A validated porcine corneal organ culture model to study the limbal response to corneal epithelial injury.

: Limbal epithelial stem cells are required for the maintenance and repair of the cornea epithelial surface. The difficulty in obtaining human corneal tissue for research purposes means that animal models for studying the corneal and limbal epithelium are extremely useful. In particular, in organ culture systems, animal models can enable the study of limbal epithelial stem cells in the context of their native niche. The similarities of the porcine limbal microanatomy and stem cell marker expression make it an attractive model, however, functional analysis of the limbal epithelial cell population is needed to validate the use of the tissue. Single cell clonal analysis revealed that holoclone-generating cells were enriched in the limbus as compared with the central cornea (38.3% vs 8.3%) and that label-retaining cells were also enriched in the limbus and compared with the central cornea (44.7 ± 6.4 vs 4.7 ± 1.5). Furthermore, it was demonstrated that in a 3D-printed organ culture system, porcine tissue was capable of maintaining and healing the corneal epithelium. Ki67 staining of corneal sections revealed a proliferative response in the limbal basal epithelium that subsequently resolved, in response to central epithelial wounding. Therefore, the authors present a comprehensively validated model system which will be a powerful tool in studying the interactions between limbal niche factors and limbal epithelial stem cell fate.