Group 6 - GTC

1. GENES & TISSUE CULTURE
By:
-ANIS ZAFIRAH-
-CHANG MAY POH-
-KUGUNESHWRY-
-ROHINI-
-THIBAN THURAI-
-YEE HUI RONG-
GROUP
6
“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”.

2. 2 Dimensional cell culture
• Cells are grown on flat dishes made of polystyrene plastic that is
stiff and unnatural.
3 Dimensional cell culture
• Cell attach to one another and form natural cell-to-cell
attachment.
• Flexible and pliable like normal tissue.
• Made of complex protein in their native configuration.
• Cells can exert forces on
one another and can move
and migrate as they do in
vivo.
HUI
RONG
Figure 1 shows the differences between a 2D and 3D cell
culture
INTRODUCTION

3.  Limited cell-cell interaction
 Disrupted cellular organization and polarity
 Inaccurate representation of the cellular
environment experienced by cells in vivo.
 Disconnect between cellular behavior in vitro
and in vivo. MAY
POH
Limitations of 2D cell culture

4. 1. To study factor that influences cells
 highlight innate variations in malignancies from diverse organs.
 how the microenvironment influences cells to produce clinically-relevant
observations.
2. Reveals a more realistic drug response
 recapitulate several mechanisms of drug resistance found in tumors in vivo
 offering the opportunity to dissect the mechanisms.
 test multidrug therapy regimens in vitro before proceeding to animal models and
ultimately clinical trials.
3. Captures phenotypic heterogeneity
 3D models is created to study the exact types of genetic changes.
4. Changes gene expression and cell behavior
5. Mimics the tumor microenvironment
 increase the understanding of their role in tumor progression a
 uncover new potential therapies that would remain undiscovered in monolayer
models(Biomatrix 2013).
MAY
POH
Why 3D models are more clinically relevant

5. 1. 3D spheroid better mimic real tumors
 Eg: Breast cancer cells grow in 2D can be easily killed by low dose of drugs or radiation.
 For the same cells that grown in 3D, they are resistant to the same doses of drugs or radiation.
 3D cell culture are more valid targets for testing and discovering new drugs to treat cancer.
2. Cells in 3D forms multilayer of cells
 2D cell culture forms a monolayer of cells.
 When testing a drug in 2D cells, it only needs to diffuse a short distance across cell membrane to
reach its target.
 In 3D, drug needs to diffuse across multilayer of cells to reach its target (mimics challenge found in
human body or in cancer)
3. Cells grown in 3D forms natural barriers to drugs
 Tight junction--- binds cells tightly together and block or slow the diffusion of drugs. HUI
RONG
Why three dimensional models are preferred?

6. 1.3D cell cultures are able to facilitate compound
profiling for target.
 effectiveness and cytotoxicity
 2D cell cultures are less similar compared to 3D cell cultures in vivo.
 3D spheroids gives more accurate results than 2D monolayer cells
ROHINI
Figure 2 :Ovarian cancer cell survival obtained by fluorescent microculture cytotoxicity
assay (FMCA), upon the treatment of four standard anticancer drugs to HCT-116 cells in
2D monolayer and 3 day and 6 day old 3D spheroids. Karlsson et al.
 Cell viability in 3D cancer cell culture
treated with 4 specific anti-cancer drugs
 Melphalen, Oxaliplatin, Irinotecan and
5-FU.(Karlsson et al).
 This gives a better effectiveness reading
level on each drug.
Potentials of using three dimensional cell cultures
for anti-cancer drug screening.

7.  Differ substantially compared to that of 2D cultures in the above mentioned
aspects.
 Namely in term of genetic material, Loessner (2010) had reported tovarian
cancer cells in 3D culture had significantly increased levels of mRNA
expression of certain cell surface receptors.
Next, from the protein expression aspect, it was also observed that from
human submandibular salivary gland (HSG) cell line in a 3D model there
was ;
 Increase in acinar protein production/secretion was observed.
 Decrease in vimentin expression
 Stable protein expression pattern
THIIBA
N

8.  Established organotypic co-culture system
 3D carcinoma cell sphere placed directly
next to the brain slice to investigate the
degree of tumour cell invasion
 Visualize morphological changes and
interactions between glial cells and
carcinoma cells (fluorescence or bright field
microscopy) – (Chuang, 2013)
 Approaches for target pharmacological
manipulations.
 Quantitative high throughput screening to
predicts in vivo efficacy
Figure 3: MCF-7 breast cancer cells
(black asterisks) on their way to
brain by time lapse sequence of an
organotypic brain slice co-curture
(University of Gottingen)
ANIS

