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Gilles Thuret, Emmanuel CROUZET, Chantal PERRACHE, Thibaud GARCIN, Marie-Caroline TRONE, Fabien FOREST, Philippe Gain, Zhiguo HE; The OBSERV platform (Ophthalmic Bioreactor Specialized in Experimental Research & Valorization): simulation of a DMEK. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4143. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Our university lab BiiGC patented 2 versions of a bioreactor (BR): one for long-term eyebanking (in the process of industrialization), the other for preclinical experimentations, called OBSERV and supported by the French Agence Nationale de Sécurité du Médicament et des produits de Santé (ANSM). Aim: to reproduce a Descemet membrane endothelial keratoplasty (DMEK) inside the BR.
By restoring IOP and medium renewal, the BR maintained corneal viability over a prolonged period and its transparency allowed characterizing the tissue with existing or customized devices without compromising sterility. A 9mm diameter Descemetorhexis was performed open-sky on the recipient human cornea. An 8mm diameter endothelial grafts was dissected with a no-touch technique and transferred using a soft contact lens. The DMEK-bearing cornea was installed in the BR with an air bubble in the endothelial chamber and endothelium facing down. IOP was set to 21mmHg. The bubble was removed after 3 days. For some experiments an adherence promoting solution was used. Implanted corneas and controls (rhexis alone) were stored for 1 to 4 weeks in a custom-made medium. Monitoring: D-1, D1, W1 to 4: transparency (slit-lamp and customized transparometer), OCT corneal thickness, endothelium/epithelium (specular microscopy). Final test: triple Hoechst/Ethidium/calcein-AM staining to determine endothelial viability (Fig.) and Alizarin red staining.
All DMEKs initially adhered but some of the group without adherence-promoting agents detached 48 hours after unbubbling, although their endothelial cells remained viable. The other grafts remained adherent. Implanted corneas with adherent grafts were thinner than controls. Endothelial cell survived normally and started to migrate outside the donor Descemet onto the bare recipient stroma from W1.
The OBSERV platform is efficient to study the biofunctionality of a DMEK. It will be useful for preclinical assessment of next generation bioengineered endothelial grafts.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
Triple Hoechst-Ethidium-Calcein-AM staining of a DMEK simulated in the corneal bioreactor after 2 weeks. The uniform central green staining indicated excellent endothelial cell survival on the graft.
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