Document Detail


Optimization of the cryopreservation and thawing protocol for human hepatocytes for use in cell transplantation.
MedLine Citation:
PMID:  20104500     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Cryopreservation of human hepatocytes is important for their use in hepatocyte transplantation. On thawing, cryopreserved hepatocytes often have reduced viability and metabolic function in comparison with fresh cells. The aim of this study was to modify the different steps in the standard cryopreservation procedure in an attempt to improve the overall outcome. Human hepatocytes with a viability of 69% +/- SD 16% were isolated from donor livers with a collagenase perfusion technique. Different cell densities, concentrations, rates, and methods of addition of dimethyl sulfoxide were tested for the freezing solution. Modified controlled-rate freezer programs were tested to obtain a linear decrease in the temperature. Once they were frozen, the storage time and thawing method for hepatocytes were investigated. The effects on thawed cell viability and attachment, lactate dehydrogenase release, cytochrome P450 1A1/2 activity, and albumin synthesis were determined. The results were used to produce an improved cryopreservation protocol suitable for good manufacturing practice conditions. With a cell density of 10(7) cells/mL in University of Wisconsin solution containing 300 mM glucose, 10% (vol/vol) dimethyl sulfoxide was added dropwise over 5 minutes, and was immediately frozen. Thawing was done rapidly at 37 degrees C, and dilution was performed with Eagle's minimum essential medium containing 300 mM glucose and 4% human serum albumin. Hepatocytes could be stored at -140 degrees C without significant further loss of function for up to 3 years. With this protocol, hepatocytes had a viability of 52% +/- 9%, an attachment efficiency of 48% +/- 8%, and lactate dehydrogenase leakage of 17% +/- 4%. This protocol is currently in use to cryopreserve hepatocytes for use in cell transplantation at our center.
Authors:
Claire Terry; Anil Dhawan; Ragai R Mitry; Sharon C Lehec; Robin D Hughes
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society     Volume:  16     ISSN:  1527-6473     ISO Abbreviation:  Liver Transpl.     Publication Date:  2010 Feb 
Date Detail:
Created Date:  2010-02-01     Completed Date:  2010-04-13     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  100909185     Medline TA:  Liver Transpl     Country:  United States    
Other Details:
Languages:  eng     Pagination:  229-37     Citation Subset:  IM    
Affiliation:
Institute of Liver Studies, King's College London School of Medicine at King's College Hospital, London SE5 9PJ, United Kingdom.
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MeSH Terms
Descriptor/Qualifier:
Adenosine / pharmacology
Allopurinol / pharmacology
Cell Count
Cell Survival
Cryopreservation / methods*,  standards
Cryoprotective Agents / pharmacology*
Dimethyl Sulfoxide / pharmacology*
Glutathione / pharmacology
Hepatocytes / cytology*,  transplantation*
Humans
Insulin / pharmacology
Organ Preservation Solutions / pharmacology
Raffinose / pharmacology
Temperature
Chemical
Reg. No./Substance:
0/Cryoprotective Agents; 0/Organ Preservation Solutions; 0/University of Wisconsin-lactobionate solution; 11061-68-0/Insulin; 315-30-0/Allopurinol; 512-69-6/Raffinose; 58-61-7/Adenosine; 67-68-5/Dimethyl Sulfoxide; 70-18-8/Glutathione

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


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