Abstract: SA-PO038
Tunable Stiffness Polyacrylamide Hydrogels with Functionalized Matrigel for Renal Tissue Culture
Session Information
- Engineering-Based Approaches to Problems in Nephrology
November 09, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Bioengineering
- 300 Bioengineering
Authors
- Fissell, William Henry, Vanderbilt University, Nashville, Tennessee, United States
- Hunter, Kuniko, Vanderbilt University, Nashville, Tennessee, United States
- Evans, Rachel C., Vanderbilt University Medical Center, Nashville, Tennessee, United States
- Love, Harold D., Vanderbilt University Medical Center, Nashville, Tennessee, United States
- Roy, Shuvo, UCSF, San Francisco, California, United States
Background
Tunable stiffness polyacrylamide (PA) based hydrogels are commonly used for mechanotransduction studies. PA gels must be functionalized with protein for cell attachment. This is commonly accomplished using sulfo-SANPAH or acrylic acid NHS ester to bind protein to the gel surface. However, these methods do not produce reliably uniform surface protein concentrations. In order to produce PA gels with highly reproducible surfaces, we modified methods used for producing methacylated gelatin (GelMA), to produce methacrylated Matrigel. The “MatrigelMA” can be added into the polymerization mix prior to casting gels, where it is covalently linked to the gel network.
Methods
5 ml of Matrigel (10 mg/ml) with phenol red, was mixed with 5 ml of ice cold 50mM HEPES pH 8.5 while stirring at 4oC. 25 ul of methacrylic anhydride was added dropwise. 1N Sodium hydroxide was added as needed to maintain an alkaline pH. After 30 minutes, this was repeated with an additional 25 ul of methacrylic anhydride. The reaction was continued overnight, then the mix was dialyzed against sterile deionized water for 5 days at 4oC, with daily changes of water. The resulting solution was aliquoted and stored at -20oC. MatrigelMA was added to PA mixes designed to produce gels with expected stiffnesses of 4.5 kPa or 40 kPa. The elastic modulus was measured using an Electroforce 3100 mechanical analyzer.
Results
Gel stiffnesses were not significantly altered by the addition of up to 100 ug/ml of MatrigelMA. Immunohistochemical staining for laminin was highly uniform wihtin and between gels. RPTEC/TERT1 cells were found to attach exceptionally well when seeded on both soft and stiff gels containing 100 ug/ml MatrigelMA. However, the cells tended to become round and detach from the soft gels after 6-8 days in culture, but persisted for several weeks on stiffer gels.
Conclusion
Extracellular basement membrane (Matrigel) was functionalized with methacrylate groups to facilitate crosslinking to polyacrylamide. The addition of 100 ug/ml to PA gels resulted in surfaces with the expected stiffnesses that promoted excellent attachment of RPTEC/TERT1 cells. However, cells tend to detach from the softer gels. We are continuing to determine if this results from degradation of matrix or if this is due to a cellular response to soft matrices.
Funding
- Other NIH Support