Three key points: 1) 3D cell cultures provide a more physiologically relevant model than 2D cultures by mimicking the in vivo microenvironment and cell-cell interactions. However, 3D cultures are more complex and expensive. 2) Studies show 3D cultures better maintain tumor dormancy states and drug resistance patterns observed in patients. Ki-67 indexes indicate higher fractions of non-proliferating cells in 3D. 3) While 3D models are improving, they do not fully replicate the in vivo tumor microenvironment and are not yet standardized for high-throughput drug screening. Further development is still needed to address challenges like customizing the microenvironment and expanding models.

1. SCT60103105008-M Genes and Tissue Culture Technology
By -
• Haziq Luqman
• Jeremy Chai
• Josiah Mendel Sim
• Mohannad Hamdeh
• Theck Ruey
The potential of using 3D in vitro models for drug
efficiency testing compared to 2D culture.
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2. 2D cell culture• 2D cell culture are monolayer cells cultured on flat and rigid substrates.
• 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 cell culture have significantly advanced our understanding of cell
behaviour such as cell differentiation, migration, growth, and mechanics
(Duval et al. 2017).
• However, 2D cultures fails to imitate the architecture and
microenvironments of in vivo perfectly (Lv et al. 2017).
• Despite that, 2D cultures are still used in most of the cell
research (Fang & Eglen 2017).
Introduction

3. 3D cell culture
• 3D culturing techniques was first proposed in the early 1980s by Mina
Bissell, a lead researcher at Lawrence Berkeley National Laboratory.
• 3D cell the culture of living cells within micro-assembled devices and
supports 3D structures, mimicking tissue and organ specific
microarchitecture (Huh, Hamilton & Ingber 2011).
• 3D culture possesses several in vivo features of tumors such as cell-cell
interaction, hypoxia, drug penetration, response and resistance, and
production/deposition of extracellular matrix (Kimlin, Casagrande &
Virador 2011).
• The advances in cell biology, enabled the development of a wide range
of 3D cell culture technologies such as spheroids, organoids, scaffolds,
hydrogels, organs-on-chips, and 3D bioprinting (Fang & Eglen 2017).
• 3D cell cultures are used more now as it promotes the understanding of
multilayer cell culture system over the 2D cell culture system in the
cancer research (Lv et al. 2017).
Murine Prostate Organoids (Karthaus
et al. 2014)
Breast Cell line (BT-549)
spheroids (IMAMURA et al. 2015)
Joint configuration scaffold
(Larson 2015)

4. Advantage of 2D cell culture
2D cell culture systems and technologies are less expensive than some other
systems.
Inexpensive
2D cell cultures have been used since the early 1900s, gaining widespread
acceptance in the 1940s and 1950s.
Its well established
Easier to compare with previous journals
Lots of comparative literature
Cells adhere and grow on a flat surface
Easier cell observation and measurement
Edmondson et al. 2014 & Kapałczyńska et al. 2016

5. Disadvantages of 2D Cell Cultures
• Does not mimic natural growth and
function of the cells in vivo.
• Does not allow for testing of drug
penetration.
• Does not showcase cell matrix
interactions.
• Not predictive, does not represent
a real cell environment.
• Rapid consumption of growth
media and waste build up
• Homogenous drug distribution.
(Adjei and Blanka, 2015).

6. Advantage of 3D cell culture
More relevant cell models that shows
interaction between different type of
cells
Better simulation of condition in living
organism
More realistic way to grow and treat
tumour cell
Shows the integration of flow
Better for study purposes
Edmondson et al. 2014 & Kapałczyńska et al. 2016

7. Disadvantages of 3D
Cell Cultures
• High cost.
• Low throughput capacity.
• Difficulty in distribution of nutrition.
• Difficulty in microscopic analysis.
• Matrices used may contain un-wanted
components, i.e. viruses.
• Difficult to assay, cells are hard to
remove.
Adjei and Blanka, (2015) & Edmondson et al., (2014).

8. Current development of drug-tests using
2D and 3D cell culture.
Why does it matter?
Due to rapid growth
of some tumors,
some regions of
some tumor are
deprived of oxygen
as they lack blood
supply, leading to
hypoxia.
The state of deficiency in oxygen reaching the cell. It has been reported that
it induces cancer cell dormancy in the G0 phase (Sullivan & Graham 2009).
Hypoxia
Reported to be positive in cells
unless they are in the G0 phase. The
study performed found that Ki-67
indexes on 2D cell cultures are
higher. Lower Ki-67 labelling index
indicates higher population in the
G0 phase.
Ki-67
2D cell culture 3D cell culture
BT- 549
BT-474
MCF- 7
Immuno-
histochemical
staining
(brown) for Ki-
67 antibodies
According to studies done by
Imamura et al., (2015), drug
resistance may also be associated
with the hypoxia in the Multicellular
Spheroids (MCS), but may not always
be associated to the increased G0-
dormant cell population.

9. Comparison of
2D and 3D cell
cultures to PDX
tumor
Study done found that tumor dormancy
and downregulation of caspase-3
observed in the original patient tumor
and/or PDX tumor was
better maintained in the 3D-primary
cultured cells than in the
2D-primary cultured cells (Imamura et
al., 2015).
Patient-derived Xenograft
-patient’s tumor are implanted into
immunodeficient mouse.
Ki-67 (index)
Caspase -3
Caspase -8
Challenges
Various 3D-culture systems are not considered to be a standard method,
and it is unclear which system is the most clinically relevant.
3D-culture of cell lines will never accurately, microenvironment in vivo,
because the latter have interactions with stromal tissues or blood
perfusions.
3D culture system has difficulty in the development and expansion of the
PDX model, and their high-cost will preclude their use in high-throughput
screening. (Imamura et al., 2015).

10. Conclusion
• Three-dimensional (3D) cell culture systems have gained increasing interest in drug discovery and tissue
engineering due to their evident advantages in providing more physiologically relevant information and more
predictive data for in vivo tests.
• 3D cell culture systems represent more accurately the actual microenvironment where cells reside in tissues.
Thus, the behaviour of 3D-cultured cells is more reflective of in vivo cellular responses.
• As compared to the two-dimensional case, the three-dimensional (3D) cell culture allows biological cells to
grow or interact with their surroundings in all three dimensions thanks to an artificial environment.
• Cells grown in a 3D model have proven to be more physiologically relevant and showed improvements in
several studies of biological mechanisms
• 3D cell cultures provide more accurate depiction of cell polarization since in 2D the cells can only be partially
polarized.
• 3D cell cultures have greater stability and longer lifespans than cell cultures in 2D.
Antoni et al. 2015 & Edmondson et al. 2014

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12. Reference
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