Assessing the Minimum Time-Period of Normoxic Preincubation for Stable Adipose Stromal Cell-Derived Vascular Networks

Pre-vascularization of tissue engineered grafts is a promising strategy to facilitate their improved viability following in vivo implantation. In this process, endothelial cells (ECs) form capillary-like networks that can anastomose with host vasculature. Adipose-derived stromal cells (ASCs) are a commonly used cell population for tissue engineering and contain a subpopulation of ECs capable of assembling into robust vascular networks and anastomosing with the host. However, their initial vascular assembly is significantly impaired in hypoxic conditions (2% O₂). In this study, we explored the minimum period of normoxic (20% O₂) pre-treatment required to enable the formation of stable vascular networks.

ASC-derived vascular structures were allowed to preassemble in fibrin hydrogels in normoxia for 0, 2, 4, or 6 days and then transplanted into hypoxic environments for 6 days. Total vascular length, pericyte coverage, cell proliferation, apoptosis rates, and ECM production was assessed.

Vascular assembly increased with time over the 6 days of culture. We found that 4 days was the minimum period of time required for stable vascular assembly. We compared the major differences in cell behavior and network structure at Days 2 and 4. Neither proliferation nor apoptosis differed, however, the Day 4 time-point was associated with a significant increase in pericyte coverage (46.1 ± 2.6%) compared to Day 2 (24.3 ± 5.3%).

These data suggest oxygen tension may be a mediator of EC-pericyte interactions during vascular assembly. Pre-vascularization strategies should incorporate a normoxic period of to enable successful vascular formation and development.