*Purpose: The chronic shortage of allografts has generated intense interest in the field of regenerative medicine. Given the limited regeneration that can be accomplished in quiescent tissues, normal recovery results in scarring and inadequate restoration of functional capacity. In vivo, the response to injury involves coagulative, inflammatory and adhesion phases that result in the scarring and loss of function. To regenerate damaged organs new cells must replace the lethally damaged cells to restore function. Optimized cell culture supports the completion of the cell cycle in ≥24 hours. The main component of regeneration is cell proliferation that involves DNA replication and mitosis initiated via receptor-mediated signal transduction. We evaluated the potential to use prolonged ex vivo acellular warm perfusion of as much as 72 hours to support regenerative pathways. We evaluated whether replacement of lethally injured cells could be accomplished during an acellular ex vivo warm perfusion environment.

*Methods: An acellular perfusion technology was employed that represents the tissue culture of a whole organ. Regenerative pathways were supported by nutrient, trophic and cytokine supplementation to the recirculating perfusate. Discarded ischemically damaged (>45 minutes WI) human kidney allografts (n=4) were used. Confirmation of renal regeneration was determined by restoration of cytoskeletal integrity (ZO-1), DNA synthesis (S phase) and mitosis (M phase). Image J software was used to objectively quantify fluorescence for ZO-1. The mean number of PCNA-positive nuclei was also quantified objectively using Image J software. The percent of positive toluidine stained cells were also determined per 40 separate fields.

*Results: Stable perfusion dynamics, oxygen consumption, metabolism and synthesis was maintained during the 2-days of ex vivo perfusion. The data listed in Table 1 demonstrates that time dependent normalization of cytoskeleton occurred. Similarly, DNA synthesis increased incrementally and was of sufficient magnitude to result in mitosis during the ex vivo perfusion.

*Conclusions: An acellular environment eliminates the normal in vivo response to injury that results in scarring and enhances regeneration. The observed DNA synthesis and mitosis demonstrates that acellular warm perfusion also holds the potential to support the generation of new cells to replace lethally damaged ones to restore normal function without scarring.

« Back to 2020 American Transplant Congress