Extended Expansion of High Quality Corneal Stromal Keratocytes on xeno free Cell RevTM Kera ExHQ for therapeutic applications

Background & Aim The corneal stromal keratocytes are important to regenerate collagen structures in a proper alignment within avascular cornea. Incorrect collagen fiber alignment leads to cloudiness which in turn can cause corneal blindness, one of the most common blindness worldwide. However, expansion of human primary corneal keratocytes is limited within the first few passages; thereby, resulting into a major barrier to corneal research in tissue regeneration, drug screening, and toxicity testing. Methods, Results & Conclusion Here, we present a xeno free solution that expands at least 15 more passages in our Kera EXHQ Cell RevTM without losing corneal keratocyte characteristics. Importantly, advanced P15 keratocytes exhibited dendritic shapes, and expressed robust levels of keratocyte phenotypic markers such as ALDH1A1, CD34, and notably essential proteoglycans of cornea like lumican as well as collagen production, at parred to freshly isolated primary stromal keratocytes (P2). They did not express alpha-smooth muscle, a marker of stromal keratocytes conversion to myoblasts. Both immunofluorescence and qPCR data showed consistent results. Of interest, they survive and proliferate better in a mimicked natural habitat, aligned PCL-collagen fiber scaffolds than non-aligned scaffolds, suggesting that their corneal stromal functional properties remain faithful. On the other hand, corneal keratocytes stopped expanding in recommended stromal growth media by P4. These results indicate that our media formulae achieve extended high quality stromal keratocytes, removing a major bottleneck of research & development in corneal tissue maintenance and regeneration.