Document Detail


l-Cysteine improves survival and growth of mesothelial cells after freezing.
MedLine Citation:
PMID:  19706334     Owner:  NLM     Status:  In-Process    
Abstract/OtherAbstract:
Enzymic isolation, cryopreservation and culture of cells places considerable demand on intracellular antioxidant thiol status. Thiol status is carefully regulated in the cell by the balance of reduced and oxidized thiol species, including glutathione (GSH/GSSG) and l-cysteine (l-Cys/l-CySS disulphide). These play a pivotal role in redox signaling, cell attachment, proliferation and differentiation. Thiol depletion exposes cells to "thiol debt", increasing their vulnerability to sustained pro-oxidant attack and injury. This study focused on the ability of l-Cys-enriched medium to enhance survival and growth of human peritoneal mesothelial cells (hPMC) after prolonged storage at -196 degrees C. HPMC derived from human omentum suspended in freezing solution (90 % foetal bovine serum, 10% dimethylsulfoxide) were cryopreserved at passage 1 for 6 years. Thawed cells cultured in medium M199 plus or minus l-Cys (0.25 mmol/L) were subjected to morphological, growth, ultrastructural studies, RT-PCR and cell signaling studies to assess cell function after cryopreservation. Viability of thawed cells was lower (85 +/- 3%) than non-frozen control cells (98 +/- 2% viable). l-Cys added to the cryopreservation fluid and the post-thaw medium increased cell viability to 94 +/- 2%. Cell growth plus l-Cys was 2-fold greater compared with the minus l-Cys controls. The former had a cobblestone appearance. Ultrastructural studies showed lamellar bodies, indicative of surfactant production not evident in cells in minus l-Cys, which were a fibroblastic appearance. l-Cys treated hPMC constitutively expressed message for growth factors, TGFbeta1, PDGF-A, PDGF-B and PDGFbeta-receptor. The functionality of the PDGFbeta-receptor was confirmed in fura-2 loaded cells with release of intracellular calcium when challenged with exogenous PDGF-BB. The addition of reduced thiols to culture media may have wider application in survival and enhance cell-based therapies.
Authors:
Stephen D Bird; Robert J Walker; Michael Legge
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2009-08-23
Journal Detail:
Title:  Cell biology international     Volume:  33     ISSN:  1095-8355     ISO Abbreviation:  Cell Biol. Int.     Publication Date:  2009 Nov 
Date Detail:
Created Date:  2009-11-09     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9307129     Medline TA:  Cell Biol Int     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  1155-64     Citation Subset:  IM    
Affiliation:
Department of Medical and Surgical Sciences, University of Otago, Dunedin School of Medicine, Dunedin, New Zealand. sdbird@hotmail.com
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