Improved Cryopreservation of Human Umbilical Vein Endothelial Cells: A Systematic Approach

Abstract

Cryopreservation of human umbilical vein endothelial cells (HUVECs) facilitated their commercial availability for use in vascular biology, tissue engineering and drug delivery research; however, the key variables in HUVEC cryopreservation have not been comprehensively studied. HUVECs are typically cryopreserved by cooling at 1 °C/min in the presence of 10% dimethyl sulfoxide (DMSO). We applied interrupted slow cooling (graded freezing) and interrupted rapid cooling with a hold time (two-step freezing) to identify where in the cooling process cryoinjury to HUVECs occurs. We found that linear cooling at 1 °C/min resulted in higher membrane integrities than linear cooling at 0.2 °C/min or nonlinear two-step freezing. DMSO addition procedures and compositions were also investigated. By combining hydroxyethyl starch with DMSO, HUVEC viability after cryopreservation was improved compared to measured viabilities of commercially available cryopreserved HUVECs and viabilities for HUVEC cryopreservation studies reported in the literature. Furthermore, HUVECs cryopreserved using our improved procedure showed high tube forming capability in a post-thaw angiogenesis assay, a standard indicator of endothelial cell function. As well as presenting superior cryopreservation procedures for HUVECs, the methods developed here can serve as a model to optimize the cryopreservation of other cells.

Preclinical Evaluation and Optimization of a Cell Therapy Using Human Cord Blood-Derived Endothelial Colony-Forming Cells for Ischemic Retinopathies

“…This also has important implications for the cell product cost of manufacture and suggests that blood from a single umbilical cord will be able to generate enough ECFCs to treat tens of patients. Therefore, ECFCs could be banked as a frozen product for allogeneic cell therapies and cell cryopreservation technology for endothelial cells is progressing rapidly , which is applicable to ECFCs. In addition, ECFCs may be primed prior to use by coculturing with mesenchymal stem/stromal cells to further improve vasculogenic potential and engraftment capacity .…”

Angiogenesis Analyzer for ImageJ — A comparative morphometric analysis of “Endothelial Tube Formation Assay” and “Fibrin Bead Assay”

“…The “Angiogenesis Analyzer” program was conceived to extract characteristic points and elements of endothelial cells network. This image analysis software was successfully used to characterize meshed and/or branched structures in more than 150 different studies ( http://image.bio.methods.free.fr/ImageJ/?Angiogenesis-Analyzer-for-ImageJ&artpage=6-6#outil_sommaire_6 ), which include endothelial in vitro ETFA cell differentiation in phase contrast 20 , 21 , fluorescence microscopy 22 , and in vivo studies using the mouse retina angiogenesis model 23 , 24 . The “Angiogenesis Analyzer” has also been used to analyze branching in alternative meshed or branched biological scaffolds unrelated to angiogenesis, as reported for the characterization of diatom silicate structures in phytoplankton research 25 , or for in vitro neuritic branching analysis in cultured neurons 26 .…”

Cryopreservation and post-thaw characterization of dissociated human islet cells

“…Based on our previous work on human umbilical vein endothelial cells in suspension, the procedure that yielded the highest post-thaw membrane integrity involves cooling cells at 1°C/min in the presence of 5% DMSO plus 6% HES [ 17 ]. The combination of 5% DMSO and 6% HES has been used in the cryopreservation of whole canine and human islets and has been shown to recover over 70% viable and fully functional islets [ 39 ].…”