Corneal opacities are a leading cause of visual impairment that affect 4.2 million people annually. The current treatment is corneal transplantation, which is limited by tissue donor shortages. Corneal engineering aims to develop membranes that function as scaffolds in corneal cell transplantation. Here, we describe a method for producing transplantable corneal constructs based on a collagen vitrigel (CVM) membrane and corneal endothelial cells (CECs). The CVMs were produced using increasing volumes of collagen type I: 1X (2.8 μL/mm2), 2X, and 3X. The vitrification process was performed at 40% relative humidity (RH) and 40 °C using a matryoshka-like system consisting of a shaking-oven harboring a desiccator with a saturated K2CO3 solution. The CVMs were characterized via SEM microscopy, cell adherence, FTIR, and manipulation in an ex vivo model. A pilot transplantation of the CECs/CVM construct in rabbits was also carried out. The thickness of the CVMs was 3.65–7.2 µm. The transparency was superior to a human cornea (92.6% = 1X; 94% = 2X; 89.21% = 3X). SEM microscopy showed a homogenous surface and laminar organization. The cell concentration seeded over the CVM increased threefold with no significant difference between 1X, 2X, and 3X (p = 0.323). The 2X-CVM was suitable for surgical manipulation in the ex vivo model. Constructs using the CECs/2X-CVM promoted corneal transparency restoration.

PurposeCollagen scaffolds are used as tissue engineering tool. The manufacturing methods often lack of reproducibility which limits their application to regenerative medicine. We aimed to set a simple and reproducible method for the production of collagen scaffolds for tissue engineering.MethodsA matryoshka system was built comprising a sealed desiccator containing a saturated K2CO3 solution for a 40% relative humidity (RH) regulation, this was placed inside a 40° C shaking incubator. Collagen gels were cast with a 1:1 ratio of Dulbecco’s Modified Eagle’s medium and 0.5 mg/mL acid collagen solution. Three gel volumes were selected: 2.2 mL (1x), 4.4 mL (2x), and 6.6 mL (3x). Collagen gels were inserted into the system and desiccation was performed over 37 days (6–12 mL was desiccated per cycle). Characterization of the surface, ultrastructure, transparency, composition, and biocompatibility of the gels was performed using optical, 3D confocal, and scanning electron microscopy; spectrophotometry; Fourier-transform infrared spectroscopy; and X-Ray diffraction. ResultsOn day 7, collagen membranes exhibited superficial fibrils of 1.3 µm (SD, ± 0.23 µm), whereas on day 37, a highly organized laminar structure was observed within a smooth homogeneous surface. Lamina density and organization and membrane width (3.65 µm [1x], 4.8 µm [2x], and 7.2 µm [3x]) increased with gel volume. Transmittance ranged from 77% to 99% and increased with wavelength at UV–vi. Gels at 1x and 2x exhibited a 99% transmittance peak at the green wavelength. The population of cells cultured on membranes was increased by threefold within 48 h; moreover, the size of cell populations cultured on 1x membranes increased by 12% compared with the control. ConclusionsThe scaffolds produced by the matrioshka system were biocompatible, non-cytotoxic, and optically transparent. These membranes can be tailored for multiple uses by modifying their thickness with the volume of the gel and its desiccation time.

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.

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 analyzed 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.