A xeno-free culture system for efficient derivation and amplification of human endothelial colony-forming cells from umbilical cord blood

Abstract

Purpose : Endothelial colony-forming cells (ECFCs) have been identified as endothelial progenitor cells with robust proliferative potential in vitro and vasculogenic properties in vivo. Thus, ECFCs have emerged as a favorable target for vascular regenerative therapies. However, potential therapeutic use of ECFCs is compromised by low frequency in both peripheral (PB) and umbilical cord blood (UCB) so samples must be expanded in vitro prior to use. Current formulations for expanding ECFCs in vitro contain fetal bovine serum (FBS), making them inappropriate for clinical use. To minimize the risks associated with exposure to animal serum, new culture media are required. Here we characterized ECFCs derived and expanded from primary UCB in EC Cult™-XF ECFC medium, a novel xeno-free (XF) medium and matrix system that does not contain FBS, to ECFCs generated in serum-containing (SC) medium.

Methods : Colonies generated by UCB ECFCs were counted on day 10, and then expanded by passaging the cells in the medium in which they were derived. The hierarchy of ECFCs was evaluated at passage (P) 4 using clonogenic assays. Expression of CD144, CD31 and CD45 as well as acetylated LDL (Ac-LDL) uptake was tested at P5. The functional capacity of ECFCs to mediate vascular repair was evaluated in a mouse model of oxygen-induced retinopathy (OIR).

Results : EC Cult™-XF ECFC medium supported an equivalent frequency and expansion of ECFC colonies as SC medium. EC-Cult™-XF ECFC supported the entire hierarchy of ECFCs, with larger colonies forming in EC-Cult™-XF ECFC than in SC medium. More than 97% of cells cultured in either media were CD144+CD31+, CD45- and incorporated Ac-LDL. In the OIR model, injection of ECFCs derived in EC Cult™-XF medium decreased the area of vascular obliteration and neovascular tufts.

Conclusions : In summary, ECFCs can be efficiently derived and expanded in xeno-free EC Cult™-XF medium, facilitating their use in translational research.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.