Pre-Clinical Evaluation of the Safety of Subconjunctivally Injected Bone Marrow Mesenchymal Stem Cells in Rodents and Rabbits

Abstract

Purpose : The anti-inflammatory and wound healing effects of Bone Marrow Mesenchymal Stem Cells (BM-MSCs) have been reported in many studies. However, the clinical application of BM-MSCs to the eye and cornea has been limited. In preparation for a phase I safety and dose-determination study, we evaluated the safety of subconjuntivally injected cryopreserved Human and Rabbit BM-MSCs in different animal models.

Methods : Human BM-MSCs (obtained and characterized under approved IRB at University of Wisconsin) and Rabbit BM-MSCs (Cyagen) were cultured in alpha-MEM plus 10% FBS or serum free media (Rooster Basal-MSC, Rooster Bio) then cryopreserved in freezing medium containing 5% DMSO. To assess viability, cryopreserved BM-MSCs were thawed and aspirated/injected through 27 and 30 gauge needles then stained with trypan blue and calcein AM/propidium iodide. In vivo studies were done in C57b/l mice, Sprague Dawley rats and New Zealand white rabbits. Both unwounded mice and those with 2mm debridement wounds were injected subconjunctivally with 40K human BM-MSCs or freezing medium. Unwounded rats were injected with 100K human BM-MSCs and unwounded rabbits were injected with three different doses of human and rabbit BM-MSCs: 1, 3 and 6 million cells. In vivo live fluorescence microscopy was used to image the injected labeled BM-MSCs in live animals. Histology was performed on all of the eyes.

Results : BM-MSCs remained viable after injection through 27g (87.26% ± 3.1%) and 30g (87.44% ± 3.09%) needles. None of the animals developed any infections, intraocular inflammation, or cornea or conjunctival scarring throughout the follow-up period. The rabbits injected with human BM-MSCs demonstrated mild conjunctival inflammation at the site of injection which resolved without any treatment (presumed to be due to xenogenic reaction since rabbit BM-MSCs did not elicit any inflammation). In vivo microscopy and histologic examination confirmed survival of MSCs without any pathological changes anywhere in the eye.

Conclusions : Cryopreserved BM-MSCs remain viable after thawing and immediate injection through small gauge needle. Subconjunctival administration of BM-MSCs appears safe in animal studies done in 3 different species while demonstrating effectiveness in promoting murine wound healing. Altogether, these results provide pre-clinical safety data to support a phase I safety study in humans.

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