9. 1.Kuraray
 Micro-Space Cell Culture plate by utilising its micro-fabrication technology.
 micrometer size compartments regularly arrayed on its surface which provide cells ‘micro-space’
to form 3D structure.
 advantageous features: it conforms to the standard microplate footprint -simple handling
 it has good observability; and there is uniformity in the size or shape of the microstructure. No
special techniques.
2.Q gel
 a synthetic hydrogel
 used as a matrix for 3D cell culture and in regenerative medicine, cancer research and drug
screening.
Majority of these developments utilise some sort of biomimetic
scaffold:
1) using synthetically derived materials to minimize the previously poor reproducibility
between batches, lack of consistency between cultures (especially primary cells).
2) design scaffold environments so cells respond in a physiologically relevant manner,
eg stem cells are thought to do better in gels rich in hyaluronic acid.
3) development of biodegradable scaffolds, to support applications in tissue engineering
and stem cell research. (Lawrence et al 2011)
KUGU
Current development

10. 1. The ability of the reagent to lyse the sphere and penetrate to it’s center.
2. Quenching of the assay signal under the conditions required to lyse larger
spheroids.
 Eg: Use stronger detergent, use longer incubation times
3. Complicated some methods of analysis
 Eg: Microscopy-- The multiple cell layers of 3D cultures scatters the
light reaching the objective (Caitlin 2013)
4. For tumor spheroids, it is hard to manipulate gradients of soluble
molecules in (3D spheroid) constructs, and to characterize cells in these
complex gradients.
(Chuang et al 2013)
KUGU
Challenges associated with 3D cell culture

11.  3D culture provided more precisely result for
anti-cancer drug screening
 Approached to drug target manipulation
 Robust the 3D culture to improve the
efficacy
 Possess several limitation
ANIS

12.  Biomatrix Inc., 2013, “5 reasons cancer researchers adopt 3D cell culture: A review of recent litreature”
https://3dbiomatrix.com/wp-content/uploads/2013/10/5-Reasons-Cancer-Researchers-Adopt-3D-Cell-Culture-White-Paper.pdf
 Blatt, NL, Mingaleeva, RN, Solovieva, VV, Khaiboullina, SF, Lombardi, VC, Rizvanov, AA, 2013 Application of Cell and Tissue Culture
Systems for Anticancer Drug Screening, World Applied Sciences Journal, Vol. 3, viewed on 17 October 2015
http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.388.1986&rep=rep1&type=pdf
 Caitlin.S 2013, Making the Switch to 3D Cell Culture, viewed on 17th October 2015, http://www.biocompare.com/Editorial-
Articles/144687- Making-the-Switch-to-3D-Cell-Culture/
 Chuang, HN, Lohaus, R, Hanisch, UK, Binder, C, Dehghani, F, Pukrop, T, 2013, Coculture system with an organotypic brain slice and 3D
spheroid of carcinoma cells, viewed on 15 October 2015 <http://www.ncbi.nlm.nih.gov/pubmed/24145580>
 Edmondson, R. et al. (2014) Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors
[online]. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/pmc4026212/#__ref-listid943358title (Accessed 2015).
 Elsevier B.V. 2015, Three-dimensional cell culture: the missing link in drug discover, viewed on 15th October 2015,
http://www.sciencedirect.com/science/article/pii/S1359644612003376
 Isobel.M. 2014, 3D Cell Culture Models: Challenges for Cell-Based Assays, viewed on 17th October 2012,
<http://www.promegaconnections.com/3d-cell-culture-models-challenges-for-cell-based-assays/>
 Lawrence, T, Hagemann, T, 2011, Tumour-Associated Macrophages, Springer, NewYork Dordrecht Heidelberg London, viewed 15
October 2015
<https://books.google.com.my/books?id=DBjLLGPL1r8C&pg=PA34&lpg=PA34&dq=brain+organotypic+co+culture+for+cancer+cell&so
urce=bl&ots=tDlUMkwDtq&sig=PuVe_fCu3EWCCbzcPHkwkyCVMDA&hl=en&sa=X&redir_esc=y#v=onepage&q=brain%20organotyp
ic%20co%20culture%20for%20cancer%20cell&f=false>
 Microtissue, 2015, Three Dimensional (3D) Cell Culture versus Two Dimensional (2D) Cell Culture , viewed on 15th October
2015,<http://www.microtissues.com/three_dimensional_3d_cell_culture_versus_two_dimensional_2d_cell_culture.htm>
 Promega Connections, 2014 Improving Cancer Drug Screening with 3D Cell Culture Available from :
http://www.promegaconnections.com/improving-cancer-drug-screening-with-3d-cell-culture/ [12th October 2015]
 US National Library of Medicine National Institutes of Health 2014 Three-Dimensional Cell Culture Systems and Their Applications in
Drug Discovery and Cell-Based Biosensors
Available from : http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4026212/[12th October 2015]