Ex vivo expansion of conjunctival and limbal epithelial cells using cord blood serum-supplemented culture medium.

PURPOSE: Conventional cell culture methods use fetal bovine serum (FBS) as a growth supplement. The purpose of this study was to develop a xenobiotic-free culture system using umbilical cord blood serum (CBS) as an alternative growth supplement for the cultivation of human conjunctival and limbal epithelial cells. METHODS: Human conjunctival and limbal epithelial cells were cultivated in varying concentrations of CBS-supplemented medium and compared with FBS-supplemented medium. Bromodeoxyuridine (BrdU) ELISA proliferation assay, colony-forming efficiency (CFE), and a number of cell generations were analyzed. Cytokeratin expression of cultured cells was evaluated (K3, K4, K12, K13, K14, K15, K19, and PanCK). The authors compared the cytokine and growth factor levels in CBS, FBS, and adult serum using antibody array assays. RESULTS: Conjunctival and limbal cells cultivated in 0.25% CBS- and 0.5% CBS-supplemented culture media demonstrated the highest proliferative capacity in terms of BrdU proliferation assay, CFE, and number of cell generations. These results were comparable to FBS-supplemented medium. Cultured epithelial cells retained their normal cytokeratin expression. Cytokines brain-derived neurotrophic factor, growth-related oncogene, and leptin and growth factors EGF, HGF, FGF-6, IGF-1, PDGF, and IGFBP were present in higher concentrations in CBS than in FBS and adult serum. CONCLUSIONS: CBS-supplemented culture medium supported the proliferation and differentiation of conjunctival and limbal epithelial cells. CBS contained a higher concentration of growth factors and cytokines than FBS and adult serum. CBS may be a viable and safer alternative to FBS as a growth supplement in the culture medium for culturing epithelial cells, which may have important clinical implications when bioengineering tissues for clinical use.

Cultivated human conjunctival epithelial transplantation for total limbal stem cell deficiency.

PURPOSE: To determine the feasibility of cultivated conjunctiva as a viable epithelial sheet for transplantation and corneal resurfacing in eyes with limbal stem cell deficiency (LSCD). METHODS: Human corneal epithelial (HCE) and human conjunctival epithelial (HCjE) cells were cultivated on human amniotic membrane (AM) to confluence and then air lifted to allow further stratification and differentiation. Denuded AM and cultivated HCE and cultivated HCjE cells were then transplanted into 18 eyes of rabbits with induced LSCD. The cultivated and engrafted epithelia were examined by transmission electron microscopy (TEM) and immunohistochemistry. Two weeks after transplantation, the eyes were examined by slit lamp biomicroscopy and scored on epithelial integrity, corneal haze, and corneal neovascularization. RESULTS: Both cultivated and engrafted HCjE sheets demonstrated confluent epithelial sheets with five to six layers of well-stratified epithelium. TEM examination of engrafted HCjE revealed numerous microvilli, desmosomes, and hemidesmosomes, identical with in vivo corneal epithelium. Immunohistochemical analysis of both HCjE and HCE cells showed the presence of CK3, CK4, and CK12, with absence of Muc5AC. Clinical outcomes for eyes receiving HCjE transplants and HCE transplants were comparable, with most having transparent, smooth corneas, free of epithelial defects. CONCLUSIONS: The study showed that microscopically, HCjE cells have features similar to HCE cells, with clinically equivalent outcomes. The ex vivo cultivation of conjunctiva to form transplantable epithelial sheets for corneal replacement is a promising new treatment modality in patients with LSCD.