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Extended Biomimetic Culture and Functional Assessment of Recellularized Human Lungs
Author(s) -
Gilpin Sarah,
Charest Jonathan,
Tapias Luis,
Ren Xi,
Wu Tong,
Ott Harald
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.1029.18
Subject(s) - decellularization , ex vivo , viability assay , cd31 , chemistry , population , flow cytometry , microbiology and biotechnology , in vivo , pathology , biology , cell , andrology , extracellular matrix , immunology , in vitro , medicine , biochemistry , environmental health
Rationale Native 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. Methods Human 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 (>350x10 6 ). 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. Results Donor‐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 pO 2, pCO 2 , and pH remained consistent. Cell distribution and viability was confirmed by Resazurin metabolism, and by histology/qPCR of serial tissue biopsies. Conclusion Intact acellular lung scaffolds can be repopulated with primary tissue‐derived cells and maintained in extended ex‐vivo biomimetic culture, supporting cell viability and function.