LAUSR

10 Introduction Along with hematological progenitors, other cell types from cord blood (CB), such as mesenchymal stromal cells (MSCs) and endothelial progenitors (EPs), are being used for new applications in cell therapy and bioengineering, such as vascular or bone repair, artificial vascular structures, or wound healing. Umbilical cord blood contains EPs in low numbers, and the isolation process is not successful in a high percentage of the attempts. Moreover, published protocols traditionally make use of human or calf serum. We wanted to investigate an EP isolation method from cryopreserved CB that is sufficiently efficient and reproducible for clinical purposes. Objective The objective of this study was to investigate the efficiency of the isolation of endothelial progenitors from cryopreserved cord blood using a xeno-free, serum-free protocol. Methods Cryopreserved CB was thawed and equilibrated with a 10% human serum albumin-containing solution. The mononuclear fraction was isolated by the Ficoll method, and in some of the experiments, the CD133+ or CD34+ population was purified with immune-magnetic beads. The negative fraction of the immune-isolation or the total mononuclear fraction was plated on coated dishes and cultured in xeno-free EC Cult XF medium (StemCell Technologies), with or without human serum, until colonies appeared. The isolated EPs were characterized by immunofluorescence and flow cytometry.