Long-term Monitoring of Oxygen Consumption Rates in Highly Differentiated and Polarized Retinal Pigment Epithelial Cultures

Summary

We introduce a novel device for measuring oxygen consumption rates (OCR) in retinal pigment epithelial (RPE) cultures. The device can measure OCR for weeks at a time on RPE grown on standard cell culture plates with standard media while the plates are in a standard cell culture incubator.

Abstract

Mitochondrial metabolism is critical for the normal function of the retinal pigment epithelium (RPE), a monolayer of cells in the retina important for photoreceptor survival. RPE mitochondrial dysfunction is a hallmark of age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world, and proliferative vitreoretinopathy (PVR), a blinding complication of retinal detachments. RPE degenerative conditions have been well-modeled by RPE culture systems that are highly differentiated and polarized to mimic in vivo RPE. However, monitoring oxygen consumption rates (OCR), a proxy for mitochondrial function, has been difficult in such culture systems because the conditions that promote ideal RPE polarization and differentiation do not allow for easy OCR measurements.

Here, we introduce a novel system, Resipher, to monitor OCR for weeks at a time in well-differentiated RPE cultures while maintaining the RPE on optimal growth substrates and physiologic culture media in a standard cell culture incubator. This system calculates OCR by measuring the oxygen concentration gradient present in the media above cells. We discuss the advantages of this system over other methods for detecting OCR and how to set up the system for measuring OCR in RPE cultures. We cover key tips and tricks for using the system, caution about interpreting the data, and guidelines for troubleshooting unexpected results.

We also provide an online calculator for extrapolating the level of hypoxia, normoxia, or hyperoxia RPE cultures experience based on the oxygen gradient in the media above cells detected by the system. Finally, we review two applications of the system, measuring the metabolic state of RPE cells in a PVR model and understanding how the RPE metabolically adapts to hypoxia. We anticipate that the use of this system on highly polarized and differentiated RPE cultures will enhance our understanding of RPE mitochondrial metabolism both under physiologic and disease states.

Introduction

The retinal pigment epithelium (RPE) is a monolayer of functionally postmitotic, highly polarized epithelial cells that form a barrier between light-sensitive photoreceptors in the retina and their blood circulation, a capillary bed termed the choriocapillaris. Like the role of glia-supporting neurons, the RPE carries out myriad functions to support photoreceptors, including phagocytosis of photoreceptor outer segments, transport of nutrients and metabolic support for photoreceptors, and secretion of essential growth factors, all critical for maintaining visual function.

Degeneration of the RPE underlies several common degenerative disorder....

Protocol

For protocols to establish human primary or iPSC-RPE cultures, see the following references^15,16,17,18. The acquisition and use of human tissue for these protocols were reviewed and permitted by the University of Michigan Institutional Review Board (HUM00105486).

1. General application of the system to RPE culture

1. Plate human induced plur.......

Discussion

Mitochondrial metabolism of the RPE plays a critical role in the pathogenesis of common blinding retinal diseases, including AMD and PVR. Modeling RPE mitochondrial metabolism in vitro allows one to isolate its metabolic state from those of surrounding tissues, along with subjecting the tissue to different disease-simulating insults in a controlled manner. Such in vitro modeling of RPE mitochondrial metabolism has been facilitated by the advent of high-fidelity human primary and iPSC-RPE cultures that a.......

Materials

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA	Gibco	#25-200-056
3,3',5-triiodo-L-thyronine sodium salt	Sigma	T5516
32-channel Resipher lid	Lucid Scientific	NS32-101A for Falcon
Antimycin A from streptomyces sp.	Sigma	A8674-25MG	Inhibitor of Complex III of the electron transport chain
BAM15	Sigma	SML1760-5MG	Uncoupling agent to increase mitochondrial respiration
DMSO, cell culture grade	Sigma-aldrich	D4540-100ML	Vehicle for reconstituting mitochondrial drugs
Extracellular matrix coating substrates: Synthemax II-SC	Corning	#3535	Extracellular matrix for hfRPE
Extracellular matrix coating substrates: Vitronectin	Gibco	A14700	Extracellular matrix for iPSC-RPE
FCCP	Sigma	C2920-10MG	Uncoupling agent to increase mitochondrial respiration
Fetal Bovine Serum (Bio-Techne S11550H)	Bio-Techne	S11550H
Hydrocortisone-Cyclodextrin	Sigma	H0396
Hypoxia chamber	Embrient Inc.	MIC-101
N1 Media Supplement	Sigma	N6530
Non-Essential Amino Acids Solution	Gibco	11140050
O2 sensor	Sensit technology or Forensics Detectors	P100 or FD-90A-O2
Penicillin-Streptomycin	Gibco	15140-122
Recombinant human TGFβ2	Peprotech	100-35B	Transforming growth factor beta-2 to induce epithelial-mesenchymal transition
Rotenone	Sigma	R8875-1G	Inhibitor of Complex I of the electron transport chain
System-compatible plate	Corning	#353072
Taurine	Sigma	T8691
αMEM (Alpha Modification of Eagle's Media)	Corning	15-012-CV