Ex vivo evaluation of intravitreal mesenchymal stromal cell viability using bioluminescence imaging

Background: Bone marrow-derived mesenchymal stromal cell (MSC) therapy is a promising treatment for several degenerative ocular diseases; however, no reproducible method of monitoring these cells into the eye has been established. The aim of this study was to describe successful bioluminescence imaging (BLI) to detect viable luciferase-expressing MSC in the eye. Methods: Human donor MSC in culture were transduced with 50 mu l luciferase lentiviral vector (three viral particles/cell) prior to intraocular injection. Twenty-one right eyes of 21 rabbits were evaluated through BLI after receiving 1 x 10(6) luciferase-expressing MSC intravitreally. Contralateral eyes were injected with vehicle (phosphate-buffered saline (PBS)) and were used as controls. At seven different time points (1 h to 60 days), D-luciferin (40 mg/ml, 300 pl PBS) was injected in subsets of six enucleated eyes for evaluation of radiance decay through BLI analysis. CD90 and CD73 immunofluorescence was studied in selected eyes. Results: Eyes injected with MSC showed high BLI radiance immediately after D-luciferin injection and progressive decay until 60 days. Mean BLI radiance measures from eyes with luciferase-expressing MSC were significantly higher than controls from 8 h to 30 days. At the thirtieth day, positive CD90- and CD73-expressing cells were observed only in the vitreous cavity of eyes injected with MSC. Conclusions: Viable MSC were identified in the vitreous cavity 1 month after a single injection. Our results confirmed BLI as a useful and reliable method to detect MSC injected into the eye globe.