Document Detail


Conjunctival epithelial cells maintain stem cell properties after long-term culture and cryopreservation.
MedLine Citation:
PMID:  19761393     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
AIM: Transplantation of tissue-engineered conjunctival epithelial cell sheets has proven to be a promising technique for conjunctival reconstruction. The ability to cryopreserve conjunctival epithelial cells and maintain their stem cell population would improve their availability for clinical use. The aim of this study was to evaluate whether cryopreservation and long-term in vitro culture has an effect on the proliferative capacity and the progenitor-like cell characteristics of conjunctival epithelial cells. METHOD: Human conjunctival cells from bulbar biopsies were isolated and expanded on a growth arrested 3T3 feeder layer. The cells were evaluated for cytokeratin (CK4/CK19) expression by immunostaining. An aliquot with half of the cells from the initial culture was frozen in liquid nitrogen and stored for 14 days and, in addition, donor cells were cryopreserved for more than 6 months (202.7 +/- 13.0 days). Both cryopreserved and noncryopreserved cells were serially cultivated over four passages. For each passage the colony-forming efficiency and the cell population doubling rates were evaluated, and expression of putative progenitor cell markers, p63alpha and ABCG2, was assessed by immunostaining and reverse transcription PCR. RESULTS: Both noncryopreserved and cryopreserved cells demonstrated a high colony-forming capacity that decreased with passage. Cells from both groups underwent approximately 20 cell population doublings before senescence. Immunoreactivity to p63alpha and ABCG2 was found in both groups until passage 4 and their presence was also confirmed by reverse transcription PCR. No difference in cell viability, colony-forming efficiency and immunoreactivity to p63alpha and ABCG2 was observed between cells cryopreserved for 14 days, and more than 6 months (202.7 +/- 13.0 days). CONCLUSION: Conjunctival epithelial cells with progenitor cell-like characteristics can be efficiently cryopreserved and can subsequently maintain their function in vitro over several culture passages. The option to cryopreserve conjunctival cells prior to in vitro expansion would be an advantage when cells have to be cultivated for clinical transplantation.
Authors:
S Schrader; M Notara; M Beaconsfield; S Tuft; G Geerling; J T Daniels
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Regenerative medicine     Volume:  4     ISSN:  1746-076X     ISO Abbreviation:  -     Publication Date:  2009 Sep 
Date Detail:
Created Date:  2009-09-18     Completed Date:  2009-12-03     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101278116     Medline TA:  Regen Med     Country:  England    
Other Details:
Languages:  eng     Pagination:  677-87     Citation Subset:  IM    
Affiliation:
Cells for Sight Transplantation & Research Programme, Department of Ocular Biology & Therapeutics, UCL Institute of Ophthalmology, EC1V 9EL, London, UK. mail@stefanschrader.de
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MeSH Terms
Descriptor/Qualifier:
Animals
Cell Culture Techniques
Cell Proliferation
Coculture Techniques
Conjunctiva / cytology
Cryopreservation*
Humans
Immunohistochemistry
Mice
Middle Aged
NIH 3T3 Cells
Reverse Transcriptase Polymerase Chain Reaction
Stem Cells / cytology*
Time Factors
Tissue Culture Techniques
Tissue Engineering

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


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