DECELLULARIZE D SCAFFOLDS RECELLULARIZING DECELLULARIZE D SCAFFOLDS Dominic Polsinelli Central Catholic High School Grade 11

Scaffolds Scaffolds are structures on which cells can grow They can be synthetic, for instance PLA scaffolds They can also be organic

Decellularized scaffolds Decellularized scaffolds are the extra cellular matrix of cells They are called decellularized because everything but this ecm is removed New cells can grow on and throughout this ecm

3T3 Cells Cell line established from Swiss mouse embryo tissue Standard fibroblast cell line Do not differentiate, produce ECM components in connective tissue Often used in the cultivation of keratinocytes, with the 3T3 cells secreting growth factors favorable to these kinds of cells.

C2C12 Cells Subclone of the mus musculus (mouse) myoblast cell line. A common TE experimental model Differentiates rapidly, forming contractile myotomes and produces characteristic muscle proteins. Useful model to study the differentiation of nonmuscle cells (stem cells) to skeletal muscle cells.

MG63 Cells Human cancer cell line Osteosarcoma cells, an aggressive form of bone cancer Useful model to test the effects of variables on cancer cell survivorship

Collagen The main connective protein in the body The most abundant protein in the body Provides a support structure for cells Main component of ligaments and cartilage

Fibronectin An important component of the extra cellular matrix Plays a role in cell adhesion, migration, and differentiation

Spin coating technology Spin coating is a method of coating a surface with a very thin coating This is done by spinning the substrate being coated very quickly and dripping a solution of whatever you want to coat the substrate

Purpose To evaluate the growth of cell on different scaffold types

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 cell types The null hypothesis is that there will be no variation in cell attachment, differentiation, or proliferation among scaffold types for each cell type

Materials Spun coat scaffolds C2C12, 3T3, and MG63 cell lines DMEM media Evos microscope 6 well plates Gloves Power pipettes Phosphate buffer solution Ethanol Toludine blue Laminar flow hood Lab coat

Procedure Scaffolds were placed in 6 well plates Each cell type was cultured to approximately 10^6 cells per milliliter in a T75 flask These cells were trypsinized and 3mL of the resulting cell suspension was put into each well of a 6 well plate 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 was changed

Fibronectin coated glass + 3mL 3T3 cell suspension Collagen coated glass + 3mL 3T3 cell suspension Glass + 3mL 3T3 cell suspension Fibronectin coated glass + 3mL MG63 cell suspension Collagen coated glass + 3mL MG63 cell suspension Glass + 3mL MG63 cell suspension Fibronectin coated glass + 3mL C2C12 cell suspension Collagen coated glass + 3mL C2C12 cell suspension Glass + 3mL C2C12 cell suspension Empty

C2C12, Fibronectin coated Glass

C2C12, Collagen coated Glass

C2C12, Uncoated Glass

Analysis for C2C12 The collagen appeared to have the most myotube formation followed by fibronectin, and lastly by uncoated glass The last set of pictures has lower quantities of cells because of media contamination but myotubes can be seen forming

3T3, Fibronectin coated Glass

3T3, Collagen coated Glass

3T3, Uncoated Glass

3T3 Analysis There didn’t seem to be significant difference in proliferation or confluency for 3T3 cells

MG63, Fibronectin coated Glass

MG63, Collagen coated Glass

MG63, Uncoated Glass

Analysis of MG63 The MG63 cells appeared to proliferate most on the fibronectin, followed by the collagen, followed by the uncoated glass

Limitations and Extensions The lack of cell counts, protein assays, or any other purely objective measure of scaffold effect on cell behavior Not using the decellularized scaffolds as I originally intended Do a protein assay or cell counts Use 3D decellularized structure Image the exact same place on the scaffold every day for a better analysis of cell growth

Conclusion The fibronectin seemed to be the best for MG63 proliferation and confluency followed by collagen and lastly by glass C2C12 differentiated the best on collagen, second best on fibronectin and worst on the uncoated glass The 3T3 cells did not seem to be effected, this may be due to them being fibroblasts and secreting their own ecm

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