Ciencias Exactas y Ciencias de la Salud

Lack of proliferative capacity of corneal endothelial cells (CEC) is associated with loss of corneal transparency, compromising visual acuity. Corneal transplantation is required to restore vision; however, the donor shortage worldwide limits this strategy. Tissue engineering is a promising approach that lacks standardization methods, making the transition from bench to bedside complex. This study sets parameters for optimal engineered construct production for endothelium regeneration based on Collagen Vitrigel Membranes (CVM) and CEC. We evaluated i)Biomaterial characterization, ii)Primary cell culture standardization Rabbit CEC (RCEC), and iii)Tissue engineering cell proliferation assay, NR (Neutral Red). This project was conducted through 3 stages: i) CVM for production, collagen concentration; 1x,2x, and 3x were tested; material transmittance, cell proliferation, cell adhesion, and degradability membrane assay. ii) For procurement of cornea (enucleation vs. cornea-scleral ring segment) and RCEC culture expansion (Opti-MEM vs. MEM) were evaluated for optimization iii) CMV-CEC engineered corneal endothelium construct; different cell densities (2500, 5000, and 7000 cells/mm2) were seeded in 2x CMV over 5 days. NR assay was performed to evaluate cell growth concentration. Paired and unpaired t-tests were performed to evaluate the significant difference between the cell growth concentrations during time. Results described per stages as previously described: i) CVM production using 2x shows a transmittance above 95% and better cell adherence and proliferation than 1x and 3x. A centrifugal method is a good way to study cell-attached populations, which allows us to test the cell adhesion capacity of the membrane. ii) For RCEC isolation, enucleation is a better method for sample isolation than cornea-scleral ring. Cell circularity is higher cultured with MEM medium than with OptiMEM. Moreover, ~1,100 cells/mm2 is the minimum to guarantee the highest cell density monolayer with a hexagonal pattern. iii) No significant difference has been shown in cell proliferation with and without CVM (p>0.05). There are significant differences in cell loss from day 2 to 3 for all cell densities (p= 0.00388, p=0.00404, p= 0.04371); however, middle density shows better cell proliferation behavior. In conclusion, for optimal tissue-engineered corneal endothelium, seed on CMV 2x of 5,000 cells/mm2 with OptiMEM for 2 days to guarantee hexagonal cells above 22,100 cellsmm2. These findings contribute by setting the parameters to produce a construct that could be used for transplantation for future pre-clinical models.

Ciencias Exactas y Ciencias de la Salud

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