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Protocol Designs to Optimize Cryopreservation of Human Umbilical Vein Endothelial Cells Open Access


Other title
ethidium bromide
interrupted cooling protocols
flow cytometry
dimethyl sulfoxide
viability assessment
propidium iodide
two-step freezing
fluorescence microscopy
hydroxyethyl starch
human umbilical vein endothelial cells
graded freezing
membrane integrity
gel red
cell culture
Type of item
Degree grantor
University of Alberta
Author or creator
Sultani, Ahmad B
Supervisor and department
McGann, Locksley E. (Laboratory Medicine & Pathology)
Elliott, Janet A. W. (Chemical & Materials Engineering and Laboratory Medicine & Pathology)
Examining committee member and department
Li, Xing-Fang (Laboratory Medicine & Pathology)
Sauvageau, Dominic (Chemical & Materials Engineering)
Acker, Jason (Laboratory Medicine & Pathology)
Hyo-Jick, Choi (Chemical & Materials Engineering)
Laboratory Medicine and Pathology
Department of Chemical and Materials Engineering
Chemical Engineering and Medical Sciences
Date accepted
Graduation date
Master of Science
Degree level
Cryopreserving cells for human health therapeutics is a multidisciplinary process with great complexity. Cryopreservation of human umbilical vein endothelial cells (HUVECs) has facilitated vascular biology research since they were first isolated in 1973 [59]; however identifying key variables to optimize HUVEC cryopreservation has been a challenge. It is hypothesized that through the use of interrupted cooling protocols, key variables to optimize cryopreservation of HUVECs can be determined. Interrupted cooling protocols, which have been well-characterized using human erythroleukemia TF-1 cells [150], [151], [152], were previously used to study HUVEC cryopreservation conditions [147]. Mazur proposed the two-factor hypothesis to explain observations of optimal cooling, which is affected by cell characteristics and solution characteristics [101]. Hydraulic conductivity, a cell characteristic related to water flow across the cell membrane [31], is very low for HUVECs [149], suggesting that slower cooling protocols may be better than rapid cooling protocols. Cryoprotectants are useful if optimal cooling is insufficient to maintain high viability; however neither DMSO nor hydroxyethyl starch (HES) have previously been used in interrupted cooling studies of HUVEC suspensions. Several methods were carefully considered to ensure high experiment repeatability: i) HUVEC culture and cell preparation methods, ii) fluorescence microscopy methods, and iii) flow cytometry methods. The HUVEC culturing and cell preparation methods that contributed directly to high experiment repeatability and high membrane integrity include: i) growing to ≤ 15 population doublings, ii) growing to 50% – 80% culture flask coverage, iii) maintaining HUVECs on ice for 2 – 4 hours prior to experiments, and iv) consistent thawing followed by immediate measurement of membrane integrity. Fluorescence microscopy image capture methods were optimized using the Viability3 cell counting program (version 3.2, Great Canadian Computer Company, Spruce Grove, Alberta, Canada) as a reference. Flow cytometry methods were optimized to record fluorescence emissions from different fluorochromes separately, resulting in separation of membrane-intact cells, membrane-damaged cells and background light scatter. Two interrupted cooling protocols were used: i) graded freezing [107], an interrupted slow cooling protocol [152], and ii) two-step freezing [108], an interrupted rapid cooling protocol. Interrupted cooling protocols were used to determine key variables to optimize HUVEC cryopreservation, namely: i) flow cytometry versus fluorescence microscopy membrane integrity measurements, ii) cooling protocol, iii) cooling rate, iv) cryoprotectant addition procedures, v) cryoprotectant composition, and vi) plunge temperature. Flow cytometry, either in the presence or absence of cryoprotectant, was determined to be the more stringent method to measure membrane integrity. Higher membrane integrities were attained after the interrupted slow cooling protocol than the interrupted rapid cooling protocol. A cooling rate of 1.0 °C/min resulted in better survival of HUVECs than a 0.2 °C/min cooling rate. It was predicted that a previously designed cryoprotectant addition procedure would be optimal [138]; however adding 20% DMSO directly to HUVECs to a final concentration of 10% DMSO for 15 minutes at 0 °C resulted in membrane integrities that were similar. Increasing the cryoprotectant concentration from 10% to 20% DMSO did not provide additional protection to HUVECs. However, adding HES with 10% DMSO was beneficial for HUVEC survival. The optimal cryopreservation procedure obtained in this work involved cooling HUVECs in suspension in the presence of 10% DMSO and 8% HES at 1.0 °C/min to −45 °C before cryogenic storage. A significant improvement in HUVEC survival was measured compared to the 64.8% ± 2.2% membrane integrity of supplied HUVECs measured in this work and the 69.2% ± 2.3% reported in the literature for the standard protocol using good manufacturing practices [140]. Importantly, we describe detailed procedures to ensure reproducible results and explore the effects of key variables required to optimize the cryopreservation of HUVECs. The careful attention to methods and the use of interrupted cooling protocols can be used to design studies to improve routine cryopreservation of HUVECs and other types of cells.
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
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