Limbal stem cell culture on amniotic membrane for corneal tissue reconstruction: a comparison of techniques
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Abstract
Objective: To grow cells on amniotic membrane corneal limbus with two different methods to assess the confluence, viability and differentiation of both.
Methods: LSC samples were obtained from 16 deceased donors. Additionally, six cultures were made from each sample, 3 by LSC suspension method and 3 by LSC explant method, all in triplicate. Furthermore, AMs without oral mucosal cells were used as negative control cultures. The cells were seeded with SHEM, 37°C, 5% CO2 , 95% humidity. Moreover, the cultures were maintained for 14 days and the culture medium was changed every other day or every 3 days. Also, on the 14th day, the cultures were analyzed to evaluate viability with trypan blue, confluence with microscopy and cell differentiation with immunohistochemistry to detect cytokeratin 3/12.
Results: As a result of the experiments mentioned above, for suspension and explant cultures, we found a viability of 98.92% and 98.32%, a confluence of 55.95% and 48.27%, a final cellular concentration of 38.83x104 cells/ml and 36.484x104 cells/ml, and a differentiation of 22.93% and 16.55%, respectively.
Conclusions: Although this study compared two methods, LSC suspension culture and LSC explant culture, both with AM as scaffold, we did find a slight difference between them. As a result, the suspension culture was better than the LSC explant culture, but the results are not conclusive. It is necessary to conduct more research in this field to reach a conclusion regarding the behavior of these two methods.
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