Experimental design of a culture approach for corneal endothelial cells of New Zealand white rabbit

Abstract

The harvesting of corneal endothelial cells (CEC) has received special attention due to its potential as a therapy for corneal blindness. The main challenges are related to the culture media formulation, cellular density at the primary isolation, and the number of passages in which CEC can retain their functional characteristics. To alternate different media formulations to harvest CEC has an impact on the cellular yield and morphology. Therefore, we analyzed four different sequences of growth factor-supplemented Stimulatory (S) and non-supplemented Quiescent (Q) media, upon passages to find the optimal S-Q culture sequence. We assessed cell yield, morphology, procollagen I production, Na + /K + -ATPase function, and the expression of ZO-1 and Na + /K + -ATPase. Our results show SQSQ and SQQQ sequences with a balance between an improved cell yield and hexagonal morphology rate. CEC cultured in the SQQQ sequence produced procollagen I, showed Na + /K + -ATPase function, and expression of ZO-1 and Na + /K + -ATPase. Our study sets a culture approach to guarantee CEC expansion, as well as functionality for their potential use in tissue engineering and in vivo analyses. Thus, the alternation of S and Q media improves CEC culture. SQQQ sequence demonstrated CEC proliferation and lower the cost implied in SQSQ sequences. We discarded the use of pituitary extract and ROCK inhibitors as essential for CEC proliferation.

Corneal tissue engineering: From research to industry, quality of life impact, and Latin American ophthalmologists' perspectives

“…19 Only the transition from blindness to sight incurs expenses, including the cost of obtaining cells from one corneal donor and generating the necessary cells for up to ten engineered corneal endothelium constructs. 20 We used a hypothetical cohort of 1000 blind patients aged 35 years and ran the program for 40 years to calculate life expectancy. A discount rate of 3.5% over a 10-year period was used in the computations.…”