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RECELLULARIZING

This study evaluates the growth of cells on different types of scaffolds, including decellularized, synthetic, collagen-coated, and fibronectin-coated scaffolds. The analysis focuses on the proliferation, differentiation, and attachment of C2C12, 3T3, and MG63 cells. Results suggest that fibronectin-coated scaffolds promote the best proliferation and confluency for MG63 cells, while collagen-coated scaffolds are more favorable for C2C12 differentiation. Further investigations using objective measures and 3D structures are recommended for a comprehensive understanding of cell growth on scaffolds.

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RECELLULARIZING

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  1. RECELLULARIZING DECELLULARIZED SCAFFOLDS Dominic Polsinelli Central Catholic HighSchool Grade11

  2. Scaffolds • Scaffolds are structures on which cellscan grow • They can be synthetic, for instance PLA scaffolds • They can also beorganic

  3. Decellularizedscaffolds • Decellularized scaffolds are the extracellular matrix ofcells • They are called decellularized because everything but this ecm isremoved • New cells can grow on and throughout this ecm

  4. 3T3Cells • CelllineestablishedfromSwissmouse embryo tissue • Standard fibroblast cell line • Do not differentiate, produceECM components inconnective tissue • Often used in the cultivation of keratinocytes, with the 3T3 cells secretinggrowthfactorsfavorableto these kinds ofcells.

  5. C2C12Cells • Subclone of the mus musculus(mouse) myoblast cellline. • A common TE experimentalmodel • Differentiates rapidly, forming contractile myotomes andproduces characteristic muscleproteins. • Useful model to study thedifferentiation of nonmuscle cells (stem cells) to skeletal musclecells.

  6. MG63Cells • Human cancer cellline • Osteosarcoma cells, an aggressive form ofbone cancer • Useful model to testthe effects of variables on cancer cellsurvivorship

  7. Collagen • The main connective protein in thebody • The most abundant protein in thebody • Provides a support structurefor cells • Main component ofligaments andcartilage

  8. Fibronectin • An important component ofthe extra cellularmatrix • Plays a role in cell adhesion, migration, anddifferentiation

  9. Spin coatingtechnology • Spin coating is a method of coating a surface with avery thincoating • This is done by spinning the substrate being coated very quickly and dripping a solution of whatever you want to coat thesubstrate

  10. Purpose • To evaluate the growth of cell on different scaffoldtypes

  11. Hypothesis • I hypothesize that the collagen coated glass scaffolds will have the best growth, followed by fibronectin, followed by the uncoated glass scaffolds for all celltypes • The null hypothesis is that there will be no variation in cell attachment, differentiation, or proliferation among scaffold typesfor each cell type

  12. Materials • Spun coatscaffolds • C2C12, 3T3, and MG63 celllines • DMEMmedia • Evosmicroscope • 6 wellplates • Gloves • Powerpipettes • Phosphate buffersolution • Ethanol • Toludineblue • Laminar flowhood • Labcoat

  13. Procedure • Scaffolds were placed in 6 wellplates • Each cell type was cultured to approximately 10^6 cells per milliliter in a T75flask • These cells were trypsinized and 3mL of the resulting cell suspension was put into each well of a 6 wellplate • These wells were stored in an incubator at 37 degrees Celsius with 5% CO2 concentration • The cells were imaged every 4 days and the media waschanged

  14. C2C12, Fibronectin coatedGlass

  15. C2C12, Collagen coatedGlass

  16. C2C12, UncoatedGlass

  17. Analysis forC2C12 • The collagen appeared to have the most myotube formation followed by fibronectin, and lastly by uncoatedglass • The last set of pictures has lower quantities of cellsbecause of media contamination but myotubes can be seenforming

  18. 3T3, Fibronectin coatedGlass

  19. 3T3, Collagen coatedGlass

  20. 3T3, Uncoated Glass

  21. 3T3Analysis • There didn’t seem to be significant differencein proliferation or confluency for 3T3cells

  22. MG63, Fibronectin coatedGlass

  23. MG63, Collagen coatedGlass

  24. MG63, UncoatedGlass

  25. Analysis ofMG63 • The MG63 cells appeared to proliferate most on the fibronectin, followed by the collagen, followed by the uncoatedglass

  26. Limitations andExtensions • The lack of cell counts, protein assays, or any other purely objective measure of scaffold effect on cellbehavior • Not using the decellularized scaffolds as Ioriginally intended • Do a protein assay or cellcounts • Use 3D decellularizedstructure • Image the exact same place on the scaffold every day for a better analysis of cellgrowth

  27. Conclusion • The fibronectin seemed to be the best for MG63 proliferation and confluency followed by collagen and lastly byglass • C2C12 differentiated the best on collagen, second beston fibronectin and worst on the uncoatedglass • The 3T3 cells did not seem to be effected, this may be due to them being fibroblasts and secreting their ownecm

  28. Sources • http://www.macroevolution.net/collagen.html • http://jcs.biologists.org/content/115/20/3861 • https://www.researchgate.net/publication/312502808_MEDIA_PREPARATION_FOR_PLANT_TISSUE_CULTURE • https://www.sciencedirect.com/science/article/pii/S0142961217300856#fig2 • https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5591503/ • https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3084613/ • https://www.ncbi.nlm.nih.gov/pubmed/29912197 • https://www.liverpool.ac.uk/~sd21/tisscult/what.htm • http://himedialabs.com/TD/PT022.pdf • https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2913783/ • https://www.atcc.org/Products/All/CRL-1772.aspx#generalinformation

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