The cultivation of endothelial cells ex vivo in the laboratory and their subsequent injection into the anterior chamber to achieve corneal re-endothelization has been the subject of numerous research works since the functionalism of the endothelial monolayer was discovered in the last century^1-4. Initially, the difficulties in achieving correct cell isolation and optimum conditions for its growth and long-term survival were raised. The classic concept of absence of mitosis that physiologically is attributed to the human corneal endothelium has been replaced by the possibility that there are peripheral endothelial cells with characteristics of stem cells, which help maintain endothelial cell homeostasis. Under appropriate laboratory conditions, endothelial cell lines have already been maintained and proliferated in culture media and transplanted, both in animals and humans⁵. The main problems such as cellular senescence, poor proliferative capacity and fibroblastic transformation during culture have been solved by using various biochemical strategies adding different substances to culture media^6-8.

Currently, the technique of cultivation and injection in the anterior chamber of human endothelial cell cultures is under intense research and development work by the Kinoshita group in Kyoto, Japan. In 2009, the use of a Rho kinase inhibitor (ROCK), Y-27632 (ripasudil, Glanantec^®) was described as an agent capable of increasing the proliferative capacity of cultured endothelial cells⁹. Subsequently it has been shown that ROCK inhibitors use both the cyclin D and p27 pathway through PI-3-kinase signaling to promote endothelial cell proliferation¹⁰. Although the first studies attempted to use ripasudil in eye drops to stimulate cell proliferation in vivo¹¹, in the latest Kinoshita studies, the ROCK inhibitor is used as a supplement to the culture medium to facilitate cell proliferation and stimulate adhesion of the transplanted cells. In addition, to facilitate the growth of endothelial cells, a culture medium has been developed which is conditioned with stem cells derived from bone marrow obtained under GMP conditions. This medium from this undifferentiated cell line counteracts the effect of decrease in endothelial density associated with the migration that is thought to be produced by cellular senescence. To avoid the fibroblastic transformation that occurs in endothelial cultures, the TGF-β pathway is inhibited, a factor that induces this transformation, maintaining the cell phenotype with growth inhibition by contact¹².

Two methods have been developed for isolated endothelial cell transplantation. In the first one, it was used in animal studies and a transport plate was used in which the cultured cells were sedimented. In this method, the main difficulty was the technique of introducing the flexible sheet in the anterior chamber. Both amniotic membrane and various type I or IV collagen sheets were used^13,14. In the second, much simpler method, a suspension of cultured endothelial cells is used and injected directly into the anterior chamber (Figure 1).

The main problem that arises with direct injection in the anterior chamber is the presence of a continuous outflow of aqueous humor that would produce a cellular wash towards the trabeculum. A method has been described by magnetic guidance using iron powder incorporated in cultured cells¹⁵ but this has not yet been applied in humans. The Kinoshita group has shown that the use of the ROCK inhibitor improves the adhesive properties of the endothelial cells, obtaining in the studies carried out both in rabbits (Figure 2) and in primates (Figure 3) a stable endothelial monolayer and a restoration of the corneal transparency by injecting the cell suspension enriched with said inhibitor.

Currently the technique developed by the Kinoshita group is under study in a human clinical trial at the University of Kyoto (clinical trial registry: UMIN000012534). Although satisfactory preliminary results have been presented in the first patients treated¹¹, the first results of the study have not yet been published. According to Kinoshita, a young cornea donor could be used to treat 243 patients, which would drastically reduce the needs of donor corneas for the treatment of endothelial pathology. Although the technique of cell culture and injection is at the dawn of its development, probably the improvements that are going to be implemented in the culture media¹⁶ and the greater knowledge of endothelial physiology post-transplant thanks to the experience accumulated with endothelial keratoplasties will allow to lay the foundations of a very simple technique and with promising results.