GMP ISOLATION AND CULTURE OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS LED TO SELECTIVELY EXPANDED CD34-POSITIVE CELLS

Therapeutic angiogenesis is a critical process in repair and regeneration that can be enhanced by perinatal human umbilical vein endothelial cells (HUVEC). The activation of quiescent endothelial cells into migrating tip cells is a key event during the sprouting phase of angiogenesis, and CD34+ HUVECs have been proposed as a critical substitute. CD34 is considered a critical marker to identify angiogenic vascular endothelial cells. The isolation and expansion of such progenitor cells are critical steps as well as ex vivo expansion in accordance with Good Manufacturing Practice (GMP). We are attempting to standardize reagents and procedures to efficiently expand CD34+ HUVEC collected from full-term placentae. As a first step, the clinical translation of cell-based approaches requires the replacement of conventional fetal bovine serum (FBS) with human serum replacement (such as human platelet lysate, hPL). We performed HUVEC extraction from 10 human umbilical cords using a mechano-enzymatic approach, and we cultured the cells in a basic endothelial medium supplemented with FBS or hPL. Fluorescent-based immunocytostainings proved characteristic expression for endothelial markers in the cell products but also highlighted a 5-10 fold increase in progenitor markers (CD34) in HUVEC cultured with hPL rather than FBS. Functional assays (angiogenesis on Matrigel) confirmed the efficiency in forming tubule-like structures in vitro for every donor analyzed so far. Furthermore, was coupled a quantitative assessment including an analysis of segment length and branching index. We measured both parameters efficiently improved in primary HUVEC exposed to hPL than standard xeno-conditions. Replacement of xeno-condition with GMPgrade reagents apparently resulted in a superior ability to generate expanded migrating CD34+ HUVECs, promoting the activity of cells with a tip cell phenotype.