Timeline of Achieving a Differentiated RPE Phenotype and Specialised Metabolism of ARPE-19 Cells Under Physiologically Relevant-Inducing Culture Conditions
- 1Ocular Molecular Biology and Mechanisms of Disease Group, School of Biological and Chemical Sciences, Manchester Metropolitan University, Manchester M1 5GD, UK
- 2Department of Biology, Edge Hill University, Ormskirk L39 4QP, UK
- 3Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PL, UK
Abstract
Background. ARPE-19 cells are widely used to investigate and model retinal pigmented epithelium (RPE). Naïve ARPE-19 cells do not fully replicate RPE features. Previous reports show that under defined conditions, ARPE-19 differentiate towards an RPE phenotype, with protocols relying on prolonged or shorter-term cultures, the latter of which do not achieve full pigmentation. In particular, bioenergetics and mitochondrial dynamics have been assessed in a limited manner to date. The present work aims to characterise ARPE-19 under long-term physiologically relevant phenotype-inducing culture conditions, monitoring the timeline of RPE phenotype acquisition, with a focus on mitochondrial function and bioenergetics alongside structural and functional markers. Methods. Low passage ARPE-19 were cultured for 4 months using low-serum, pyruvate and nicotinamide-supplemented DMEM. Monthly assessments measured pigmentation, TER, gene/protein expression, mitochondrial parameters, and metabolism. Results. ARPE-19 acquired relevant features early (1 month), including polarisation, expression of visual cycle and mitochondrial dynamic drivers, as well as increases in mitochondrial potential, mass/area and branching. Melanin accumulation was increased beginning at 1 month. Bioenergetic assays revealed a shift towards increased reliance on oxidative phosphorylation compared to glycolysis. Conclusions. Under these conditions, ARPE-19 acquired specific RPE features supporting functional assays after 1 month. Time-dependent accumulation of RPE-like features suggests that, for certain applications, later differentiation times are suitable. Appropriately differentiated ARPE-19 are a viable model to study key RPE physiological functions when accompanied by evidence of specific phenotypic and metabolic characterization.
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BibTeX
@article{matei2026timeline,
title = {{Timeline of Achieving a Differentiated RPE Phenotype and Specialised Metabolism of ARPE-19 Cells Under Physiologically Relevant-Inducing Culture Conditions}},
author = {Ioan V. Matei and Daniel Grimes and Gunticha Suwanmanee and James Bagnall and David G. Spiller and Luminita Paraoan},
journal = {Cells},
year = {2026},
month = oct,
doi = {10.3390/cells15201846},
url = {https://www.mdpi.com/2073-4409/15/20/1846},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Timeline of Achieving a Differentiated RPE Phenotype and Specialised Metabolism of ARPE-19 Cells Under Physiologically Relevant-Inducing Culture Conditions
AU - Ioan V. Matei
AU - Daniel Grimes
AU - Gunticha Suwanmanee
AU - James Bagnall
AU - David G. Spiller
AU - Luminita Paraoan
PY - 2026
DA - 2026/10/12
JO - Cells
DO - 10.3390/cells15201846
UR - https://www.mdpi.com/2073-4409/15/20/1846
AB - Background. ARPE-19 cells are widely used to investigate and model retinal pigmented epithelium (RPE). Naïve ARPE-19 cells do not fully replicate RPE features. Previous reports show that under defined conditions, ARPE-19 differentiate towards an RPE phenotype, with protocols relying on prolonged or shorter-term cultures, the latter of which do not achieve full pigmentation. In particular, bioenergetics and mitochondrial dynamics have been assessed in a limited manner to date. The present work aims to characterise ARPE-19 under long-term physiologically relevant phenotype-inducing culture conditions, monitoring the timeline of RPE phenotype acquisition, with a focus on mitochondrial function and bioenergetics alongside structural and functional markers. Methods. Low passage ARPE-19 were cultured for 4 months using low-serum, pyruvate and nicotinamide-supplemented DMEM. Monthly assessments measured pigmentation, TER, gene/protein expression, mitochondrial parameters, and metabolism. Results. ARPE-19 acquired relevant features early (1 month), including polarisation, expression of visual cycle and mitochondrial dynamic drivers, as well as increases in mitochondrial potential, mass/area and branching. Melanin accumulation was increased beginning at 1 month. Bioenergetic assays revealed a shift towards increased reliance on oxidative phosphorylation compared to glycolysis. Conclusions. Under these conditions, ARPE-19 acquired specific RPE features supporting functional assays after 1 month. Time-dependent accumulation of RPE-like features suggests that, for certain applications, later differentiation times are suitable. Appropriately differentiated ARPE-19 are a viable model to study key RPE physiological functions when accompanied by evidence of specific phenotypic and metabolic characterization.
SN - 2073-4409
ER -