Abstract
Purpose :
More than 1000 transplantation of ex vivo cultured limbal epithelial cells (LECs) have been performed worldwide. Strict regulations promote centralization of culture units and the treatment remains limited to a few centres of expertise, thus, definition of reliable transportation strategies is vitally important. The aim of the study was to optimise the transportation of uncultured limbal biopsies (LBs) and cultured limbal epithelial cells (LECs).
Methods :
Cadaveric tissue was obtained from 3 donors with ethics consent. To assess the best transport conditions for LBs, they were stored at room temperature (RT) and 4°C for 30 minutes and 24h in simulated transportation conditions. LBs were ex vivo expanded on human amniotic membrane (HAM). The best transport conditions for cultured LECs on HAM were investigated by comparison of 24h storage in culture media with prolonged 4 and 7 days storage in serum-free media at 23°C. The best transport conditions were defined by comparison of explants growth rates, cell number and viability, colony forming efficiency (CFE), the expression of stem cell marker ΔNp63 and markers of corneal differentiation Cytokeratin 3 (CK3) and Connexin 43 by immunohistochemistry.
Results :
LBs transported at 4°C showed later and slower growth throughout the culture. While the cell number and viability were not affected, 24h transport at 4°C led to significantly lower CFE (p<0.05) compared to RT transports. 30min and 24h transport of LBs at 4°C caused significant decrease in ΔNp63 expression (p<0.01 and p<0.05, respectively) and significantly higher expression of CK3 (p<0.001) compared to RT. Cell number and viability of LECs did not differ significantly between 24h and 4 and 7 days storage, but a trend of increased cell death with longer storage was observed. ΔNp63 and Connexin 43 expression did not differ, as well as CK3 expression, but the latest showed a trend of increase with time spent in storage.
Conclusions :
RT transport of LBs is significantly superior to 4°C, preserving better cell proliferation and growth, CFE and gene expression. 24h RT transport was equally efficient as 30 min RT. LECs may be stored in serum-free media and transported up to 7 days at 23°C without any negative effect on cell number, viability, CFE or gene expression profile.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.