Comparative Toxicity of Trypan Blue and Mono-Azo-Red on Human Corneal Endothelial Cells and Clinical Outcomes After Descemet Membrane Endothelial Keratoplasty Using Different Trypan Blue Dyes

Purpose: To characterize early cellular stress responses induced by short-term exposure to commercially available trypan blue (TB) dyes in human corneal endothelial cells (HCEC), to assess whether isolated Mono-Azo-Red derivatives reproduce these effects, and to evaluate whether in vitro findings are reflected in clinical outcomes after Descemet membrane endothelial keratoplasty (DMEK). Methods: Human corneal endothelial cells (HCEC-B4G12) were exposed for 30 seconds to 4 TB solutions (RS-Blue, VisionBlue, Vioron, AJL Blue) or to Mono-Azo-Red at 3 concentrations (0.6%, 2.4%, 3.6%). Metabolic activity (ATP-based assay), proliferation (BrdU), and membrane integrity (LIVE/DEAD) were assessed after a 1-hour recovery period. In addition, a single-center retrospective clinical cohort of 338 DMEK procedures performed with Vioron (n = 148) or VisionBlue (n = 190) was analyzed over 12 months. Results: All tested TB formulations induced a significant reduction in ATP-dependent luminescence compared with balanced salt solution, indicating early metabolic stress, whereas proliferation and membrane integrity remained unaffected. In contrast, isolated Mono-Azo-Red derivatives did not induce significant metabolic, proliferative, or viability changes at any tested concentration. In the clinical cohort, DMEK performed with either dye showed comparable postoperative outcomes. Conclusions: Short-term exposure to TB induces a reproducible, sublethal metabolic stress response in HCEC without evidence of cell death or impaired proliferation. This effect is not replicated by isolated Mono-Azo-Red derivatives, suggesting that TB-related cellular stress cannot be attributed to this impurity alone. In the exploratory clinical cohort, no clinically relevant differences in postoperative outcome were observed between the 2 TB dyes under routine surgical conditions.