Abstract: SA-PO552
Electrospun Polyacrylamide Nanofiber Mats for Renal Cell Tissue Culture
Session Information
- Bioengineering
October 26, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Bioengineering
- 400 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
We have previously used soft polyacrylamide (PA) based hydrogels for renal cell tissue culture. PA gels functionalized with amino groups promote robust attachment and differentiation of primary proximal tubule cells under fluid shear stress. We have now developed a method of electrospinning (ES) PA nanofibers with 50-200 nm diameters. When functionalized with ECM protein, renal cells attach on the ES nanofiber mats and express increased levels (up to 10X) of several transporters, exceeding those cultured on non-permeable PA gels.
Methods
Linear PA was prepared by polymerizing a 3.75% solution of acrylamide monomer without bis-acrylamide at room temperature. The linear PA was precipitated with isopropanol and redissolved at ~10% concentration in water. Glutaraldehyde and HCl were added to crosslink the linear PA into stable fibers. Crosslinking required incubation at 60oC overnight after spinning. ES was performed at 0.1-0.2 ml/hour at 15KV. Atomic force microscopy was used to measure the elastic modulus of the resulting mats. The crosslinked mats were mounted in transwell supports, functionalized using Sulfo-SANPAH and Geltrex and sterilized with 70% ethanol. PA gels incorporating N-(3-Aminopropyl) methacrylamide (APMA) were used as non-permeable controls. Primary proximal tubule cells (Lonza) were then seeded on mats and gels. Cultures were placed on oscillating platforms at 72 RPM for 2-4 weeks.
Results
ES mats consisted of randomly oriented fibers 50-200 nm diameter with an elastic modulus of ~0.9 kPa. Cells attached rapidly to fibers and APMA gels and maintained attachment up to 4 weeks. Cells formed confluent patches on the mats but did not form complete monolayers as seen on APMA gels. The expression (qPCR) of several renal markers (LRP2, NHE3, OAT1-3, NKCC2 and SGLT2) were 3-10 fold higher in cells on ES mats than on APMA gels.
Conclusion
Primary human renal cells were found to attach to PA nanofiber mats functionalized with ECM. Cells formed confluent patches and remained attached for at least 4 weeks, under fluid shear stress (~2 dyn/cm2). RNA levels for several renal genes in cells on mats were increased 3-10 fold compared to levels seen on non-permeable APMA gels. We conclude that our linear PA spun nanofiber mats can significantly increase expression levels of several important genes in cultured renal cells in standard media over those seen on non-permeable PA gels.
Funding
- Private Foundation Support