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R. Tandon, N. Singh, T. Saxena, H. Sharma, S. Kashyap, R.B. Vajpayee; Culture of Limbal Stem Cells–Measures to Reduce Cost and Widen Scope for Developing World . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2095.
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Purpose: Limbal stem cells (LSC) have been expanded ex–vivo using growth media containing Dulbecco–modified Eagle’s medium (DMEM) and various additives. Some of the adjuvants are not readily available and others too expensive or practically inaccessible in developing countries. Limbal stem cells are known to proliferate easily. The substantia propria of human amniotic membrane (AM) contains many of the factors used in the culture media. The aim of this study is to try ex–vivo expansion of LSC on AM using minimal adjuvants in the culture media and other cost effective modifications. Methods: Cadaveric or autologous limbal tissue was divided in pieces of 1–2mm and placed on AM. To stabilize the AM, various techniques were tried i.e. wrapping around a sterile glass slide and suturing the edges, placing AM and denuded AM over a cover slip, on nitrocellulose paper and directly on the surface of the culture plate. Limbal tissue was placed on the plate without AM for comparison. The tissue pieces were allowed to adhere on the surface and then submerged in the specially prepared culture medium containing DMEM, fetal bovine serum, insulin, adenine, hydrocortisone, gentamicin and amphotericin B and incubated. The medium was changed every 2 days and extent of outgrowth monitored. After standardization of technique, clinical application was done. Cells cultured on AM were transplanted in 5 patients with stem cell deficiency. Three had localized stem cell deficiency in the form of pterygium, two had total stem cell deficiency. Patients with allografts, were given topical 2% Cyclosporine A and systemic corticosteroids for 6 months. Impression cytology was done after 6 months in all cases. Results: Visible outgrowth of epithelial cells was seen in 2–3 days. The growth reached confluence and spread uniformly on the AM in 14–21 days except in the well in which the limbal tissue was kept without AM. The donor tissue kept on the denuded amniotic membrane showed initial slow rate of growth (5–6 days) as compared to that grown on non– denuded AM, but reached confluence in 14–21 days. None of the patients with pterygium had recurrence in the follow up period. Both patients of chemical burn had a decrease in corneal scarring, vascularization and improvement in vision. In all cases impression cytology done after 6 months revealed absence of goblet cells on the cornea. Conclusions: Limbal epithelial cells can be cultured on human AM for successful clinical use with minimal adjuvants in the culture media.
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