Evaluation of human MRC-5 cells as a feeder layer in a xenobiotic-free culture system for conjunctival epithelial progenitor cells.
OBJECTIVE
Aim of this study was to evaluate the efficiency of human MRC-5 cells to act as a feeder layer for conjunctival epithelial cells in order to develop a complete xenobiotic-free culture system for the expansion of conjunctival epithelial progenitor cells for clinical applications.


METHODS
Human conjunctival epithelial cells were expanded from a bulbar biopsy, in a completely xenobiotic-free culture system using growth arrested MRC-5 cells as a feeder layer, without (MRC-5/No Serum) and in the presence of 5% (MRC-5/HS 5%) or 10% (MRC-5/HS 10%) human serum. The total cell count, the surface area as well as the total colony-forming efficiency (CFE), the percentage of aborted colonies and the expression of putative progenitor cell markers p63α, ABCG2, CK15 was compared to the gold standard culture system (GS) in which growth arrested 3T3 feeder cells and feotal calf serum were used.


RESULTS
The epithelial cell count revealed significantly less proliferation in the MRC-5/No Serum group compared to the GS conditions. All groups showed immunoreactivity to CK19; however, more differentiated epithelial cells were observed in the MRC-5/No Serum- and MRC-5/HS 10%-group and less immunoreactivity to p63 α and ABCG2 was found in these groups compared to GS and MRC-5/HS 5% conditions. This was in accordance with CFE results, were the MRC-5/HS 5% group showed similar CFE results compared to the GS group, while in the MRC-5/No Serum- and MRC-5/HS 10%-group significantly lower CFE's were observed.


CONCLUSIONS
Our results indicate that a completely xenobiotic-free culture system using MRC-5 cells as a feeder layer in combination with human serum can be successfully used to expand conjunctival epithelial cells with progenitor cell characteristics and might be a useful tool for the safe expansion of these cells for clinical use.
Cells for Sight Transplantation and Research Programme Ocular Biology and Therapeutics, UCL Institute of Ophthalmology, London, UK. mail@stefanschrader.de
Stefan
S
Schrader
Stephen J
SJ
Tuft
Michele
M
Beaconsfield
Maria
M
Borrelli
Gerd
G
Geerling
Julie T
JT
Daniels
D016475:3T3 Cells; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002452:Cell Count; D049109:Cell Proliferation; D002469:Cell Separation; D002474:Cell-Free System; D018920:Coculture Techniques; D003228:Conjunctiva / Q000166:cytology* / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D061252:Feeder Cells / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007633:Keratins / Q000378:metabolism; D051379:Mice; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D015415:Biomarkers; D009363:Neoplasm Proteins; C115051:TP63 protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D007633:Keratins