Expansion and characterization of human limbus-derived stromal/mesenchymal stem cells in xeno-free medium for therapeutic applications

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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13287-023-03299-3.