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Edgar M. Espana, Marcony R. Santhiago, William J. Dupps, Jr.; Viability of Corneal Endothelial Cells Following a Combined Air/Enzymatic Technique that Facilitates Stroma /Descemet’s Membrane Separation. Invest. Ophthalmol. Vis. Sci. 2011;52(14):793.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the viability of corneal endothelial cells after using a novel enzymatic technique that facilitates air separation of Descemet’s membrane from the corneal stroma and in isolated Descemet’s membrane endothelial cells sheets.
Seven fresh human pairs of corneoscleral donors were mounted on an artificial anterior chamber with Optisol medium filling the endothelial side. A group of eyes (n=7) received an intrastromal injection of 2.5 mg/ml Collagenase type 2 in BSS followed by an injection of air into the stroma after incubation at 37º C for 1 hour. The contralateral eye, without any enzymatic or air treatment, was used as a control to evaluate the effect of collagenase and air injection in endothelial cell viability. All injections were performed using a 27G needle beveled down. Histological examination of the endothelial cells sheets and after stromal/Descemet's membrane separation was performed. Trypan blue cell viability assay was used to study endothelial cells viability in isolated endothelial cells sheets after collagenase incubation and compared to the viability of endothelial cells in the control corneas. The percentage of dead cells was estimated by counting the percentage of blue stained cells in a 20x magnification once isolated Descemet ‘s membrane flat mounts were placed on a glass slide.
Descemet's membrane and the underlying endothelial cells can be reproducibly separated from corneal stroma using a combination of intrastromal collagenase and air injection. Histological examination of the separated Descemet’s membrane and underlying endothelial cells appeared to be of healthy appearance after collagenase incubation and when evlauated as isolated Descemet's membrane sheets. Trypan blue evaluation of the separated Descemet’s flat mounts demonstrated a mean viability of endothelial cells following collagenase incubation of 89.7% (82.1% to 94.1%) compared to a mean viability of 91.2% (87.5% to 92.7%) in the control group. (p= 0.1; not statistically significant).
This technique facilitates the separation of Descemet’s membrane from the stroma without affecting endothelial cells viability. This technique may be useful for DALK and DMEK procedures.
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