Phenotypic and Functional Characterization of human Umbilical Vein Endothelial Cells (hUVEC) Cultures Established under Physiologic vs. Atmospheric Oxygen Tension
Authors: Smith Anna Gray , Cheu Jessica , Presnell Sharon Collins 
Published: April 2020

Primary cultures of human umbilical vein endothelial cells (hUVEC) have historically been established at atmospheric oxygen (O2) tension (21%) and their surface marker profiles and angiogenic potential in are understood. However, physiologic O2 tension ranges from ~1% in cartilage to ~13% in major arteries, thus 21% O2 represents a supraphysiologic O2 exposure. We conducted experiments to establish and characterize hUVEC at 21% O2 and physiologically relevant oxygen tension (5% O2) from three independent donors.hUVEC were isolated by standard methods with dissociation and culture steps conducted at 21% or 5% O2. Cells were maintained in standard endothelial cell growth media and were cryopreserved at the end of passage 1. Cells were thawed, used to establish live passage 2 cultures, and confirmed to be sterile and free of mycoplasma and endotoxin. hUVEC established at 5% O2 (hUVEC‐LO) were compared to hUVEC established at 21% O2 (hUVEC‐HO) with respect to population doubling time, surface marker expression by flow cytometry, and angiogenic potential by tube formation assay.Both hUVEC‐LO and hUVEC‐HO were immunopositive for CD31, VE‐cadherin, CD146, CD202b, and vEGFR1, and were negative for CD45 and CD90. hUVEC‐LO uniquely expressed a greater percentage of markers associated with an endothelial progenitor phenotype, including CD117, Slug, CD133, and vEGFR2. The calculated population doubling time of hUVEC‐LO was on average 10% faster than the donor‐matched hUVEC‐HO. Angiogenic potential was confirmed by tube formation assay for each lot at the oxygen tension at which they were established. Crossover experiments were conducted to assess tube formation in hUVEC‐LO and hUVEC‐HO under assay conditions of 21% or 5% O2. No statistical differences were noted after 6 hours of assay duration with respect to: number of junctions, segments, meshes, and total length of segments, branches, or extremities. Significant differences were observed beyond 6 hours between hUVEC‐LO and hUVEC‐HO in mean mesh size and number of branches, but the magnitude and pattern of differences varied from donor‐to‐donor. In contrast, the early phase of angiogenesis (0–6 hours) revealed notable differences between hUVEC‐LO and hUVEC‐HO, with hUVEC‐LO generally exhibiting greater variance with respect to number of junctions, mesh area, number of meshes, and number of segments.Taken together, these results suggest that hUVEC‐LO may have a more stem‐like phenotype characterized by the retention of progenitor‐associated surface markers and a faster doubling time compared to hUVEC‐HO from the same donor. While the ultimate capacity for tube formation did not differ between hUVEC‐LO and hUVEC‐HO assayed at 21% or 5% O2, the preliminary data suggest that hUVEC‐LO respond more actively to adapt to changes in oxygen tension, undergoing significant fluctuations in multiple measures at early time points. Further experiments are needed to elucidate the processes and mechanism(s) that lead to differential phenotype and/or performance of hUVEC‐LO and hUVEC‐HO, in particular during the early initiating events of angiogenesis.