Trypsin for Dissociation of Limbal Cells for Engineering of Grafts May Induce DNA Strand Breaks in the Harvested Cells

Aim: Cultures for engineering of transplantable limbal epithelial grafts for treatment of ocular surface disorders may be initiated using dissociation of limbal epithelial cells by trypsin-EDTA or dispase or by a sequential incubation with these enzymes. The safety of such procedures is debated, and in the present study we examined levels of DNA damage in cells dissociated by a commonly used concentration of trypsin. Limbal samples subjected to the dissociation procedure were subsequently cultivated and monitored for outgrowth of cells. Methods: Corneo-limbal rings were retrieved after transplant surgery, divided into samples measuring approx. 2×2 mm (n=32), and incubated in 0.05% trypsin-EDTA for one or three hours in either 250 μl or in 3 ml of the enzyme solution at 37 °C. DNA damage (strand breaks plus alkali-labile sites) was assessed using single cell gel electrophoresis (Comet assay) and evaluation of tail intensity (TI). Outgrowths from the cultivated samples were monitored by phase contrast microscopy and cells were subjected to Hoechst. Results: Noticeable levels of DNA damage were seen regardless of incubation time and volume of enzyme solution. There was a trend towards increased levels of damage in cells when using 3 ml compared to values recorded in cells dissociated in 250 μl of the enzyme solution. Outgrowth of cells was observed from all of the 32 cultivated samples. Conclusion: Dissociation of human limbal epithelial cells by a commonly used concentration of trypsin-EDTA may induce evident DNA damage in the cell population destined for graft production. The current methods for cell dissociation should be examined more closely for induction of damage to essential molecular constituents of the cells including to the stem cell population. Procedural steps and components of the ex vivo system that may reduce such damage and/or facilitate repair should be identified.