Purpose: The purpose of this study was to establish a simple, xeno-feeder-free method for cultivating human corneal epithelial cells. Methods: Limbal tissue explants from a cadaver were cultured in Iscove's modified Dulbecco's medium and low-calcium Panserin 801 medium in a 1:1 ratio. The outgrowing cells were characterized by flow cytometry, immunohistochemistry and real-time PCR (rtPCR). Limbal epithelial cells were expanded in a xeno-feeder-free, low-calcium medium and airlifted for 2 weeks each. Results: Migration of fibroblast-like stromal cells initially occurred from the limbal explants, and then epithelial cells migrated and grew on the stromal cells as an autofeeder layer. After airlifting, the cultured epithelium consisted of two to three layers. The cultured cells expressed stem cell-associated markers (ABCG2 and ΔNp63), differentiation markers (CK3 and CK12) and extracellular matrix-associated markers (lumican and decorin). rtPCR showed increased expression of markers for epithelial progenitor cells compared to fresh limbal tissue. Side population cells comprised 0.43 ± 0.04% of the cells (n = 5) in the primary culture. Flow cytometry showed that 49.12, 40.44 and 44.55% of the cells from the explants expressed E-cadherin, ΔNp63 and ABCG2, respectively. Conclusion: This explant culture system using stromal cells as an autofeeder layer was useful in expanding human corneal epithelial cells. This system may offer clinical insight for the expansion of limbal progenitor cells for the reconstruction of the ocular surface.

Chen JJ, Tseng SC: Abnormal corneal epithelial wound healing in partial-thickness removal of limbal epithelium. Invest Ophthalmol Vis Sci 1991;32:2219-2233.
Sun TT, Tseng SC, Lavker RM: Location of corneal epithelial stem cells. Nature 2010;463:E10-E11.
Lavker RM, Tseng SC, Sun TT: Corneal epithelial stem cells at the limbus: looking at some old problems from a new angle. Exp Eye Res 2004;78:433-446.
Thoft RA, Friend J: The X, Y, Z hypothesis of corneal epithelial maintenance. Invest Ophthalmol Vis Sci 1983;24:1442-1443.
Buck RC: Measurement of centripetal migration of normal corneal epithelial cells in the mouse. Invest Ophthalmol Vis Sci 1985;26:1296-1299.
Schermer A, Galvin S, Sun TT: Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol 1986;103:49-62.
Cotsarelis G, Cheng SZ, Dong G, Sun TT, Lavker RM: Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells. Cell 1989;57:201-209.
Shapiro MS, Friend J, Thoft RA: Corneal re-epithelialization from the conjunctiva. Invest Ophthalmol Vis Sci 1981;21:135-142.
Kinoshita S, Adachi W, Sotozono C, Nishida K, Yokoi N, Quantock AJ, Okubo K: Characteristics of the human ocular surface epithelium. Prog Retin Eye Res 2001;20:639-673.
Pellegrini G, Traverso CE, Franzi AT, Zingirian M, Cancedda R, De Luca M: Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet 1997;349:990-993.
Shortt AJ, Secker GA, Rajan MS, Meligonis G, Dart JK, Tuft SJ, Daniels JT: Ex vivo expansion and transplantation of limbal epithelial stem cells. Ophthalmology 2008;115:1989-1997.
Koizumi N, Inatomi T, Quantock AJ, Fullwood NJ, Dota A, Kinoshita S: Amniotic membrane as a substrate for cultivating limbal corneal epithelial cells for autologous transplantation in rabbits. Cornea 2000;19:65-71.
Nishida K, Yamato M, Hayashida Y, Watanabe K, Maeda N, Watanabe H, Yamamoto K, Nagai S, Kikuchi A, Tano Y, Okano T: Functional bioengineered corneal epithelial sheet grafts from corneal stem cells expanded ex vivo on a temperature-responsive cell culture surface. Transplantation 2004;77:379-385.
