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Updated: May 24, 2025
Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty
Published on: September 17, 2014
Optimized laboratory techniques for assessing the quality of pre-stripped DMEK grafts
Tomy Sagnial1, Sandrine Ninotta1,2, Paul Goin1,3
1Laboratory of Biology, Engineering and Imaging for Ophthalmology (BiiO), Faculty of Medicine, Health Innovation Campus, Jean Monnet University, Saint-Étienne, France.
Abstract:
This study addressed limitations in calcein-AM-based endothelial viability assays, specifically focusing on pre-stripped DMEK grafts. Key challenges included the suboptimal calcein staining and the incompatibility of the viability assay with subsequent immunofluorescence (IF). Using human corneal grafts, we employed two strategies to optimize calcein staining. Firstly, we improved calcein staining in corneal endothelium by adjusting calcein-AM concentration and diluent, resulting in a threefold increase in fluorescence intensity with 4 µM calcein in Opti-MEM compared to the conventional 2 µM calcein in PBS. Secondly, introducing the trypan blue (TB) post-viability assay greatly reduced non-specific fluorescence, enhancing the contrast of calcein staining. This improvement significantly and importantly decreased both inter-operator's variability and the time required for viability counting. For the subsequent double IF, an extensive wash is recommended on the fixed and permeabilized graft after the viability assay, which was carried out using Hoechst-Calcein (HC) labeling. The simple technical tips outlined in this study are not only effective for pre-stripped DMEK grafts but may also prove beneficial for other types of corneal grafts, such as PK and DSAEK.
Insights
Optimized endothelial viability assays for corneal grafts improve calcein staining and reduce variability. New methods enhance fluorescence and compatibility with immunofluorescence, benefiting DMEK, PK, and DSAEK graft assessment.
Area of Science:
- Ophthalmology
- Cell Biology
- Transplantation Science
Background:
- Calcein-AM assays for endothelial cell viability have limitations, particularly with pre-stripped Descemet membrane endothelial keratoplasty (DMEK) grafts.
- Suboptimal staining and incompatibility with immunofluorescence (IF) hinder accurate assessment.
Purpose of the Study:
- To optimize calcein-AM staining for endothelial viability assays in human corneal grafts.
- To improve assay compatibility with subsequent immunofluorescence.
- To reduce variability and time in viability counting.
Main Methods:
- Adjusted calcein-AM concentration and diluent (4 µM in Opti-MEM) for enhanced corneal endothelium staining.
- Incorporated post-assay trypan blue (TB) staining to reduce non-specific fluorescence and improve contrast.
- Developed an extensive wash protocol for fixed and permeabilized grafts after Hoechst-Calcein (HC) labeling for subsequent IF.
Main Results:
- Achieved a threefold increase in fluorescence intensity compared to conventional methods.
- Significantly decreased inter-operator variability and time required for viability assessment.
- Successfully enabled subsequent double immunofluorescence after viability assay.
Conclusions:
- Optimized calcein-AM staining and trypan blue counterstaining enhance endothelial viability assessment in corneal grafts.
- The improved method is effective for pre-stripped DMEK grafts and potentially applicable to PK and DSAEK.
- These technical improvements facilitate more reliable and efficient graft evaluation.
