Abstract
Purpose :
To investigate the stability of cultured limbal stem cells (cLSCs) over a 48-hour storage period at a controlled temperature.
Methods :
Limbal explants obtained from a total of 6 human sclerocorneal tissues were cultured on denuded human amniotic membrane (hAM) in modified supplemented hormonal epithelium medium (mSHEM) using xeno-free and cGMP-compliant components. Once the limbal explant outgrowths reached 13mm in diameter, they were either harvested immediately (t=0) or stored in an air-tight cell culture-grade container at 20oC. Two different storage media (M1 and M2) were evaluated and the cells were harvested at 12, 24 and 48 hours (t=12, t=24 and t=48, respectively). At each time point, glucose, lactate, glutamine and glutamate concentration and pH of the storage media were measured. Cell phenotype was determined using immunocytochemistry. Endotoxin, mycoplasma and sterility tests were also conducted.
Results :
Temperature was stable during the storage period (20.5oC ± 0.003) as well as pH (7.34 ± 0.01). Sterility was confirmed and validated through bacteriostasis/fungistasis testing. Endotoxin level was below 0.1 EU/mL in storage medium. Lactate and glutamine concentration at each time point remained the same (0.06 ± 0.016 g/L-1 and 2.03 ± 0.038 mM, respectively, p > 0.05), but a significant decrease in glucose concentration in M2 after 48 hours of storage (3.37 ± 0.078 g/L-1) when compared to t=0 (2.96 ± 0.034 g/L-1, p < 0.05). Glutamate concentration was very close to zero for both M1 and M2. In terms of phenotype, no significant differences were observed after the storage period when compared to non-stored cells (i.e. t=0). Indeed, putative LSC markers, Keratin 14 (K14) and p63α (bright population) maintained an expression level ≥ 90% and ≥ 3%, respectively. Keratin 12 (K12), a marker for mature corneal epithelial cells, showed an expression below 2%. Pan-cytokeratin (PanK) was expressed in more than 90% of the cells and Vimentin (Vim) in less than 5%, demonstrating the overall composition of the culture.
Conclusions :
In the present study, the cLSCs are able to maintain their phenotype after 48 hours of incubation at 20oC in comparison to t=0.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.