductive tract and 28-day menstrual cycle. Nature Communications, [online] 8.
Available at: https://www.nature.com/articles/ncomms14584 [Accessed 24 Apr. 2019].