The vision impairments suffered by millions of people worldwide and the shortage of corneal donors show the need of substitutes that mimic native tissue to promote cell growth and subsequent tissue regeneration. The current study focused on the in vitro assessment of protein-based biomaterials that could be a potential source for corneal scaffolds. Collagen, soy protein isolate (SPI), and gelatin films cross-linked with lactose or citric acid were prepared and physicochemical, transmittance, and degradation measurements were carried out. In vitro cytotoxicity, cell adhesion, and migration studies were performed with human corneal epithelial (HCE) cells and 3T3 fibroblasts for the films’ cytocompatibility assessment. Transmittance values met the cornea’s needs, and the degradation profile revealed a progressive biomaterials’ decomposition in enzymatic and hydrolytic assays. Cell viability at 72 h was above 70% when exposed to SPI and gelatin films. Live/dead assays and scanning electron microscopy (SEM) analysis demonstrated the adhesion of both cell types to the films, with a similar arrangement to that observed in controls. Besides, both cell lines were able to proliferate and migrate over the films. Without ruling out any material, the appropriate optical and biological properties shown by lactose-crosslinked gelatin film highlight its potential for corneal bioengineering.

The transplantation of expansions of limbal epithelial stem cells (LESC) remains one of the most efficient therapies for the treatment of limbal stem cell deficiency (LSCD) to date. However, the available donor corneas are scarce, and the corneas conserved for long time, under hypothermic conditions (after 7 days) or in culture (more than 28 days), are usually discarded due to poor viability of the endothelial cells. To establish an objective criterion for the utilisation or discarding of corneas as a source of LESC, we characterized, by immunohistochemistry analysis, donor corneas conserved in different conditions and for different periods of time. We also studied the potency of LESCs isolated from these corneas and maintained in culture up to 3 cell passages. We hoped that the study of markers of LESCs present in both the corneoscleral histological sections and the cell cultures would show the adequacy of the methods used for cell isolation and how fit the LESC enrichment of the obtained cell populations to be expanded was. Thus, the expressions of markers of the cells residing in the human limbal and corneal epithelium (cytokeratin CK15 and CK12, vimentin, Collagen VII, p63α, ABCG2, Ki67, Integrin β4, ZO1, and melan A) were analysed in sections of corneoscleral tissues conserved in hypothermic conditions for 2-9 days with post-mortem time (pmt) < 8 h or for 1 day with pmt > 16 h, and in sclerocorneal rims maintained in an organ culture medium for 29 days. Cell populations isolated from donor corneoscleral tissues were also assessed based on these markers to verify the adequacy of isolation methods and the potential of expanding LESCs from these tissues. Positivity for several putative stem cell markers such as CK15 and p63α was detected in all corneoscleral tissues, although a decrease was recorded in the ones conserved for longer times. The barrier function and the ability to adhere to the extracellular matrix were maintained in all the analysed tissues. In limbal epithelial cell cultures, a simultaneous decrease in the melan A melanocyte marker and the putative stem cell markers was detected, suggesting a close relationship between the melanocytes and the limbal stem cells of the niche. Holoclones stained with putative stem cell markers were obtained from long-term, hypothermic, stored sclerocorneal rims. The results showed that the remaining sclerocorneal rims after corneal transplantation, which were conserved under hypothermic conditions for up to 7 days and would have been discarded at a first glance, still maintained their potential as a source of LESC cultures.

The objective of this study was to develop a reliable, cost-effective, and rapid in vitro model employing real-time PCR to assess inflammatory responses in hydrogel-based systems, and to comparatively evaluate the anti-inflammatory efficacy of human serum (HS), serum derived from plasma rich in growth factors (sPRGF), and human amniotic membrane extracts (HAMe) incorporated into gelatin-based hydrogels. An in vitro model of corneal inflammation was established by quantifying IL-1β expression via qPCR in TNFα-stimulated SV-40 immortalised human corneal epithelial (HCE) cells. Hydrogels functionalised with HS, sPRGF, or HAMe sourced from proximal, medial, distal, or pooled amniotic regions were evaluated for their anti-inflammatory potential. In vivo validation was conducted in a rabbit anterior stromal keratectomy model, assessing epithelial wound closure and clinical signs of irritation following application of unmodified hydrogels or hydrogels functionalised with autologous serum (AS) or HAMe. In vitro, hydrogels incorporating HS, followed by sPRGF and pooled HAMe, significantly attenuated IL-1β expression, whereas unmodified hydrogels exacerbated the inflammatory response; region-specific HAMe hydrogels demonstrated inconsistent effects. In vivo, AS-functionalised hydrogels facilitated complete re-epithelialisation by day 7 and achieved the lowest irritation scores, indicating both therapeutic efficacy and high tolerability. All hydrogel formulations were found to be biocompatible throughout the study period. These findings underscore the significant anti-inflammatory and regenerative potential of gelatin-based hydrogels functionalised with blood-derived products and support their development as bioactive platforms for ocular surface therapy. Furthermore, the in vitro model provides a robust preclinical screening tool, contributing to the refinement and reduction of animal use in biomedical research.