| Summary: | Abstract Background Mesenchymal stem cells (MSCs) have been proven to prevent and clear corneal scarring and limbal stem cell deficiency. However, using animal-derived serum in a culture medium raises the ethical and regulatory bar. This study aims to expand and characterize human limbus-derived stromal/mesenchymal stem cells (hLMSCs) for the first time in vitro in the xeno-free medium. Methods Limbal tissue was obtained from therapeutic grade corneoscleral rims and subjected to explant culture till tertiary passage in media with and without serum (STEM MACS XF; SM), to obtain pure hLMSCs. Population doubling time, cell proliferation, expression of phenotypic markers, tri-lineage differentiation, colony-forming potential and gene expression analysis were carried out to assess the retention of phenotypic and genotypic characteristics of hLMSCs. Results The serum-free medium supported the growth of hLMSCs, retaining similar morphology but a significantly lower doubling time of 23 h (*p < 0.01) compared to the control medium. FACS analysis demonstrated ≥ 90% hLMSCs were positive for CD90 +, CD73 +, CD105 +, and ≤ 6% were positive for CD45 −, CD34 − and HLA-DR − . Immunofluorescence analysis confirmed similar expression of Pax6 +, COL IV +, ABCG2 +, ABCB5 +, VIM +, CD90 +, CD105 +, CD73 +, HLA-DR − and CD45 −, αSMA − in both the media. Tri-lineage differentiation potential and gene expression of hLMSCs were retained similarly to that of the control medium. Conclusion The findings of this study demonstrate successful isolation, characterization and culture optimization of hLMSCs for the first time in vitro in a serum-free environment. This will help in the future pre-clinical and clinical applications of MSCs in translational research.
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