Fluorescence-Based Biochemical Evaluation of Endothelium

(alamarBlue assay)

Research Scientist Sung Lee was awarded an Eye Bank Association of America Pilot Research Grant for his study, “Evaluation of alamarBlue as an ‘in-situ’ Endothelial Cell Viability Assay in Preloaded DMEK grafts.”

In addition to the current microscopic approach, Lions Gift of Sight research staff have developed a new way to evaluate transplant DMEK grafts. Using a non-toxic, highly sensitive cell viability dye, alamarBlue (Fig 1) and a perfusion system, we are able to measure the total biochemical output of viable endothelial cells of the entire DMEK grafts while they are kept inside the DMEK storage/injector device (Fig 2 video).

Utilizing this method in conjunction with existing methods of endothelial cell analysis (spectral microscopy and slit-lamp evaluation) can offer a further comprehensive assessment of transplant corneal grafts. The video visualizes the alamarBlue signal, representing the biochemical readout from viable endothelial cells, flushed out of the modified Jones tube.

alamarBlue Graphic — Fig 1. AlamarBlue is a non-toxic, cell-permeable compound that functions as a cell health indicator. The active ingredient, resazurin, is blue in color and has low fluorescence. As the compound enters living cells, resazurin is reduced to resorufin (conversion of NADH to NAD+) which is a red/pink color with high fluorescence.

Direct In-Situ Viability Assessment of DMEK Endothelial Cells in a Modified Jones Tube

Fig 2. A DMEK graft inside a modified Jones tube was perfused to collect the fluorescent alamarBlue assay product under a blue light illumination. The inset: the bright field image of the graft.