Optimization of Storage Temperature for Cultured ARPE-19 Cells

Abstract

Purpose. The establishment of future retinal pigment epithelium (RPE) replacement therapy is partly dependent on the availability of tissue-engineered RPE cells, which may be enhanced by the development of suitable storage methods for RPE. This study investigates the effect of different storage temperatures on the viability, morphology, and phenotype of cultured RPE. Methods. ARPE-19 cells were cultured under standard conditions and stored in HEPES-buffered MEM at nine temperatures (4°C, 8°C, 12°C, 16°C, 20°C, 24°C, 28°C, 32°C, and 37°C) for seven days. Viability and phenotype were assessed by a microplate fluorometer and epifluorescence microscopy, while morphology was analyzed by scanning electron microscopy. Results. The percentage of viable cells preserved after storage was highest in the 16°C group (48.7% ± 9.8%; P < 0.01 compared to 4°C, 8°C, and 24°C–37°C; P < 0.05 compared to 12°C). Ultrastructure was best preserved at 12°C, 16°C, and 20°C. Expression of actin, ZO-1, PCNA, caspase-3, and RPE65 was maintained after storage at 16°C compared to control cells that were not stored. Conclusion. Out of nine temperatures tested between 4°C and 37°C, storage at 12°C, 16°C, and 20°C was optimal for maintenance of RPE cell viability, morphology, and phenotype. The preservation of RPE cells is critically dependent on storage temperature.

The Impact of Storage Temperature on the Morphology, Viability, Cell Number and Metabolism of Cultured Human Conjunctival Epithelium

“…Following culture, each multiwell plate was sealed and randomly selected for storage in custom-built temperature-regulating cabinets 13 at one of nine different temperatures (4°C, 8°C, 12°C, 16°C, 20°C, 24°C, 28°C, 32°C and 37°C) for four or seven days. The standard deviation of the temperature in each temperature cabinet was ±0.4°C, as reported elsewhere.…”

“…The standard deviation of the temperature in each temperature cabinet was ±0.4°C, as reported elsewhere. 13 The storage medium consisted of 1 mL MEM with 12.5 mM Hepes, 22.3mM sodium bicarbonate and 50µg/ml gentamycin. Cultured HCjE that were not subjected to storage served as controls.…”

Impact of Storage Temperature on the Expression of Cell Survival Genes in Cultured ARPE-19 Cells

“…This is in line with earlier studies from our group, showing increased cell death in both ARPE-19 cultures and human conjunctival epithelial cultures stored at 37°C. 19,56 Collectively, these findings indicate that inflammatory and oxidative stress responses are, at most, only slightly activated during cell storage at 16°C.…”

“…In cultures stored at 4°C, using a similar culture/ storage protocol as described herein, the number of viable cells dropped to less than 4% of the cultured, but unstored control group. 19 This finding can have at least two explanations: First, the cultures stored at 4°C contain a large number of dead and dying cells, which have a tendency to detach and be washed away, for example when rinsing the cell layer with PBS prior to addition of the QIAzol Lysis Reagent. Second, cellular adhesion seems to be severely affected during 4°C storage, resulting in the loss of otherwise well-functioning cells from the monolayer.…”

“…19 To determine how the storage temperature impacts on cellular function and gene expression, we performed a DNA microarray analysis to investigate the changes in gene expression profiles of stored ARPE-19 cells.…”