A simple and efficient method for cultivation of limbal explant stem cells with clinically safe potential.

BACKGROUND: Several methods to cultivate limbal epithelial stem cells (LESCs) in vitro with the support of feeder layers and different growth medium formulations have been established for several years. The initial green medium consists of various ingredients that exhibit a non-optimal level of biosafety, therefore, different modifications have been made to suit it to safe clinical applications. However, the question of which formulation is the most appropriate remains to be answered. AIMS: This study evaluated the outgrowth kinetics and stemness of cells cultured from human limbal explants with the aim of preserving LESC characteristics in the human-derived platelet-rich fibrin (HPRF)-conditioned medium with no feeder cell layer or carrier for the first time. The final composition of the cell culture system included only human-derived products without any xenobiotic or chemical substances to minimize the potential risk for human health, which will be useful for clinical purposes. METHODS: To test our hypothesis, limbal explants were incubated with either Dulbecco's Modified Eagle's Medium (DMEM)/F12-10% human serum (HS), human-derived amniotic membrane (HAM)-conditioned DMEM/F12-10% HS or HPRF-conditioned DMEM/F12-10% HS to determine whether outgrowth kinetics and stemness of cells show any differences among groups. RESULTS: The results showed that the HPRF-conditioned medium showed higher concentration levels of growth factors, which may be involved in the promotion of LESC expansion while preserving the stem cell characteristics. HPRF-conditioned medium had significantly superior capacity to enhance the cell growth rate, the stem/progenitor cell phenotype and the expressions of putative stem cell markers. CONCLUSION: This novel xeno-feeder-chemical-free, completely human-derived and biologically safe culture system including HPRF and HS would be of interest to replace conventional cell culture strategies to meet safety requirements mandatory for clinical use in humans.

A completely human-derived biomaterial mimicking limbal niche: Platelet-rich fibrin gel.

Platelet-rich fibrin (PRF) is a natural biomaterial and has excellent biochemical and physical properties with a history of proven biocompatibility in the field of tissue engineering and regenerative medicine. Recent reports of fibrin-based matrices have offered new opportunities to apply PRF as a supplement for in vitro cell culture. Here, custom-modified human-derived PRF (HPRF) was produced via different centrifugation protocols, then, characterized by morphologically and chemically and utilized as a substrate and as a conditioned medium for limbal explant culture for the first time. It was found that the HPRF released significantly higher levels of growth factors which are essential for epithelial cell growth. The enhanced physicochemical properties of the HPRF were also proven in the limbal explant cultures in terms of cell growth, migration, viability, and stemness in comparison with the conventional limbal explant culture on human-derived amniotic membrane. Consequently, HPRF hydrogels are appealing natural biomaterials for the purpose of mimicking limbal niche and the discovery elucidates this new, xeno-chemical-free, completely human-derived biomaterial can be utilized as a supplement to promote epithelial cell behaviour in vitro.