Document Detail


Cryopreservation of vascular endothelial cells as isolated cells and as monolayers.
MedLine Citation:
PMID:  12061847     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
This paper reports the cryopreservation of an immortalized human endothelial cell line (ECV304), either as a single cell suspension or as a confluent layer on microcarrier beads. Cell suspensions were exposed to 10% w/w dimethyl sulfoxide in a high-potassium solution (CPTes) at 0 degrees C. The cells were then cooled to -60 degrees C at controlled rates between 0.3 and 500 degrees C/min and stored below -180 degrees C. Samples were thawed in a 37 degrees C water bath and the cryoprotectant was removed by serial dilution at 22 degrees C over 6 min. The recovery of cell suspensions was assayed by culturing aliquots in 24-well plates for 7-9 days and counting the number of colonies that contained >25 cells. Maximum survival was 45-50% at cooling rates of 0.3, 1.0, and 10 degrees C/min, but decreased to 20% at 50 degrees C/min and to <1% at 500 degrees C/min. Biosilon microcarrier beads were used for the attached cells. Confluent beads were cryopreserved by exactly the same technique and cell function was assayed by measuring active amino acid (leucine) transport at 37 degrees C. Control, untreated confluent beads gave approximately 73% of control uptake and negative controls (frozen without cryoprotectant) gave approximately 4% uptake. The cells attached to beads showed percentage uptakes that were numerically similar to the survival of cells in suspension at cooling rates between 10 and 500 degrees C/min, but at lower cooling rates the recovery of attached cells increased to 70% at 1 degrees C/min and to 85% at 0.3 degrees C/min. These results indicate a marked difference in the effect of cooling rate on ECV304 cells depending upon attachment.
Authors:
David E Pegg
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Cryobiology     Volume:  44     ISSN:  0011-2240     ISO Abbreviation:  Cryobiology     Publication Date:  2002 Feb 
Date Detail:
Created Date:  2002-06-13     Completed Date:  2002-08-19     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0006252     Medline TA:  Cryobiology     Country:  United States    
Other Details:
Languages:  eng     Pagination:  46-53     Citation Subset:  IM    
Copyright Information:
(c) 2002 Elsevier Science (USA).
Affiliation:
Medical Cryobiology Unit, Department of Biology, University of York, York YO1 5YW, UK. dep1@york.ac.uk
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MeSH Terms
Descriptor/Qualifier:
Cell Adhesion
Cell Culture Techniques
Cell Line
Colony-Forming Units Assay
Cryopreservation / methods*
Cryoprotective Agents
Dimethyl Sulfoxide
Endothelium, Vascular / cytology*,  metabolism
Humans
Leucine / metabolism
Mannitol / metabolism
Microspheres
Chemical
Reg. No./Substance:
0/Cryoprotective Agents; 61-90-5/Leucine; 67-68-5/Dimethyl Sulfoxide; 69-65-8/Mannitol

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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