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T. Utheim, S. Raeder, Ø. Utheim, Y. Cai, B. Roald, T. Noren, K. Haug, L. Drolsum, T. Lyberg, B. Nicolaissen; Storage of Cultured Limbal Epithelial Cells at 23ºC and 31ºC in Organ Culture Medium and at 5ºC in Optisol-GS: A Light Microscopy and Phenotypic Study. Invest. Ophthalmol. Vis. Sci. 2007;48(13):460.
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© ARVO (1962-2015); The Authors (2016-present)
To compare 31°C organ culture storage and hypothermic storage of cultured limbal epithelial cells with our novel and recently reported method using 23°C organ culture preservation.
Limbal epithelial cells were cultured for three weeks on amniotic membranes fastened to polyester membrane carriers. The cultured epithelia were stored for one week at 31°C in organ culture medium and at 5ºC in Optisol-GS and subsequently examined using light microscopy and immunohistochemistry.
At 23 ºC the cell borders were maintained, and the epithelia attached well to the amniotic membrane. Mild intercellular oedema was occasionally observed. In contrast, at both 31ºC and 5 ºC storage, detachment of epithelial cells and enlarged intercellular spaces occurred regularly. Pronounced detachment of the basal layer was observed at 5 ºC storage. Occurrence of fibroblasts was disclosed in 3/8 of the epithelia stored at 31ºC. Expression of p63, K19, K5, and vimentin was maintained in all groups. K3 was not expressed in either of the experimental groups. Furthermore, Integrin beta-1, E-cadherin, Connexin 43, K14, and Ki 67 demonstrated minor changes in immunostaining in the three groups.
31ºC organ culture storage and in particular hypothermic storage were less satisfactory in preserving cultured limbal epithelial cells than organ culture preservation at 23ºC. However, the original undifferentiated phenotype was maintained irrespective of the organ culture medium and temperature.
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