POS-476 OPTIMIZATION OF SDS-BASED KIDNEY DECELLULARIZATION RESULTS IN ECM SCAFFOLDS APPLICABLE FOR 3D RECELLULARIZATION

Abstract

Kidney decellularization is used to obtain extracellular matrix (ECM) scaffolds as a platform for 3D culturing and kidney tissue regeneration. ECM contains biological molecules such as growth factors necessary for cell survival, proliferation, and maturation during organogenesis. While decellularization allows for the complete removal of cells, it also causes the removal of specific growth factors from the ECM, which could have deleterious effects on cell survival, proliferation, and differentiation during recellularization. We hypothesize that using a mild decellularization protocol which employs the use of sodium dodecyl sulphate (SDS) below its critical micelle concentration (CMC) of 0.2% in water at room temperature will allow for the greater retention of bioactive molecules which will improve recellularization efficiencies. Porcine kidney cortex was cut into 1mm x 6mm slices and decellularized using 0.05%, 0.075%, or 0.1% SDS in a soak and agitation protocol for 24 hours at room temperature. Decellularization was determined through histological and immunofluorescent staining, and DNA quantification. The retention of ECM proteins and biomolecules was determined by histological and immunofluorescent staining, mass spectrometry, and protein array analysis. Four different cell lines were used for recellularization; human-induced pluripotent stem cells (hiPSC), mesoderm derived from hiPSC, intermediate mesoderm derived from hiPSC, and primary human renal epithelial cells. ECM scaffolds were dried on Transwell inserts and cells were pipetted on the surface of the scaffold. ECM-cell constructs were grown for two weeks in culture at air-liquid interface. Recellularization efficiency, cell organization, and differentiation were determined using whole mount staining. Complete decellularization was achieved when SDS was used in concentrations below its CMC. Decellularizing with 0.05% SDS resulted in the greatest retention of glycosaminoglycan chains (GAGs), revealing that GAGs are still significantly removed from the ECM when using SDS below its CMC. Upon protein quantification, scaffolds decellularized with higher SDS concentrations were more enriched with matrisome proteins compared to scaffolds decellularized with lower SDS concentrations, which correlated to a decrease in growth factors and cytokines present on the scaffolds as determined by protein array. Following 14-day recellularization, hiPSCs and kidney progenitor cells proliferated on the ECM and expressed markers such as LIM1, WT1, PBX1 and CD31. Primary renal epithelial cells maintained a mature phenotype, expressing LTL, cytokeratin, and KSP. Herein, we describe a novel protocol for the decellularization of kidney cortex scaffolds that allows for greater retention of biomolecules. This retention of growth factors and cytokines aids in the proliferation and directed differentiation of hiPS and kidney progenitor cells toward mature renal lineages. Thus, we determined that our ECM scaffolds are able to support the growth of both hiPS and organ-matched cells. This can be applied to other organ decellularization/ recellularization protocols, as well as for the generation of kidney tissue for further in vitro studies.