Improvement of Storage Medium for Cultured Human Retinal Pigment Epithelial Cells Using Factorial Design

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.

Transcriptomic Profiling of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium over Time

“…ATP1B1 encodes an apical Na + /K + ATPase, whose expression reduces with age and in age-related macular degeneration (AMD) [38] . Defects in CTNNB1 are linked to abnormalities in RPE development and pigmentation [39] , whereas GSTP1 is a survival factor for RPE cells, whose expression increases as cells mature [40] ; CD63 is a late endosome/exosome marker known to be released by RPE cells [41] ; and HNRNPH1 levels are associated with improved survival of RPE cells in culture [42] . Hence, expression of these markers indicates functional RPE cells.…”

Clever Experimental Designs: Shortcuts for Better iPSC Differentiation

“…The use of DOE is not limited to cell cultivation and can also be applied for other cell-related purposes ( Table 8 ), such as cell storage, cell transportation, and related materials and devices, which are especially important issues for industrial cell usage. The refrigerated storage conditions for retinal pigment epithelial cells [ 118 ] and epithelial cell sheets [ 119 ] were optimized through full and fractional factorial screening. In addition, the virus inactivation process of Vero cells [ 120 ] was investigated using an orthogonal array.…”

Transcriptomic Profiling of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium Over Time

“…"Young" Subpopulation One (1,873 cells; 58 conserved markers) was characterised by gene expression of CTNNB1, NOG, ATP1B1, GSTP1, CD63, HNRNPH1. All play fundamental roles for the homeostasis and functions of the RPE: ATP1B1 encodes an apical Na + /K + ATPase which expression reduces with age and in AMD [38]; defects in CTNNB1 are linked to abnormalities in RPE development and pigmentation [39]; GSTP1 is a survival factor for RPE cells, which levels increase as cells mature [40]; CD63 is a late endosome/exosome marker known to be released by RPE cells [41] and HNRNPH1 levels are associated with improved survival of RPE cells in culture [42]. Hence, those markers indicate cellular functions suggestive of functional RPE cells.…”