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Evaluating different methods for kidney recellularization Luque Badillo, Ana Cecilia

Abstract

End-stage renal disease (ESRD) is a condition where the kidneys fail to effectively filter waste and excess fluids from the blood. In Canada, the number of ESRD patients has increased by 24% over the past decade. While dialysis is a treatment option, kidney transplantation remains the gold standard due to higher survival rates. Unfortunately, there is a shortage of available organs for transplantation. Tissue engineering has emerged as a potential solution to address this shortage. The extracellular matrix (ECM) obtained from decellularized organs retains the organ's intricate microarchitecture and bioactive molecules crucial for successful recellularization. In order to regenerate a kidney, it must be reseeded with new cells after the decellularization process. However, achieving complete organ repopulation remains a challenge. Therefore, the current study aims to evaluate different methods for kidney recellularization that can potentially be functional for transplantation. Pig kidneys were decellularized using a meticulous protocol involving detergent perfusion through the renal artery and vein. The resulting ECM was analyzed histologically and characterized using proteomic analysis, ensuring the preservation of growth factors and structural proteins. Primary porcine renal cells, labeled as whole kidney pig cells (WKPC), were isolated, cultured, and extensively characterized using RT-qPCR, immunofluorescence, and flow cytometry. Recellularization of a whole pig kidney was performed using an in vitro bioreactor and negative pressure over a 12-day period, with WKPC perfusion and co-perfusion of human erythrocytes to enhance oxygenation. Successful decellularization of a pig kidney was achieved within 45 hours, preserving the ECM's integrity and important biomolecules. The isolated WKPC population exhibited the presence of native cell markers, confirming their suitability for kidney recellularization. Histological evaluation revealed a high density of cells distributed throughout the ECM matrix, extending to the organ's periphery. Analysis of the culture media and immunohistochemistry showed cells proliferating and no signs of cell death. Moreover, the presence of cell colonies expressing kidney cell markers in different kidney sections confirmed improvement in the recellularization process. This research serves as a proof-of-concept, laying the foundation for the potential translation of this technology to recellularize human kidneys, ultimately addressing the organ shortage and improving outcomes for ESRD patients.

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Attribution-NonCommercial-NoDerivatives 4.0 International