REVASCULARIZATION OF ACELLULAR RAT LUNG SCAFFOLDS USING THE ENDOTHELIAL COLONY FORMING CELLS

Abstract

s S157 revascularization of acellular lung scaffolds using ECFCs as a critical first step in engineering truly functional grafts. OBJECTIVES: 1) Compare decellularization Methods to achieve optimal decellularization with maintained vascular perfusion. 2) Regenerate vascular endothelium using ECFCs. METHODS: Acellular rat lung scaffolds were generated by static incubation and perfusions Methods of decellularization. For static incubation, lungs were submerged into Triton-X 100 (0.1%) and sodium deoxycholate (2%) based decellularization buffer. For perfusion decellularization, isolated rat lungs were perfused using CHAPS buffer (8mM CHAPS, 1M NaCl, 25mM EDTA in 1X PBS) or SDS-Triton-X buffer (0.1% SDS followed by 1% Triton-X). Decellularization of lungs was evaluated by tissue histology, total DNA measurement and immuno-blotting. For revascularization, ECFCs were generated from human peripheral blood or rat bone marrow derived mononuclear cells and characterized for ECFCmarkers. Further, we also studied initial engraftment of human ECFCs in the rat lung scaffolds. RESULTS: Static incubation method of decellularization generated acellular lung scaffolds with no evidence of intact cells; however, presence of intracellular protein (GAPDH) was evident. Vascular perfusion could not be established in these scaffolds. On the other side, perfusion decellularization using CHAPS buffer produced acellular lung scaffolds with maintained extracellular structure. Moreover, perfusion decellularization with CHAPS buffer consistently demonstrated complete absence of cells, below detectable DNA remnants and intracellular protein, with more optimal vascular perfusion. ECFCs were successfully derived from human peripheral blood and rat bone-marrow mononuclear cells. ECFCs were positive for endothelial specific markers such as Ac-LDL uptake, lectin binding and expressed CD31, von Willbrand Factor (vWF) and VEGFR2. Moreover, these cells did not express pan-leukocyte marker CD45. Initial engraftment study of human ECFCs in rat lung scaffolds showed engraftment of human ECFCs in vascular compartment of the lung scaffolds. CONCLUSION: We successfully prepared acellular lung scaffolds and identified ECFC, which can be utilized to generate truly functional grafts. HSF, CIHR 204 GENERATION AND UTILIZATION OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS-DERIVED-IPS FOR STUDYING ENDOTHELIAL GENE REGULATION M Nakhaei-Nejad, AG Murray, N Jahroudi