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Limited oxygen availability in standard cell culture alters metabolism and function in terminally differentiated cells

ABSTRACT:

Cell culture is generally considered to be hyperoxic. However, the importance of cellular oxygen consumption is often underappreciated, with rates of oxygen consumption often sufficient to cause hypoxia at cell monolayers. We initially focused on cultured adipocytes as a terminally differentiated cell-type with substantial oxygen consumption rates to support diverse cellular functions. Under standard conditions, cultured adipocytes are hypoxic and highly glycolytic. Increasing oxygen diverted glucose flux toward mitochondria and resulted in thousands of gene expression changes that pointed toward alleviated physiological transcriptional responses to hypoxia. Phenotypically, providing more oxygen increased adipokine secretion and rendered adipocytes more sensitive to insulin and lipolytic stimuli. The functional benefits of increasing pericellular oxygen were transferable to other cellular systems including hPSC-derived hepatocytes and cardiac organoids. Our findings suggest that oxygen is limiting in many terminally-differentiated cell culture systems, and that controlling oxygen availability can improve the quality and translatability of cell models.

INSTRUMENT(S): Liquid Chromatography MS - alternating - hilic

SUBMITTER: Joycelyn Tan

PROVIDER: MTBLS6677 | MetaboLights | 2024-02-21

REPOSITORIES: MetaboLights

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Project description:The intestinal epithelium is an immunologically active barrier adapted for a low oxygen environment. Its role is implicated in the pathophysiology of various diseases, including inflammatory bowel disease (IBD) and colorectal cancer. Patient-derived intestinal epithelial organoids (IEOs) mimic the architecture and cell type composition of the intestine and can be used for disease modeling and personalized drug screening. IEOs are usually cultured in atmospheric oxygen concentrations (20% oxygen) without accounting for the physiological hypoxia in the colonic epithelium, where the oxygen concentration range from 3% to <1%. Previously, we showed that human colon derived IEOs (colonoids) are viable and respond to the pro-inflammatory cytokines TNF and IL17 after short-term (40 hours) cultivation in low (2%) oxygen. We suggested that recapitulating the in vivo physiological oxygen environment (i.e., physioxia) will enhance the translational value of intestinal organoids as pre-clinical models. Here we evaluate whether human colonoids can be established and cultured in physioxia for the entire in vitro culture period and compare growth, differentiation, and immunological responses in colonoids at 2% and 20% oxygen. Growth from single cells to differentiated organoids was monitored by brightfield images captured throughout cultivation and evaluated with a linear mixed model. Cell composition in undifferentiated vs. differentiated colonoids was identified by immunofluorescence staining of cell markers and single-cell RNA-sequencing. Enrichment analysis was used to identify transcriptomic differences within cell populations. Pro-inflammatory stimuli induced chemokines and Neutrophil gelatinase-associated lipocalin (NGAL) release were analyzed by Multiplex profiling and ELISA. Direct response to a lower oxygen level was analyzed by enrichment analysis of bulk RNA sequencing data. Colonoids established in a 2% oxygen environment acquired a significantly larger cell mass compared to a 20% oxygen environment. There were significantly more cells with proliferation potential (KI67 positive) in undifferentiated than in differentiated colonoids; and significantly higher expression of cell markers for goblet cells (MUC2) and absorptive cells (MUC2 negative, CK20 positive) in differentiated colonoids. Enteroendocrine cells (CGA positive) were only present in differentiated colonoids. No differences in expression of any of the cell marker proteins were found between colonoids cultured in 2% and 20% oxygen, but the single-cell RNA-sequencing analysis identified differences in the transcriptome within stem-, progenitor- and differentiated cell clusters. Both colonoids grown at 2% and 20% oxygen secreted CX3CL1, CXCL2, CXCL5, CXCL6, CXCL10, CXCL12, CCL20, CCL25, and NGAL upon TNF + poly(I:C) treatment. However, there appeared to be a lower pro-inflammatory response in 2% oxygen. Reducing the oxygen environment from 20% to 2% in differentiated colonoids altered the expression of genes related to differentiation, metabolism, mucus lining, and immune networks. Our results suggest that colonoids studies can and should be performed in physioxia when the resemblance to in vivo conditions is important.

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Limited oxygen availability in standard cell culture alters metabolism and function in terminally differentiated cells

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