Comparison between serum-free and fibroblast-cocultured single-cell clonal culture systems: evidence showing that epithelial anti-apoptotic activity is present in 3T3 fibroblast-conditioned media.
OBJECTIVE
To compare the supporting mechanism between the serum-free and the fibroblast-cocultured single-cell clonal culture systems.


METHODS
Clonal growth, measured by colony forming efficiency (CFE) and size, was compared between rabbit corneal and limbal epithelial cells in a previously-established serum-free MCDB medium supplemented with growth factors, and in a coculture system with a feeder layer of mitomycin C-treated mouse 3T3 fibroblasts grown in the MCDB or DMEM medium plus 20% fetal bovine serum (FBS).


RESULTS
Limbal epithelial cells in the serum-free MCDB medium had a significantly lower CFE than corneal epithelial cells (p < 0.001), suggesting that this system promoted more clonal growth of corneal progenitor cells. In contrast, with cocultured 3T3 fibroblasts limbal CFE was significantly increased (p < 0.001), while corneal CFE was not changed, indicating that the 3T3 system promoted more clonal growth of limbal progenitor cells. Addition of 20% FBS in the MCDB medium cocultured with 3T3 fibroblasts significantly promoted both limbal and corneal CFEs (p < 0.001). For both cultures, switching the serum-containing MCDB medium to the serum-containing DMEM medium produced clonal growth only with cocultured fibroblasts. This epithelial growth-promoting activity was not present on the cell surface or in the extracellular matrix, but present in pre-centrifuged and prefiltered 3T3 fibroblast-conditioned media. Both growth-promoting and anti-apoptotic activities were present in fibroblast-derived serum-free conditioned media. In the presence of this anti-apoptotic activity, serum addition promoted clonal growth, and the expression of cornea-type K3 keratin in limbal colonies was negative using AE-5 monoclonal antibody.


CONCLUSIONS
Further purification and characterization of this fibroblast-derived anti-apoptotic survival factor will facilitate understanding of the mechanism by which epithelial stem cells are regulated via epithelial-mesenchymal interactions.
Tseng
S C
SC
Department of Ophthalmology, Bascom Palmer Eye Institute, William L. McKnight Vision Research Center, Miami, FL 33136, USA.
Kruse
F E
FE
Merritt
J
J
Li
D Q
DQ
D016475:3T3 Cells / Q000502:physiology*; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001685:Biological Factors / Q000502:physiology; D002454:Cell Differentiation; D002478:Cells, Cultured; D002999:Clone Cells; D018920:Coculture Techniques; D003315:Cornea / Q000166:cytology / Q000502:physiology*; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D004848:Epithelium / Q000502:physiology; D016850:Limbus Corneae / Q000166:cytology / Q000502:physiology*; D051379:Mice; D011817:Rabbits
D001685:Biological Factors; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free