Background The harvesting of corneal endothelial cells (CECs) has received special attention given its potential as therapy for corneal blindness. The main challenges to overcome for this purpose are related to the culture media formulation, cellular density at the primary isolation, and the number of passages in which CECs can retain their functional characteristics. The alternance of different media formulations to harvest CECs has an impact on the cellular yield and morphology. We herein

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... yzed eight different sequences of growth factor-supplemented proliferative (P) and non-supplemented resting (R) media upon passages to find the optimal P/R culture media sequence in regards of cell yield, morphology, procollagen I production, ATPase function, and the expression of ZO-1 and ATPase. Results PRPR and PRRR sequences showed the higher cell yield and hexagonal morphology rate. CECs cultured in the PRRR sequence produced procollagen I, showed Na/K-ATPase function, and expression of ZO-1 and Na/K-ATPase by immunocytochemistry. Our study sets a culture approach to guarantee CECs expansion, as well as functionality for their potential use in tissue engineering and in vivo analyses. Conclusions Alternation of P and R culture media improves CECs culture. PRRR sequence demonstrated to be effective and for CECs proliferation lowering the cost implied in PRPR sequences. We discarded the use of pituitary extract and ROCK inhibitors as essential for CECs proliferation.