Cultivated limbal mesenchymal stromal cells (L-MSC): a new opportunity for eye banks?

Abstract

Purpose : To educate those working in eye banks about the properties and clinical potential of mesenchymal stromal cell cultures established from donor corneal-limbus (L-MSC). Guidance is provided concerning suitability of sourced tissue (hypothermic vs. normothermic storage), cell isolation methods and choice of vehicle for delivering L-MSC to the ocular surface.

Methods : We grow L-MSC in DMEM with 10% fetal bovine serum (FBS) and 2 mM L-glutamine. For cultures established from tissue digests, tissue samples are minced using a scalpel and incubated with 1 mg/mL collagenase type I for 12-48 hr before seeding into culture. For cultures established from explants, a commercial tissue attachment reagent (Attachment Factor; Life Technologies) has been compared to serum-treated tissue culture plastic and collagen gels (1 mg/mL prepared from medical grade type I porcine collagen; Nitta-Gelatin, Japan). Expanded stocks of L-MSC are stored in liquid nitrogen while suspended in FBS with 10% DMSO. Potential vehicles for L-MSC implantation that have been tested include amniotic membrane, contact lenses manufactured from silicone hydrogels and scleral lenses manufactured from rigid gas permeable (RGP) materials. A rabbit corneal wound model has been used.

Results : L-MSC display a typical MSC phenotype when examined by flow cytometry (<5% CD34/CD45; >90% CD73/CD90/CD105). No difference is observed between cultures established from tissue digests or tissue explants. Collagen gels provide the most reliable method for initiating cultures from tissue explants. Source tissue may be stored under either hypothermic or normothermic conditions. Cultures remain viable when stored in liquid nitrogen. L-MSC attachment to silicone hydrogel lenses is poor, with best results being observed using Balafilcon A or Lotrafilcon. Optimal L-MSC attachment and growth is acheived on amniotic membrane or scleral lenses prepared from Hexafocon A or Tisilfocon A. L-MSC increase re-epithelialization of wounded rabbit corneas.

Conclusions : Banked cultures of L-MSC can be readily established from donor eye tissue using a variety of methods. Amniotic membrane and lenses prepared from RGP materials provide best options as a potential vehicle for L-MSC implantation. Our preclinical data encourages use of L-MSC as a treatment for persistent epithelial defects.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.