Table 1.
Basic donor demographics of corneas used for small diameter DMEK graft preparation.
Fig 1.
Image collage showing the different steps of preparing three small diameter grafts from one donor cornea.
(A) Local DM separation from the stoma by hydro-dissection using a bent 30G needle. (B, C) Separation of the DM along with the corneal endothelium by a bubble spanning the full diameter of the cornea. (D) Complete detachment of the peripheral DM with its adjacent TM using a hockey stick blade. (E) The anterior remnant was replaced on a soft contact lens. (F, G) Transfer of the soft contact lens holding the DM with its TM to a punch block. (H) Preparation of three small diameter grafts by carefully punching out the grafts using a 4 mm diameter biopsy punch. (J) Schematic representation of the trephination pattern for the three grafts. (K, L) Remaining part of the DM sheet after punching out three 4mm grafts and the three resulting grafts. (M) Light microscopy image of a graft showing the endothelial cells on the graft and a very thin denuded band along the graft edge caused by trephination.
Fig 2.
In vitro surgery performed with the small diameter DMEK graft.
(A) Graft staining with 0.06% trypan blue. (B) Graft loading into a straight DMEK injector. (C) Descemetorhexis performed on a cornea mounted on an artificial anterior chamber. (D) Graft unfolding and positioning in the descemetorhexis area performed in a whole globe. Purple dashed line indicates the outline of the descemetorhexis, and the blue dashed line indicates the position of the 4mm DMEK graft. (E) AS-OCT graft imaging after 30–45 minutes of a fully pressurized anterior chamber.
Fig 3.
Graft viability after preparation.
(A) Light microscopy image of a flattened 4 mm DMEK graft. The insert in the right top corner represents an area on the graft (white square) that may be perceived as a bare area in the overview image but is populated by cells. Please note that areas on the graft that may be perceived as bare areas in are artefacts from the mounting the flat tissue and keeping the graft moist to avoid tissue drying during imaging, which in turn causes some areas to be out of focus. (B) Fluorescence microscopy image of the same graft showing Calcein-AM staining for cell viability. The fluorescence image in (B) shows that all those areas that appeared to be devoid of cells in (A) are indeed covered by viable cells. (C) Higher magnification images from the areas marked by * and ** in overview image (B) to illustrate the viable cells (green) and the corresponding segmentation images (red). Scale bar: 100 μm.
Fig 4.
Light and fluorescence microscopy images of two 4mm DMEK grafts before and after in vitro surgery.
(A, D) Light microscopy images of the grafts before in vitro surgery already showing some areas denuded of cells (red asterisk mark) directly after preparation which is probably related to the low-quality of the corneas ineligible for transplantation that were used for preparation. (B, E) Light microscopy images and (C, F) fluorescence microscopy images of the grafts after in vitro surgery showing again the same areas devoid of cells in addition to some other small areas on the graft that do not show any Calcein-AM fluorescence signal (indicative for the presence of viable cells). Note that one image tile each if missing in (C, F). The graft surface area within these missing image tiles was calculated (0.7% and 1.9%, respectively) and cell viability percentages were corrected for the missing graft area to avoid a potential overestimation.
Fig 5.
Example of in vitro endothelial cell migration from a gel-cultured 4mm graft.
(A) Light microscopy overview collage (50x magnification) of a 4 mm DMEK graft after 17 days in gel culture showing uniform cell migration from all around the graft. (B) Light microscopy image showing central graft endothelium and (C) graft edge (bottom of the image) from where migration was directed as a confluent cellular monolayer. (D, E) Fluorescence microscopy images showing expression of ZO-1 (red signal) counterstained with DAPI (blue signal) in the graft center and in the migrated monolayer. The absence of ZO-1 stained cell borders in the lower part of image 5E can be attributed to the fact that cells in this area reside on the graft and are elevated as compared to the new cell monolayer. Therefore, the cells on the graft have an elevation of about 10 μm (on the Descemet membrane) when compared to the migrated cells on the glass cover slide and therefore appear out of focus. Scale bars: 100 μm.