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GENES & TISSUE CULTURE

By:-ANIS ZAFIRAH--CHANG MAY POH- -KUGUNESHWRY- -ROHINI--THIBAN THURAI--YEE HUI RONG-GROUP 6The 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 Dimensional cell cultureCells are grown on flat dishes made of polystyrene plastic that is stiff and unnatural.3 Dimensional cell cultureCell 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 RONGFigure 1 shows the differences between a 2D and 3D cell cultureINTRODUCTION

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Limited cell-cell interactionDisrupted cellular organization and polarityInaccurate representation of the cellular environment experienced by cells in vivo.Disconnect between cellular behavior in vitro and in vivo.

MAY POHLimitations of 2D cell culture

1. To study factor that influences cellshighlight 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 behavior5. Mimics the tumor microenvironmentincrease the understanding of their role in tumor progression a uncover new potential therapies that would remain undiscovered in monolayer models(Biomatrix 2013).

MAY POHWhy 3D models are more clinically relevant

1. 3D spheroid better mimic real tumorsEg: 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 cells2D 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 drugsTight junction--- binds cells tightly together and block or slow the diffusion of drugs.

HUI RONG

Why three dimensional models are preferred?

1.3D cell cultures are able to facilitate compound profiling for target.effectiveness and cytotoxicity2D cell cultures are less similar compared to 3D cell cultures in vivo. 3D spheroids gives more accurate results than 2D monolayer cellsROHINI

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. Karlssonet al.Cell viability in 3D cancer cell culture treated with 4 specific anti-cancer drugs Melphalen, Oxaliplatin, Irinotecan and 5-FU.(Karlssonet al). This gives a better effectiveness reading level on each drug.Potentials of using three dimensional cell cultures for anti-cancer drug screening.

2. Cell receptor, protein and gene expression in 3D cultures

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

THIIBAN

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3. 3D culture visualize the interaction of carcinoma cells with resident cellsEstablished organotypic co-culture system3D 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

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)

KUGUCurrent development

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1. The ability of the reagent to lyse the sphere and penetrate to its center.2. Quenching of the assay signal under the conditions required to lyse larger spheroids.Eg: Use stronger detergent, use longer incubation times3. Complicated some methods of analysisEg: 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

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Summary3D culture provided more precisely result for anti-cancer drug screeningApproached to drug target manipulationRobust the 3D culture to improve the efficacy Possess several limitation

ANIS

ReferencesBiomatrix 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.pdfBlatt, 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 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/S1359644612003376Isobel.M. 2014, 3D Cell Culture Models: Challenges for Cell-Based Assays, viewed on 17th October 2012, Lawrence, T, Hagemann, T, 2011, Tumour-Associated Macrophages, Springer, NewYork Dordrecht Heidelberg London, viewed 15 October 2015 Microtissue, 2015, Three Dimensional (3D) Cell Culture versus Two Dimensional (2D) Cell Culture, viewed on 15th October 2015,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]

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