Nishida K, Yamato M, Hayashida Y, Watanabe K, Yamamoto K, Adachi E, Nagai S, Kikuchi A, Maeda N, Watanabe H, Okano T, Tano Y: Corneal reconstruction with tissue-engineered cell sheets composed of autologous oral mucosal epithelium. N Engl J Med 2004;351:1187-1196.
Murakami D, Yamato M, Nishida K, Ohki T, Takagi R, Yang J, Namiki H, Okano T: The effect of micropores in the surface of temperature-responsive culture inserts on the fabrication of transplantable canine oral mucosal epithelial cell sheets. Biomaterials 2006;27:5518-5523.
Mariappan I, Maddileti S, Savy S, Tiwari S, Gaddipati S, Fatima A, Sangwan VS, Balasubramanian D, Vemuganti GK: In vitro culture and expansion of human limbal epithelial cells. Nat Protoc 2010;5:1470-1479.
Kim HS, Jun Song X, de Paiva CS, Chen Z, Pflugfelder SC, Li DQ: Phenotypic characterization of human corneal epithelial cells expanded ex vivo from limbal explant and single cell cultures. Exp Eye Res 2004;79:41-49.
Varghese VM, Prasad T, Kumary TV: Optimization of culture conditions for an efficient xeno-feeder free limbal cell culture system towards ocular surface regeneration. Microsc Res Tech 2010;73:1045-1052.
Koizumi N, Sakamoto Y, Okumura N, Okahara N, Tsuchiya H, Torii R, Cooper LJ, Ban Y, Tanioka H, Kinoshita S: Cultivated corneal endothelial cell sheet transplantation in a primate model. Invest Ophthalmol Vis Sci 2007;48:4519-4526.
Umemoto T, Yamato M, Nishida K, Yang J, Tano Y, Okano T: Limbal epithelial side-population cells have stem cell-like properties, including quiescent state. Stem Cells 2006;24:86-94.
Spurr SJ, Gipson IK: Isolation of corneal epithelium with Dispase II or EDTA. Effects on the basement membrane zone. Invest Ophthalmol Vis Sci 1985;26:818-827.
Meyer-Blazejewska EA, Kruse FE, Bitterer K, Meyer C, Hofmann-Rummelt C, Wünsch PH, Schlötzer-Schrehardt U: Preservation of the limbal stem cell phenotype by appropriate culture techniques. Invest Ophthalmol Vis Sci 2010;51:765-774.
O'Callaghan AR, Daniels JT, Mason C: Effect of sub-atmospheric oxygen on the culture of rabbit limbal epithelial cells. Curr Eye Res 2011;36:691-698.
Notara M, Shortt AJ, Galatowicz G, Calder V, Daniels JT: IL6 and the human limbal stem cell niche: a mediator of epithelial-stromal interaction. Stem Cell Res 2010;5:188-200.
Montzka K, Führmann T, Wöltje M, Brook GA: Expansion of human bone marrow-derived mesenchymal stromal cells: serum-reduced medium is better than conventional medium. Cytotherapy 2010;12:587-592.
Kawakita T, Espana EM, He H, Yeh LK, Liu CY, Tseng SC: Calcium-induced abnormal epidermal-like differentiation in cultures of mouse corneal-limbal epithelial cells. Invest Ophthalmol Vis Sci 2004;45:3507-3512.
Kruse FE, Tseng SC: Proliferative and differentiative response of corneal and limbal epithelium to extracellular calcium in serum-free clonal cultures. J Cell Physiol 1992;151:347-360.
Baylis O, Figueiredo F, Henein C, Lako M, Ahmad S: 13 years of cultured limbal epithelial cell therapy: a review of the outcomes. J Cell Biochem 2011;112:993-1002.
Shortt AJ, Secker GA, Notara MD, Limb GA, Khaw PT, Tuft SJ, Daniels JT: Transplantation of ex vivo cultured limbal epithelial stem cells: a review of techniques and clinical results. Surv Ophthalmol 2007;52:483-502.
Kruse FE, Tseng SC: Serum differentially modulates the clonal growth and differentiation of cultured limbal and corneal epithelium. Invest Ophthalmol Vis Sci 1993;34:2976-2989.
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