A Complete Retina Patch to Treat Irreversible Retinal Injuries

Abstract

Purpose : To develop a cellular therapy for irreversible eye injuries by co-grafting polarized retinal pigment epithelium (RPE) supported by an artificial Bruch’s membrane (ultrathin parylene), together with sheets of retina organoids (RO) that will mature into photoreceptors and other retinal cells.

Methods : The CSC14 (AIVITA Biomedical; NIH registry 0284) and the H9 derived CRX-GFP expressing hESC cell lines (Collin et al. 2016, Stem Cells 34:311–321) were used to differentiate retina organoids (modified after the protocol of Zhong et al 2014). hESCs at 80% confluency are aggregated to embryoid bodies (EBs). From day 21-38, eye field (“horseshoe”) structures are cut out and the resulting retinal spheres (containing retinal progenitors and RPE) are maintained in suspension culture (d30 – 93). For the preparation of RPE implants, H9 hESC were cultured and spontaneously differentiated to RPE cells. The hESC-RPE obtained from the primary cultures were isolated and further cultured. Passage 3 of hESC-RPE were seeded and cultured around 30 days on ultrathin parylene substrates to produce polarized monolayer RPE implants. For the preparation of hESC-RPE+RO co-grafts, RO sheets were dissected out and combined with RPE implants with the help of suitable bio-adhesive agents. Matrigel, gelatin and alginate were tested in 7 different embedding trials. The co-grafts were surgically placed into the subretinal (SR) space of immunodeficient Royal College of Surgeon (RCS) rats (46-55 day old males and females, n=5). Implant placement was evaluated by Optical Coherence Tomography (OCT). Histological assessments were performed using H&E staining and immunohistochemistry.

Results : Retina co-grafts can be prepared by combining RPE implants and RO sheets. Alginate appeared to have the best adhesive properties and provided gentle encapsulation. Successful surgical implantation of the co-grafts into the SR space of immunodeficient RCS rats is demonstrated by OCT imaging. Histological evaluation confirmed the survival of the co-grafts and maintenance of its laminar structure until the end of the experimental period (up to 1 month post-implantation).

Conclusions : The study supports our ability to combine RPE+RO into a co-graft that can be surgically placed into the rat’s SR area. This novel tissue engineering technique can enable replacement of an area of a diseased retina.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.