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H.N. Madhavan, B. Sudha, G. Sitalakshmi, S. KrishnaKumar, Y. Mori, H. Yoshioka, S. Abraham; Comparative Study on Growth Characteristics of Cadaveric Human Corneal Limbal Stem Cells in Mebiol Gel (a Synthetic Polymer) and on Human Amniotic Membrane . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3033.
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© ARVO (1962-2015); The Authors (2016-present)
To compare the growth characteristics of cells of human corneal limbal tissue (HCLT) embedded in Mebiol Gel (MG), a thermo–reversible gelation polymer with its growth on Human Amniotic Membrane (HAM).
Explant cultures of HCLT from eight human donor eyes, embedded in MG and on HAM were performed in tissue culture medium – equal volumes of Dulbecco's minimum essential medium and HAM's F–12, fetal calf serum and antibiotics. Growth of HCLT in MG and on HAM was documented photomicrographically at intervals. Corneal stem cell immunomarkers (p63, ABCG2, Connexin43, Integrin ß1) were studied on cultured cells. Growth rate of three HCLTs was measured by 3H thymidine uptake studies. Reverse transcriptase polymerase chain reaction (RT–PCR) was carried out initially on two HCLT cultivated cells harvested on alternate days till day 10 and subsequently on three HCLT cultivated cells harvested at intervals of 4 days till day 20 of incubation for expression of p63, ABCG2, Connexin43, Integrinß1, K3 and K12.
3–dimentional growth of HCLT in MG and patches of monolayer cells on HAM occurred. Cells cultivated in MG expressed immunomarkers p63, ABCG2, Connexin43 and Integrinß1. 3H thymidine uptake studies indicated 4 – 5 fold more rapid multiplication of cells in MG than on HAM. RT–PCR on the initial two HCLT cells cultivated in MG expressed p63, Connexin43 and K3 till day 10; ABCG2 and Integrinß1 on days 6, 8 and 10 and K12 was absent. Comparative expression of stem cell markers by RT–PCR on the subsequent three HCLT cells cultivated in MG and over HAM were as follows: Cells cultivated in MG expressed p63, ABCG2 and Integrinß1till day 20; Connexin43 and K3 from days 12 to 20 and K12 was not expressed. Cells cultivated on HAM expressed p63, Connexin43 and Integrinß1 till day 20; ABCG2 only on day 4; K3 and K12 from days 12 and 16 respectively till day 20.
Since HCLT cells cultured in the Mebiol Gel showed 3–dimensional growth; expressed corneal stem cell markers indicating their multiplication in the forms of transient amplifying cells and pluripotent cells with growth charecteristics similar as on HAM and 3H thymidine uptake studies indicated more rapid multiplication than on HAM, their transplantation on to prospective recipients is a distinct possibility; thus avoiding human tissue in the form of HAM.
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