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Sayena Jabbehdari, Ghasem Yazdanpanah, Ilham Putra, Khandaker N Anwar, Xiang Shen, Behnam Rabiee, Medi Eslani, Peiman Hematti, Ali R Djalilian; Efficient expansion of corneal mesenchymal stromal cells with preserved therapeutic effects. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4112.
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© ARVO (1962-2015); The Authors (2016-present)
Mesenchymal Stromal/Stem cell (MSC) therapy is a promising approach for the treatment of ocular surface disorders. Production of sufficient number of MSCs with preserved therapeutic effects is crucial for clinical applications. We investigated the explant-method for efficient derivation of MSCs from human cadaveric corneas followed by cell expansion to reach high number of cells for conducting clinical trials using the same batch of cells.
Thirty-four human cadaver corneas (50-65 years) that had been stored in Optisol (average of 25.5±4.5 days) were used to derive MSCs by the explant method. After removing the Descemet’s membrane, the central 7mm of the cornea was trephined and the resultant corneoscleral rims were split into 4, 6, or 8 equal pieces. Each explant was placed epithelial side up in a plate and was covered with MEM-α plus 10% fetal bovine serum. After adequate confluency (80-90%) the explants were removed and the cells were detached and subcultured into T175 flasks. Following 80-90% confluency, the cells were passaged with a ratio of 1 to 10 up to 4 times. Using a scratch assay, the in vitro effects of MSC conditioned media (CM) was examined while the in vivo effects of MSCs was assessed using a mouse epithelial wound healing model.
The ideal explants were found to be the 1/4 sizes since the smaller explants had a higher likelihood of detachment from the plate. All of the quarter size explants gave rise to cell outgrowth by 7 days. While the initial outgrowth included a mixture of both epithelial and mesenchymal cells, by the end of passage (P)1 only MSCs remained. By the end of P1 the total MSC yield from each quarter explant was approximately 3-4x106(80-90% confluency in T175 flask). The MSCs maintained their normal spindle morphology from P2 to P5. Both P2 and P5 MSC CM induced faster wound closure in a scratch assay of human corneal epithelial cells compared to control CM (82.2±6.1,P2; 86.3±7.2%,P5 vs 49.1±9.3%, p<0.05). In vivo, following 2mm epithelial debridement wounds, the ratio of healed area was significantly higher in mice subconjunctivally injected with P5-MSCs compared to controls (67.7±34.1% vs 42.2±21.6%, P<0.05).
Human cadaveric corneas can provide a high yield of MSCs using the explant method while maintaining their therapeutic effects. Cornea-derived MSCs are a promising source for cell-therapy applications in ocular surface diseases.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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