title: Beyond membrane integrity: Assessing the functionality of human umbilical vein endothelial cells after cryopreservation.
abstract: Assessment of cell membrane integrity is one of the most widely used methods to measure post-cryopreservation viability of cells such as human umbilical vein endothelial cells (HUVECs). However, an evaluation of cell function provides a better measure of cell quality following cryopreservation. The tube formation assay mimics angiogenesis in vitro and can be used to quantitate the ability of endothelial cells to form capillary-like tubular structures when cultured on reconstituted basement membrane (Matrigel). We compared the membrane integrity (measured by flow cytometry) and tube forming ability of HUVEC suspensions exposed to 10% dimethyl sulfoxide (Me2SO), cooled at 1 °C/min to various sub-zero temperatures, plunged directly into liquid nitrogen, stored for an hour, and thawed rapidly. We found that as membrane integrity increased so did the various parameters associated with the extent of in vitro angiogenesis; however, in comparison to fresh cells with a similar percentage of membrane-intact cells, the extent of tube formation, expressed as total tube length, is significantly lower in previously frozen cells for the lower range of post-thaw membrane integrities. Our findings underscore the value of an assay that quantifies a specific function that a cell is known to perform in vivo to measure the success of cryopreservation protocols.
authors.affiliation: Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta, Canada; Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Leah A
authors.initials: LA
authors.lastname: Marquez-Curtis
authors.affiliation: Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta, Canada; Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: A Billal
authors.initials: AB
authors.lastname: Sultani
authors.affiliation: Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Locksley E
authors.initials: LE
authors.lastname: McGann
authors.affiliation: Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta, Canada; Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada. Electronic address: janet.elliott@ualberta.ca.
authors.forename: Janet A W
authors.initials: JA
authors.lastname: Elliott
mesh_terms: D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology*; D015925:Cryopreservation / Q000379:methods*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005434:Flow Cytometry; D005615:Freezing; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D018919:Neovascularization, Physiologic
chemical_list: D004121:Dimethyl Sulfoxide
keywords: Angiogenesis; Cryopreservation; Dimethyl sulfoxide; Flow cytometry; Graded freezing; Human umbilical vein endothelial cells (HUVECs); Membrane integrity; Tube formation assay