Purchase this article with an account.
T. Kawakita, E.M. Espana, H. He, M. Di Pascuale, C.–Y. Liu, S.C. G. Tseng; In Vitro Growth and Differentiationof MouseCorneal Epitheliumon Plastic Dependson Extracellular Calcium Concentration . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1450.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: Unlike human and rabbit counterparts, mouse corneal epithelial cells surprisingly have not been isolated, cultured and characterized. We speculate that one important reason is that its growth and differentiation is highly susceptible to extracellular Ca2+ concentrations. Methods: Intact and viable corneal epithelial sheets were isolated from CD–1 albino mouse eyes by incubating with 15 mg/ml dispase II and sorbitol in supplementary hormonal epithelial medium (SHEM) or defined keratinocyte serum–free medium (KSFM) for 18 hours at 4 oC. These sheets were trypsinized into single cells, and cultured on plastic in either SHEM (0.9 mM Ca+2) or KSFM (0.07 mM Ca+2), resulting in 4 different experimental conditions, i.e., SHEM/SHEM, SHEM/KSFM, KSFM/SHEM, and KSFM/KSFM (digestion medium/culture medium). Epithelial differentiation was assessed by immnofluorescence staining and/or immunoblotting by antibodies against keratin K12, connexin 43 (Cx43) and ZO–1. The growth was compared between SHEM/SHEM and KSFM/KSFM by continuous passage at 1:3 split and by crystal violet staining of confluent dishes. Epithelial growth and differentiation were further studied by manipulating Ca2+ concentrations in KSFM. Results: Unlike human and rabbit eyes, our digestion protocol resulted in isolation of corneal rather than limbal epithelium. Intact and viable corneal epithelial sheets were consistently isolated from more than 100 mouse eyes. Unlike human or rabbit corneal sheets, isolated mouse corneal sheets could not yield any outgrowth in SHEM. Indeed, epithelial growth derived from single cells was limited to passage 1 in SHEM/SHEM. In contrast, continuous passage to P3 could be achieved in KSFM/KSFM. Gradual increases in cell sizes and expression of keratin K12, Cx43 and ZO–1 were noted from KSFM/KSFM to SHEM/KSFM, KSFM/SHEM and SHEM/SHEM at passage 0. Immunoblotting analysis revealed that keratin K12 expression was the highest in SHEM/SHEM and gradually reduced from passage 0 to 2 in KSFM/KSFM. Conclusions: Mouse corneal epithelial sheets can be successfully isolated. Mouse corneal epithelial growth and differentiation are greatly affected by Ca2+ concentrations even during dispase digestion. Growth potential is promoted by lowering Ca2+ concentrations. Differentiation is facilitated by increasing Ca2+ concentrations, but in the absence of serum, we speculate that normal differentiation marked by K12 keratin expression is declined due to squamous metaplasia.
This PDF is available to Subscribers Only