Freezing/Thawing without Cryoprotectant Damages Native but not Decellularized Porcine Renal Tissue

Abstract

abstractWhole organ decellularization of porcine renal tissue and recellularization with a patient's own cells would potentially overcome immunorejection, which is one of the most significant problems with allogeneic kidney transplantation. However, there are obstacles to achieving this goal, including preservation of the decellularized extracellular matrix (ECM), identifying the proper cell types, and repopulating the ECM before transplantation. Freezing biological tissue is the best option to avoid spoilage; however, it may damage the structure of the tissue or disrupt cellular membranes through ice crystal formation. Cryoprotectants have been used to repress ice formation during freezing, although cell toxicity can still occur. The effect of freezing/thawing on native (n = 10) and decellularized (n = 10) whole porcine kidneys was studied without using cryoprotectants. Results showed that the elastic modulus of native kidneys was reduced by a factor of 22 (P < 0.0001) by freezing/thawing or decellularization, while the elastic modulus for decellularized ECM was essentially unchanged by the freezing/thawing process (p = 0.0636). Arterial pressure, representative of structural integrity, was also reduced by a factor of 52 (P < 0.0001) after freezing/thawing for native kidneys, compared to a factor of 43 (P < 0.0001) for decellularization and a factor of 4 (P < 0.0001) for freezing/thawing decellularized structures. Both freezing/thawing and decellularization reduced stiffness, but the reductions were not additive. Investigation of the microstructure of frozen/thawed native and decellularized renal tissues showed increased porosity due to cell removal and ice crystal formation. Orcein and Sirius staining showed partial damage to elastic and collagen fibers after freezing/thawing. It was concluded that cellular damage and removal was more responsible for reducing stiffness than fibril destruction. Cell viability and growth were demonstrated on decellularized frozen/thawed and non-frozen samples using human renal cortical tubular epithelial (RCTE) cells over 12 d. No adverse effect on the ability to recellularize after freezing/thawing was observed. It is recommended that porcine kidneys be frozen prior to decellularization to prevent contamination, and after decellularization to prevent protein denaturation. Cryoprotectants may still be necessary, however, during storage and transportation after recellularization.