The Effects of Prolonged Storage on ARPE-19 Cells Stored at Three Different Storage Temperatures

This study aimed to investigate how prolonged storage of adult retinal pigment epithelial (ARPE-19) cell sheets affects cell metabolism, morphology, viability, and phenotype. ARPE-19 cell sheets were stored at three temperatures (4 degrees C, 16 degrees C, and 37 degrees C) for three weeks. Metabolic status and morphology of the cells were monitored by sampling medium and examining cells by phase-contrast microscopy, respectively, throughout the storage period. Cell viability was analyzed by flow cytometry, and phenotype was determined by epifluorescence microscopy after the storage. Lactate production and glucose consumption increased heavily, while pH dropped considerably, through storage at 37 degrees C compared to 4 degrees C and 16 degrees C. During storage, morphology started to deteriorate first at 4 degrees C, then at 37 degrees C, and was maintained the longest at 16 degrees C. Viability of the cells after three weeks of storage was best preserved at 16 degrees C, while cells stored at 4 degrees C and 37 degrees C had reduced viability. Dedifferentiation indicated by reduced expression of retinal pigment epithelium-specific protein 65 (RPE65), zonula occludens protein 1 (ZO-1), and occludin after three weeks of storage was noticed in all experimental groups compared to control. We conclude that storage temperature affects the metabolic status of ARPE-19 cells and that 16 degrees C reduces metabolic activity while protecting viability and morphology.