Abstract: SA-PO017
Formulating Customizable Extracellular Matrix Scaffolds From Decellularized Mouse Kidneys
Session Information
- Bioengineering
November 05, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Bioengineering
- 300 Bioengineering
Authors
- Cintron Pregosin, Nina, Stony Brook Medicine, Stony Brook, New York, United States
- Mallipattu, Sandeep K., Stony Brook Medicine, Stony Brook, New York, United States
Background
Current cell culture methods are not adequate for recapitulating the physiology of mature, differentiated kidney cells in vivo. Podocytes plated on plastic tissue culture dishes differ from podocytes within the glomerulus due to their lack of foot processes or slit diaphragms, making human podocytopathies difficult to accurately study in this system. Several researchers have shown that growing podocytes in 2D culture systems that better resemble their native environment, the glomerular basement membrane, can help promote their differentiation. Rather than using expensive commercially available products, here we formulate a cell culture matrix derived from decellularized mouse kidneys. By removing the cellular content from mouse kidneys while leaving the extracellular matrix intact, we create a customizable, non-immunogenic scaffold for cell growth in-house.
Methods
Wild-type C57BL/6 mice were initially perfused with D-PBS and kidneys were dissected and sliced into 300µm sections then washed in D-PBS on a shaker overnight. The slices were decellularized by washing in 1% TritonX-100 for 5 hours, then 0.1% SDS for 1 hour. SDS was washed off in D-PBS overnight and kidneys were sterilized by washing in 1% normal saline. The tissue was then lyophilized, crushed, and digested in 3M urea for 36 hours. The decellularized solution was then diluted to a final concentration of 1-3% in Geltrex for coating cell culture plates or mixed with GelMA bioink for 3D bioprinting applications.
Results
Decellularized kidney slices had an 89% reduction in DNA content compared to control slices with preserved histological structure. Loss of cellular content was also observed after counting DAPI+ cells in ImageJ. Both glomerular endothelial cells and iPSCs showed adhesion, proliferation, and expression of mature differentiation markers on the decellularized extracellular matrix scaffold as compared to no matrix or commercially available matrix under differentiating conditions.
Conclusion
We generated a more accessible, sustainable, and reproducible protocol for generating decellularized extracellular matrix scaffolds in-house. Future work includes measuring the viability of various cells, such as renal epithelial cells and podocytes, and their ability to differentiate on these scaffolds.
Funding
- NIDDK Support