RESUMEN
Current research on healthy corneal stromal cells will typically use primary cells as they are the most representative of in vivo behaviour. Primary cells are normally isolated from the limbus of discarded donor peripheral corneal tissue left over from transplantation (due to its relative abundance). Therefore, the central part of the cornea is less used in research as this tissue is usually used for transplantation. In some cases, although rare, the whole cornea, can become available for research. It is important to keep in mind that these corneas often have longer storage time, but the use of the central tissue for research is even more interesting, as knowing what cells are being transplanted into recipients would be highly relevant. To this end, stromal cells were extracted from both the limbus and central button of healthy corneas donated for research. This allowed for important comparison between central and limbal cells in culture. Of interest here was the extraction method of stromal cells from the donor tissue. The two most common methods of extraction are enzyme digestion and explant migration. However, no work has been done to understand how each method relatively affects the extracted cells. The extraction method and location from which stromal cells are harvested seems to have a significant effect on the cell adherence, survival, and gene expression of the stromal cells in culture. Enzyme digested cells showed that limbal and central cells had different gene expressions prior to culture, with gene such as ALDH3A1 being much more expressed in limbal cells. Enzyme digesting the limbal ring seems to yield the hardiest populations of stromal cells, a desirable trait in the culture of primary cells.
Asunto(s)
Separación Celular/métodos , Queratocitos de la Córnea/fisiología , Sustancia Propia/citología , Limbo de la Córnea/citología , Técnicas de Cultivo de Célula , Supervivencia Celular/fisiología , Medio de Cultivo Libre de Suero , Proteínas del Citoesqueleto/genética , Regulación de la Expresión Génica/fisiología , Humanos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Donantes de TejidosRESUMEN
AIM: Inflammation is a process that underlies sight-threatening ocular surface diseases, and gene supplementation with the plasmid that encodes for p-IL10 will allow the sustained de novo synthesis of the cytokine to occur in corneal cells, and provide a long-term anti-inflammatory effect. This work describes the development of solid lipid nanoparticle systems for the delivery of p-IL10 to transfect the cornea. RESULTS: In vitro, vectors showed suitable features as nonviral vectors (size, ζ-potential, DNA binding, protection and release), and they were able to enter and transfect human corneal epithelial cells. Ex vivo, the vectors were found to transfect the epithelium, the stroma and the endothelium in rabbit corneal explants. Distribution of gene expression within the cell layers of the cornea depended on the composition of the four vectors evaluated. CONCLUSION: Solid lipid nanoparticle-based vectors are promising gene delivery systems for corneal diseases, including inflammation.