The in vitro and in vivo proliferative capacity of serum-free cultivated human conjunctival epithelial cells.
OBJECTIVE
To investigate the in vitro and in vivo proliferative capacity of human conjunctival epithelial cells cultured in serum-free media, and to compare this with current methods that utilize serum-containing media and 3T3 feeder layers.


METHODS
Human conjunctival epithelial cells were cultivated in serum-free media alone, serum-free media with a 3T3 feeder layer, and serum-containing media with a 3T3 feeder layer. The areas of outgrowth, colony-forming efficiencies and number of population doublings were compared. The in vivo proliferative potential was assessed by analyzing the number of cells generated by the implantation of cultured cells into athymic mice. Cultured cells were evaluated for the expression of cytokeratins K3, K4, K12, K19, as well as the gel-forming goblet cell mucin, MUC5AC.


RESULTS
Cells cultivated in serum-free media, serum-free media and feeder cells, and serum-containing media and feeder cells achieved colony-forming efficiencies of 14.5 +/- 4.1%, 10.1 +/- 3.1%, and 20.4 +/- 6.7%, respectively, and number of population doublings of 24.8 +/- 4.3, 14.8 +/- 3.6, and 30.0 +/- 5.0, respectively. Nine-day old athymic mice conjunctival cysts derived from serum-free cultures comprised 1.29 x 10(6) +/- 0.46 x 10(6) cells, while cysts derived from serum-containing cultures comprised 1.30 x 10(6) +/- 0.53 x 10(6) cells. The degree of epithelial stratification was similar in both conditions. Serum-free cultivated conjunctival cells retained their in vivo characteristics and expressed K4, K19 and MUC5AC. The presence of MUC5AC mRNA in these cells was confirmed by RT-PCR.


CONCLUSIONS
Conjunctival epithelial cells propagated in serum-free media demonstrated a similar in vivo proliferative capability, as compared to serum-containing media with 3T3 feeder cells. This has important clinical implications, as the serum-free ex vivo expansion of cells for clinical transplantation overcomes the problems associated with the use of animal serum and cells.
Singapore National Eye Center, Singapore. leopk@pacific.net.sg
Leonard P K
LP
Ang
Donald T H
DT
Tan
Toan Thang
TT
Phan
Jing
J
Li
Roger
R
Beuerman
Robert M
RM
Lavker
D016475:3T3 Cells; D000818:Animals; D018929:Cell Culture Techniques; D002455:Cell Division; D017690:Cell Transplantation; D018920:Coculture Techniques; D003228:Conjunctiva / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D016895:Culture Media, Serum-Free; D004814:Epidermal Cyst / Q000473:pathology; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007633:Keratins / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D055271:Mucin 5AC; D055257:Mucin-5B; D009077:Mucins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D016895:Culture Media, Serum-Free; C524678:MUC5AC protein, human; C524578:MUC5B protein, human; C524679:Muc5ac protein, mouse; D055271:Mucin 5AC; D055257:Mucin-5B; D009077:Mucins; D012333:RNA, Messenger; D007633:Keratins