Extended Biomimetic Culture and Functional Assessment of Recellularized Human Lungs

Abstract

RationaleNative lung scaffolds prepared by decellularization provide a novel platform for organ engineering. Recellularization with specific cell types requires ex vivo culture for evaluation and restoration of organ function prior to transplantation.MethodsHuman lung‐derived epithelial and endothelial cells were isolated and expanded in vitro. Immunostaining, flow cytometery, and qPCR confirmed cell identity. Epithelial and endothelial cells were delivered to lung scaffold airways and vasculature respectively (>350x106).Constructs were cultured for 10 days in a custom automated, closed‐circuit bioreactor, providing physiologic perfusion and negative pressure ventilation. Perfusate was analyzed daily by iSTAT. Tissue was analyzed by histology and qPCR.ResultsDonor‐derived epithelium was a population of primarily basal‐cell phenotype (70% p63+), with 20% CC10+, and minor pneumocytes (<10% SP‐B/C) by flow cytometry. Basal cell ciliogenesis was confirmed by ALI. Endothelium was sorted to CD31 purity.Biocompatibility to lung matrix confirmed maintenance of cell phenotype, viability, and proliferation (Ki67+) in slice co‐culture.Recellularized constructs were cultured for 6 days with constant perfusion, plus 4 days with additional fluid or air ventilation. Increasing rate of glucose metabolism and lactate production in the perfusate was quantified over time. Measured pO2, pCO2, and pH remained consistent.Cell distribution and viability was confirmed by Resazurin metabolism, and by histology/qPCR of serial tissue biopsies.ConclusionIntact acellular lung scaffolds can be repopulated with primary tissue‐derived cells and maintained in extended ex‐vivo biomimetic culture, supporting cell viability and function.image