Cultured Human Retinal Pigment Epithelial (hRPE) Sheets: A Search for Suitable Storage Conditions

Abstract

The advancement of human retinal pigment epithelial cell (hRPE) replacement therapy is partly dependent on optimization of cell culture, cell preservation, and storage medium. This study was undertaken to search for a suitable storage temperature and storage medium for hRPE. hRPE monolayer sheets were cultured under standard conditions at 37°C and then randomized for storage at six temperatures (4, 16, 20, 24, 28, and 37°C) for 7 days. After revealing a suitable storage temperature, hRPE sheets were subsequently stored with and without the silk protein sericin added to the storage medium. Live/dead assay, light microscopy, pH, and phenotypic expression of various proteins were used to assess cell cultures stored at different temperatures. After 7 days of storage, hRPE morphology was best preserved at 4°C. Addition of sericin to the storage medium maintained the characteristic morphology of the preserved cells, and improved pigmentation and levels of pigmentation-related proteins in the cultured hRPE sheets following a 7-day storage period at 4°C.

Sericin-Induced Melanogenesis in Cultured Retinal Pigment Epithelial Cells Is Associated with Elevated Levels of Hydrogen Peroxide and Inflammatory Proteins

“…In a previous study on preservation of cultured primary human fetal RPE, we showed that the fraction of pigmented cells was inversely related to storage temperature [ 7 ]. The precise mechanisms behind these results were not fully understood.…”

“…The exposure length and gain were maintained at a constant level for all samples and the fluorescence intensities of the FITC or Cy3 fluorochromes, which were conjugated to the secondary antibodies, were within the dynamic range of the camera. Immunofluorescence per cell was quantified by custom-made macros for ImageJ, as described previously [ 7 , 19 ].…”

“…At the end of the culture period, cell medium was aspirated and cells were incubated at 37 °C for 30 min with PBS containing 1.0 μM CAM. After washing the cultures thrice with PBS, photomicrographs were captured at 200× magnification and subsequently analyzed using custom-made macros with ImageJ, as reported elsewhere [ 7 ]. In brief, unevenly transmitted light (rolling = 50) was subtracted from all 16-bit photomicrographs using the “Subtract Background”-command in ImageJ before they were converted to binary photos, Then, the culture well area covered by calcein-stained cells was automatically measured using the “Area Fraction”-command.…”

“…We have previously reported that the addition of sericin to cell preservation medium improves RPE phenotype following a seven-day preservation period [ 7 ]. We have also shown that sericin has pro-pigmentation effects on cultured RPE, and that these effects are associated with the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway [ 8 ].…”

The Silk Protein Sericin Promotes Viability of ARPE-19 and Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelial Cellsin vitro

“…After washing the cultures three times with PBS, photomicrographs were captured at 200x magnification and subsequently analyzed using custom-made macros with ImageJ (National Institutes of Health, Bethesda, MD), as described previously. 6…”