Presentation Authors: Adarsh Manjunath*, Katherine Hekman, Kyle Koss, Christina Im, Jason Wertheim, Chicago, IL
Introduction: Bioartificial replacement renal tissues may alleviate the shortage of transplantable kidneys and may minimize the risk of rejection. In order to measure the durability of stem cell derived tissues in vivo, an animal model must be developed in which a kidney scaffold can be sustained by the flow of recipient blood. However, activation of circulating platelets occurs once exposed to type IV collagen in the scaffold's vasculature, resulting in blood clots and graft failure. We hypothesize that scaffold vascular recellularization can be optimized by altering endothelial cell number, the route of recellularization, and perfusion rate and time within a bioreactor.
Methods: Kidneys were extracted from donor rats and a series of weak detergents administered into the organ via the renal artery. In a 26-hour protocol, these weak detergents removed all native cells while leaving the extracellular matrix intact. Human umbilical vein endothelial cells were cultured from 5 to 20 million in quantity and injected via syringe pump into these kidney scaffolds via the artery, vein, or both artery and vein. These recellularized scaffolds were perfused in a bioreactor, a tissue culture device that pumps cell culture media through the scaffold, at various perfusion rates. Time within the bioreactor varied from 2 to 7 days. Resazurin assays were performed to quantify cell proliferation within the scaffolds while in the bioreactor. Scaffolds were then sectioned and immunostained for CD31, an endothelial cell marker, and elastin, a marker for blood vessels.
Results: Results from the resazurin assay and immunostaining indicate the greatest coverage of the vasculature was seen with injection of 10 million endothelial cells, injection via the artery, perfusion started at 0.5 mL/minute and incrementally increased up to 4 mL/minute within 24 hours, and 2 days within the bioreactor (Figure 1).
Conclusions: Altering several variables can optimize recellularization of the kidney scaffold vasculature. This will ultimately allow us to use a synthetic heparin derivative linked to a collagen binding peptide, with the goal of preventing clots and graft failure, to cover any exposed type IV collagen in the endothelial system prior to transplantation into recipient rats.
Source of Funding: Hartwell Foundation Grant