The use of human serum in supporting the in vitro and in vivo proliferation of human conjunctival epithelial cells.
OBJECTIVE
To evaluate the use of human serum (HS) in supporting the in vitro and in vivo proliferation of human conjunctival epithelial cells, and compare it with fetal bovine serum (FBS) and bovine pituitary extract (BPE).


METHODS
Conjunctival epithelial cells were cultivated in media supplemented with HS (5%, 10%), FBS (5%, 10%), and BPE (70 microg/ml, 140 microg/ml). The colony forming efficiency (CFE), bromodeoxyuridine (BrdU) ELISA proliferation assay, and cell generations were analysed. Cells were evaluated for keratin (K4, K19, and K3) and MUC5AC expression by immunostaining and RT-PCR. Conjunctival equivalents constructed on amniotic membranes were transplanted onto severe combined immune deficient (SCID) mice for 10 days and analysed histologically.


RESULTS
The proliferation assays of HS supplemented cultures (CFE, 6.7% (SD 1.8%); BrdU absorbance, 0.86 (0.16)) were comparable to FBS supplemented (CFE, 9.3% (1.8%); BrdU absorbance, 1.11 (0.18)) and BPE supplemented cultures (CFE, 5.9 (1.5); BrdU absorbance, 0.65 (0.12)). Goblet cell densities for HS, FBS, and BPE supplemented media were 52 cells/cm(2), 60 cells/cm(2), and 50 cells/cm(2), respectively. HS supplemented cultures formed stratified epithelial sheets in vivo following transplantation.


CONCLUSIONS
The proliferative capacity of conjunctival epithelial cells cultivated in HS supplemented cultures was comparable to FBS and BPE supplemented cultures. The elimination of animal material from the culture system is advantageous when cultivating cells for clinical transplantation.
Ang
L P K
LP
Singapore National Eye Center, 11 Third Hospital Avenue, Singapore 168751. leopk@pacific.net.sg
Tan
D T H
DT
Seah
C J Y
CJ
Beuerman
R W
RW
D000818:Animals; D001973:Bromodeoxyuridine; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002455:Cell Division; D048429:Cell Size; D003228:Conjunctiva / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D003470:Culture Media; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D020397:Goblet Cells / Q000166:cytology; D006801:Humans; D007633:Keratins / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D008858:Microscopy, Phase-Contrast; D055271:Mucin 5AC; D009077:Mucins / Q000378:metabolism; D044967:Serum; D013234:Stem Cells; D023822:Tissue Engineering / Q000379:methods*
D003470:Culture Media; C524678:MUC5AC protein, human; C524679:Muc5ac protein, mouse; D055271:Mucin 5AC; D009077:Mucins; D007633:Keratins; D001973:Bromodeoxyuridine