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Abstract: PO0499

Tunable Stiffness Amino Functionalized Polyacrylamide-Based Hydrogels for Renal Cell Tissue Culture

Session Information

Category: Bioengineering

  • 300 Bioengineering

Authors

  • Love, Harold D., Vanderbilt University Medical Center, Nashville, Tennessee, United States
  • Roy, Shuvo, University of California San Francisco, San Francisco, California, United States
  • Fissell, William Henry, Vanderbilt University Medical Center, Nashville, Tennessee, United States

Group or Team Name

  • The Kidney Project
Background

Tunable stiffness polyacrylamide (PA) based hydrogels have been utilized for tissue engineering studies. PA gels require functionalization for cell attachment. Reagents such as sulfo-SANPAH or acrylic acid NHS ester are often used to attach protein to the gel surface. However, these methods do not provide adequate binding to maintain cell attachment for studies involving fluid shear stress or long-term culture. In order to produce PA gels with uniform surfaces and robust cell attachment, we tested gels incorporating N-(3-Aminopropyl) methacrylamide (APMA) to create a positively charged polylysine-like biocompatible surface.

Methods

APMA was substituted in varying amounts in PA mixes previously reported to produce gels with expected stiffnesses of 2.6 kPa or 40 kPa. Gels were produced by free radical polymerization under nitrogen, using TEMED (1:300) and 10% ammonium persulfate (1:100). Gels were cast on glass coverslips soaked overnight in 2M NaOH, dried and treated with a 5% solution of 3-aminopropyltrimethoxy silane in isopropanol, then 1% glutaraldehyde. Gels were sterilized with 70% ethanol for 30 minutes, and then placed in sterile PBS. Different amounts of APMA were tested using primary human renal tubule cells (Lonza). The elastic modulus of the modified gels was measured using an Electroforce 3100 mechanical analyzer.

Results

Cells attached rapidly to gels in standard medium with 10-20% APMA substitution at both stiffness levels, and maintained excellent attachment for at least 6 weeks, under both static and shaking conditions. Cells proliferated on gels until confluent. Higher APMA amounts were less effective with softer gels. Cells initially attached to 5% APMA gels, but detached after 2-3 days. The addition of APMA decreased the stiffness of the softer gels by ~25%, while it increased by ~25% for the harder gels.

Conclusion

Primary human renal tubule cells were found to attach rapidly and robustly to polyacrylamide hydrogels containing 10-20% APMA. Cells proliferated well on APMA based gels, and remained attached for at least 6 weeks, even under fluid shear stress (~2 dyn/cm2). We conclude that the addition of APMA to PA gels provides a very simple and reproducible method of functionalizing PA gels for renal cell attachment, and allows for the testing of soft, tissue-like substrates under physiological fluid flow conditions.

Funding

  • Private Foundation Support