Development of a conjunctival epithelial equivalent with improved proliferative properties using a multistep serum-free culture system.
OBJECTIVE
To investigate the use of a multistep serum-free culture system in developing a conjunctival epithelial equivalent with improved in vitro and in vivo proliferative properties and to evaluate the effect of serum supplementation and culture conditions on the proliferative capacity of these cells.


METHODS
Conjunctival epithelial cells were cultivated on human amniotic membrane (HAM) in a multistep serum-free culture system, under submerged and air-lifted conditions. The bromodeoxyuridine (BrdU) ELISA proliferation assay, colony-forming efficiency (CFE), and number of cell generations were compared with those in serum-containing medium. The in vivo proliferative capability of the tissue-constructs were evaluated by xenotransplantation to SCID mice. Cultured cells were evaluated for the expression of keratin-4, -19, and -3, as well as MUC5AC goblet cell mucin.


RESULTS
The epithelial cells cultivated in serum-free medium (BrdU absorbance, 1.91 +/- 0.08; cell generations, 25.6 +/- 4.5) were more proliferative than those cultivated in serum-containing medium (BrdU absorbance, 1.06 +/- 0.08; cell generations, 12.1 +/- 3.0). The serum-free-derived epithelial equivalents demonstrated a significant increase in proliferation and stratification after transplantation. Cells that were air lifted for 6 and 12 days had a reduced proliferative capacity in vitro and in vivo compared with submerged cultures. Cultured cells expressed keratin-4 and -19, and MUC5AC mRNA was detected by RT-PCR. Electron microscopy demonstrated a basal lamina with numerous hemidesmosomes.


CONCLUSIONS
This is a multistep serum-free culture system for developing a conjunctival epithelial equivalent with improved proliferative and structural properties, which are crucial for enhancing graft survival and regeneration of the conjunctival surface after clinical transplantation.
Ang
Leonard P K
LP
Singapore National Eye Center, Singapore. leopk@pacific.net.sg
Tan
Donald T H
DT
Beuerman
Roger W
RW
Lavker
Robert M
RM
D000328:Adult; D000650:Amnion; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D001973:Bromodeoxyuridine / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002455:Cell Division; D002469:Cell Separation; D017690:Cell Transplantation; D003228:Conjunctiva / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D016895:Culture Media, Serum-Free; D062109:Dermatologic Surgical Procedures; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D006801:Humans; D007633:Keratins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D008875:Middle Aged; D055271:Mucin 5AC; D009077:Mucins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism
D015415:Biomarkers; D016895:Culture Media, Serum-Free; C524678:MUC5AC protein, human; C524679:Muc5ac protein, mouse; D055271:Mucin 5AC; D009077:Mucins; D012333:RNA, Messenger; D007633:Keratins; D001973:Bromodeoxyuridine