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

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

3. 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

4. 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.

5. 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.

6. 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

7. 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

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

9. 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

10. Purpose
To evaluate the growth of cell on different scaffold types

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 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

12. 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

13. 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

14. 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

15. C2C12, Fibronectin coated Glass

16. C2C12, Collagen coated Glass

17. C2C12, Uncoated Glass

18. 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

19. 3T3, Fibronectin coated Glass

20. 3T3, Collagen coated Glass

21. 3T3, Uncoated Glass

22. 3T3 Analysis
There didn’t seem to be significant difference in proliferation or confluency for 3T3 cells

23. MG63, Fibronectin coated Glass

24. MG63, Collagen coated Glass

25. MG63, Uncoated Glass

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

27. 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

28. 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

29. Sources http://www.macroevolution.net/collagen.html