Abstract
Purpose :
Retinal pigment epithelium (RPE) metabolic dysfunction is a key contributor to the pathogenesis of many retinal diseases. To characterize disease-relevant metabolic and morphological phenotypes, RPE cells derived from induced pluripotent stem cells (iPSC) of patients with AMD and similar ocular diseases are commonly studied in cell culture. However, the nutrient composition of culture media has been shown to alter the metabolism and phenotype of stem cells and cancer cells. Selecting several of the commonly used RPE media, we systematically investigate the metabolic impact of nutrient composition on RPE.
Methods :
Fetal (fRPE) and iPSC RPE derived from healthy subjects were cultured for 8 weeks in three commonly used culture media and a human plasma-like media (HPLM). B27, an optimized serum-free culture supplement, was substituted for FBS in two formulations. The media groups were as follows: 1) MEMα with 1% FBS, 2) MEMα with 2% B27, 3) HPLM with 1% FBS, 4) HPLM with 2% B27, 5) DMEM-HG/Ham’s F-12 with B27, 6) X-VIVO 10. To perform targeted metabolomics on extracellular and intracellular components, conditioned media and RPE lysate were collected 48h post media change. Blank media was ran in parallel to obtain media profile at 0h.
Results :
While RPE in all media groups consumed glucose from the media, both fRPE and iPSC RPE in DMEM/F12-based media consumed the highest amount of glucose and produced the most lactate. Substitution of FBS for B27 reduced glucose consumption, glycolysis intermediates and UDP-glucose in fRPE. DMEM/F12 and X-VIVO 10 media contained the highest glucose concentrations, and RPE in these cultures had the most intracellular glucose after 48h, but showed modest increases in intracellular glycolysis intermediates with no change in UDP-glucose. RPE cultured in HPLM-based media had the most acetyl-CoA and oxaloacetate, however B27 substitution reduced levels of other intracellular TCA intermediates in the fRPE. Lastly, intracellular lipid metabolites were altered based on media type. The highest intracellular and extracellular acylcarnitines were seen in RPE cultured in HPLM-based media, which was unaffected by B27 substitution in the iPSC RPE. RPE cultured in X-VIVO 10 media had the lowest intracellular acylcarnitines.
Conclusions :
Nutrient composition of culture media alters RPE metablism, and understanding these alterations can help inform the choice of specific media types for future studies.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.