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H.A. Martinez–Osorio, C. García–Vázquez, V. Sáez, I. Fernández, M. Calonge, Y. Diebold; Effect of Calcium Concentration on the Proliferation of IOBA–NHC Cells Grown on Two Different Substrates . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4933.
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© ARVO (1962-2015); The Authors (2016-present)
Primary cultures of epithelial cells (keratinocytes, corneal, limbal and conjunctival epithelium) cultured in low Ca2+ concentration [0.05–0.3 mM] are known to increase their proliferation, whereas higher Ca2+ concentrations [0.9–1.8 mM] are used to induce initial substrate adhesion and further stratification. The effect of extracellular calcium concentration is less well known in epithelial cell lines. We have tested the effect of extracellular Ca2+concentration on the proliferative capacity of an epithelial cell line plated onto different substrates.
IOBA–NHC (normal human conjunctiva) cell line, spontaneously immortalized, was used. Cells (103/well) were seeded onto PermanoxTM or amniotic membrane (AM). Three different culture media were used: medium 1 [Ca2+ 0.09 mM]= serum–free DK–SFM supplemented with EGF, bFGF and insulin; medium 2 [Ca2+ 1.16 mM]= DMEM/F12 supplemented with 2 ng/ml EGF, 1 µg/ml insulin, 0.1 µg/ml cholera toxin, 0.5 µg/ml hydrocortisone, 10% FBS; medium 3 [Ca2+ 1.2 mM]= DMEM/F12 supplemented with 10 ng/ml EGF, 5 µg/ml insulin, 0.1 µg/ml cholera toxin, 0.5 µg/ml hydrocortisone, 20% FBS. Cell proliferation was assessed in days 1, 2, and 5 using the Calcein/EthDIII kit by fluorescence microscopy. Three independent experiments were performed in duplicate for each condition. Repeated measures analysis of variance was performed for statistical analysis.
Culture medium with low Ca2+ concentration (medium 1) delayed cell proliferation and kept it significantly lower during the 5 days than that achieved with the other two media. No significant differences in proliferation were observed between high Ca2+ concentration media (medium 2 and 3). IOBA–NHC cells grown onto AM had lower proliferation regardless of the media used.
DK–SFM (low Ca2+ concentration) seems not to favor proliferation of this differentiated epithelial cell line (IOBA–NHC), neither did AM use as support. Further experiments using more cell lines, culture media and substrates are warranted.
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