Abstract
Purpose:
Limbal epithelial stem cells (LESCs) are responsible for renewal of the corneal epithelium. Their dysfunction cause corneal blindness and chronic pain. Current treatments include the transplantation of in vitro cultured LESCs. Unfortunately, the culture media (CM) used for LESC cultivation often contain supplements, like cholera toxin, dimethylsulfoxide (DMSO) and fetal bovine serum (FBS) that could induce toxicity and side effects. To overcome this limitation, we developed a CM in which potential harmful compounds were replaced by biosafe supplements.
Methods:
We tested three different CM to establish limbal primary cultures (LPCs) from limbal explants: (1) CnT20, a commercially available, yet undefined CM, 2) IOBA-FBS, a CM with FBS in which cholera toxin and DMSO were replaced by isoproterenol and transferring-selenite, respectively, and 3) IOBA-HS, IOBA-FBS CM with human serum (HS) instead of FBS. Cell morphology, the percentage of confluent LPCs, and time to confluence were analyzed. LESC (ABCG2, p63, K14, and K15), corneal epithelial cell (K3 and K12) and fibroblast (S100A4) markers were analyzed by immunofluorescence and real time RT-PCR. Markers of endothelial cells (PECAM), melanocytes (MART-1), and dendritic cells (CD11c) were analyzed by immunofluorescence. Additionally, the same limbal explant was consecutively cultured with IOBA-HS CM, obtaining LPC1 and LPC2.
Results:
All LPCs cultured with the three CM presented cuboidal cell morphology without differences in the percentage of cells positive for ABCG2, p63, K14, K3, and K12 proteins. Conversely, except for K12, mRNA expression of these markers was higher in CnT20-cultured cells. Expression of the LESC marker K15 was significantly higher with the IOBA-HS. PECAM, MART-1, and CD11c proteins were not detected, while fibroblast-protein S100A4 was mildly detected with the three CM. The LPC1 obtained with IOBA-HS CM showed similar characteristics to the LPC0, while an elongated cell morphology and a decrease in the expression of some LESC marker expression was observed in the LPC2.
Conclusions:
IOBA-HS CM enables the culture of two similar LPCs that maintain the LESC phenotype. The routine use of this CM could improve both the biosafety and the number of available LPCs intended for transplantation to regenerate the corneal epithelium.
Keywords: 479 cornea: clinical science •
482 cornea: epithelium