Objective: To study the effects of different matrices and co-culture systems on cultured limbal stem cells (LSCs). Materials and Methods: Limbal explants were co-cultured with limbal fibroblasts (LF) and/or mouse embryonic fibroblasts (MEF) on filter inserts coated with amniotic membrane (AM), matrigel (MAT) and collagen type I (COL). Results: This study revealed that AM facilitated the cell migration and expansion significantly in comparison with other matrices. However, the gene expression profile of stemness markers of LSCs showed no significant differences among the experimental groups. The data indicated that at least in two-dimensional culture systems, the mentioned matrices have no significant effect on switching the expression of genes involved in differentiation process. In addition, the results of the two co-culture systems in case of different feeders, including MEF and LF were similar in growth rate and also preserving stemness quality of cultured limbal cells. Conclusion: To exclude the pollution of transplantable cultivated cells with probable mouse viruses, LF with human origin is recommended as feeder. Hence, limbal explants grown on AM in co-culture with LF will promise a quick and safe model for preparing undifferentiated epithelial sheets suitable for transplantation.