Abstract
Storage of human retinal pigment epithelium (hRPE) can contribute to the advancement of cell-based RPE replacement therapies. The present study aimed to improve the quality of stored hRPE cultures by identifying storage medium additives that, alone or in combination, contribute to enhancing cell viability while preserving morphology and phenotype. hRPE cells were cultured in the presence of the silk protein sericin until pigmentation. Cells were then stored for 10 days in storage medium plus sericin and either one of 46 different additives. Individual effects of each additive on cell viability were assessed using epifluorescence microscopy. Factorial design identified promising additive combinations by extrapolating their individual effects. Supplementing the storage medium with sericin combined with adenosine, L-ascorbic acid and allopurinol resulted in the highest cell viability (98.6 ± 0.5%) after storage for three days, as measured by epifluorescence microscopy. Flow cytometry validated the findings. Proteomics identified 61 upregulated and 65 downregulated proteins in this storage group compared to the unstored control. Transmission electron microscopy demonstrated the presence of melanosomes after storage in the optimized medium. We conclude that the combination of adenosine, L-ascorbic acid, allopurinol and sericin in minimal essential medium preserves RPE pigmentation while maintaining cell viability during storage.
Highlights
Worldwide[17], an upcoming need for improved storage and transportation methods for cultured RPE is anticipated
After testing nine different storage temperatures between 4 °C and 37 °C, we found that human retinal pigment epithelium (hRPE) cultures stored at 4 °C in a storage medium containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)- and sodium bicarbonate-buffered Minimum Essential Medium (MEM) preserved the greatest number of viable cells
Effect of Individual and Combined Storage Medium Additives on Viability of hRPE. hRPE were seeded in complete epithelial cell medium (EpiCM) on Nunclon Δ surface plates and cultured for two days before replacing EpiCM with modified DMEM containing 1% sericin for 14 days
Summary
Worldwide[17], an upcoming need for improved storage and transportation methods for cultured RPE is anticipated. We investigated the effects of supplementing this medium with many different additives, including sericin to preserve the differentiated state of the cells. The effects of the 46 individual supplements on viability of hRPE cell cultures were analyzed after ten days of storage at 4 °C. Some additives were selected based on their known or proposed effects on viability or antioxidant function in cultures of RPE or other cell types[21,22,23,24,25,26,27,28,29,30,31,32], while others were chosen based on effects demonstrated in pilot experiments. The single best combination of additives was selected for further study by additional experiments to assess its effects on phenotype and morphology
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