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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.

SUBMITTER: Pasovic L

PROVIDER: S-EPMC5890280 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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

Pasovic L L Utheim T P TP Reppe S S Khan A Z AZ Jackson C J CJ Thiede B B Berg J P JP Messelt E B EB Eidet J R JR

Scientific reports 20180409 1

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 ser ...[more]

PMID: 29632395

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Project description:I. Exp Design 1. Type of experiment: Comparison of native versus cultured RPE cells 2. Experimental factors: Native RPE versus ARPE-19 cells grown on different matrices 3. How many hybridizations in exp: 21 4. If a common reference used for all the hybs: no 5. Quality control steps: three independent arrays for each condition 6. Description: The expression profile of ARPE-19 cells grown on different matrices were compared to morphologically normal native macular RPE cells that were laser capture microdissected from 3 donors. II. Samples used, extract prep, and labeling 1. Biosource: Human donor globes from NDRI (63, 71, 74 years old) and ARPE-19 cells. 2. Manipulations: Human donor globes were cryopreserved, and morphologically normal RPE cells from the macula were laser capture micodissected. ARPE-19 cells were grown on different matrices (plastic, Matrigel, collagen I, collagen IV, laminin, and fibronectin). 3. Extract preparation: Total RNA from cells were extracted with the RNeasy kit (Qiagen) using the manufacturerâs instructions. 4. Labeling protocol: Total RNA from cells was reverse transcribed with 33P-dCTP and 33P-dATP, and second strand cDNA was labeled with 33P-dCTP and 33P-dATP. 5. No external controls were added. III. Hybridization procedures and parameters 1. Sample, array type, batch and serial # used 2. Hybridization protocol: Hybridization was carried out using the manufacturerâs recommendations. Arrays were prehybridized with Microhyb solution containing denatured Cot-1 DNA and poly dA at 42oC for two hours. Hybridization was carried out at 42oC overnight using a hybridization oven set at 8-10 rpm. Arrays were washed twice at 50oC for 20 minutes using 2x SSC, 1%SDS and once at room temperature for 15 minutes using 0.5x SSC, 1%SDS. IV. Measurement data and specifications of data processing 1,2. Arrays were exposed to a phosphorimaging screen for 3 days and scanned at 50 mm resolution with a BioRad FX Pro-Plus phosphorimager. TIFF images from the phosphorimager were exported into ResGen Pathways 3 software for analysis. 3. Data processing: A gene was expressed if its background subtracted intensity was greater than 1.4 fold background. The data were normalized using a simple global scaling procedure, and Cluster/Treeview and Statistical Analysis of Microarrays (SAM version 1.12) programs were used for analysis.

Project description:I. Exp Design 1. Type of experiment: Comparison of native versus cultured RPE cells 2. Experimental factors: Native RPE versus ARPE-19 cells grown on different matrices 3. How many hybridizations in exp: 21 4. If a common reference used for all the hybs: no 5. Quality control steps: three independent arrays for each condition 6. Description: The expression profile of ARPE-19 cells grown on different matrices were compared to morphologically normal native macular RPE cells that were laser capture microdissected from 3 donors. II. Samples used, extract prep, and labeling 1. Biosource: Human donor globes from NDRI (63, 71, 74 years old) and ARPE-19 cells. 2. Manipulations: Human donor globes were cryopreserved, and morphologically normal RPE cells from the macula were laser capture micodissected. ARPE-19 cells were grown on different matrices (plastic, Matrigel, collagen I, collagen IV, laminin, and fibronectin). 3. Extract preparation: Total RNA from cells were extracted with the RNeasy kit (Qiagen) using the manufacturer’s instructions. 4. Labeling protocol: Total RNA from cells was reverse transcribed with 33P-dCTP and 33P-dATP, and second strand cDNA was labeled with 33P-dCTP and 33P-dATP. 5. No external controls were added. III. Hybridization procedures and parameters 1. Sample, array type, batch and serial # used 2. Hybridization protocol: Hybridization was carried out using the manufacturer’s recommendations. Arrays were prehybridized with Microhyb solution containing denatured Cot-1 DNA and poly dA at 42oC for two hours. Hybridization was carried out at 42oC overnight using a hybridization oven set at 8-10 rpm. Arrays were washed twice at 50oC for 20 minutes using 2x SSC, 1%SDS and once at room temperature for 15 minutes using 0.5x SSC, 1%SDS. IV. Measurement data and specifications of data processing 1,2. Arrays were exposed to a phosphorimaging screen for 3 days and scanned at 50 mm resolution with a BioRad FX Pro-Plus phosphorimager. TIFF images from the phosphorimager were exported into ResGen Pathways 3 software for analysis. 3. Data processing: A gene was expressed if its background subtracted intensity was greater than 1.4 fold background. The data were normalized using a simple global scaling procedure, and Cluster/Treeview and Statistical Analysis of Microarrays (SAM version 1.12) programs were used for analysis. Keywords: other

Dataset Information

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

Publications

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

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