Tissue Engineering for Ex Vivo Repair of Ischemically Damaged Tubule Epithelium

Abstract

Introduction: The ability to repair warm ischemically damaged renal tubule epithelium prior to transplantation would provide the potential to expand organ donor criteria to include uncontrolled DCD (uDCD) patients. We have demonstrated that an ex vivo acellular, near-normothermic perfusion can resuscitate oxidative metabolism after warm ischemia sufficiently for there to be new synthesis that restores cytoskeletal integrity. However, during the period of ex vivo perfusion, the tubule epithelium is not regenerated because rather than repair, cell replacement is needed. We now describe the ability to deliver progenitor renal epithelial cells (REC) during ex vivo perfusion to the renal tubule epithelium with homing to the sites of damage. Methods: Human REC were fluorescently labeled with PKH26 red fluorescent cell linker (Sigma-Aldrich, St. Louis, MO). Porcine kidneys were damaged by 60-minutes of postmortem warm ischemia. The damaged kidneys were then flushed and placed on exsanguinous metabolic support perfusion at 32°C for 60-minutes to restore oxidative metabolism and normalize perfusion pressures and vascular flow rates. 5.0 × 107 labeled human REC were then infused into the renal artery at the rate of 0.5 × 106 per minute. The perfusion was then continued for an additional 8-hours. Results: During the administration of the REC there were no adverse vascular reactions in terms of there being no rise in perfusion pressures nor diminution in the vascular flow rate. Post-perfusion the labeled human REC were only detected within the renal tubule epithelium. By using an ex vivo closed perfusion system we were able to determine the number of human REC that were introduced intra-arterially, the number of REC remaining in the perfusate post-perfusion and the number of cells removed from the vascular space by copious flushing post-perfusion. More than 90% of the fluorescently labeled human REC were taken up by the kidney and could be detected predominantly in the tubules of the outer medulla (Figure 1a & 1b). The human REC were not found in the vascular compartment.Figure: [Tissue Engineering Tubule Epithelium]Conclusions: These results demonstrate the ability to deliver REC to the renal parenchyma during an ex vivo perfusion with homing to the site of damaged tubule epithelium and the corresponding ability to quantify the number of REC retained within the renal tissue. The ability to target progenitor cell delivery with corresponding determination of the number of cells deposited could provide opportunities for enhanced ex vivo repair of ischemically damaged kidney allografts.