Metabolomics-on-a-chip and metabolic flux analysis for label-free modeling of the internal metabolism of HepG2/C3A cells

Abstract

In vitro microfluidic systems are increasingly used as an alternative to standard Petri dishes in bioengineering and metabolomic investigations, as they are expected to provide cellular environments close to the in vivo conditions. In this work, we combined the recently developed "metabolomics-on-a-chip" approach with metabolic flux analysis to model the metabolic network of the hepatoma HepG2/C3A cell line and to infer the distribution of intracellular metabolic fluxes in standard Petri dishes and microfluidic biochips. A high pyruvate reduction to lactate was observed in both systems, suggesting that the cells operate in oxygen-limited environments. Our results also indicate that HepG2/C3A cells in the biochip are characterized by a higher consumption rate of oxygen, presumably due to a higher oxygenation rate in the microfluidic environment. This leads to a higher entry of the ultimate glycolytic product, acetyl-CoA, into the Krebs cycle. These findings are supported by the transcriptional activity of HepG2/C3A cells in both systems since we observed that genes regulated by a HIF-1 (hypoxia-regulated factor-1) transcriptional factor were over expressed under the Petri conditions, but to a lesser extent in the biochip.

Real-time monitoring of metabolic function in liver-on-chip microdevices tracks the dynamics of mitochondrial dysfunction

“…ATP production in glycolysis was estimated to be two molecules per molecule of glucose. We assumed that any glucose left over was directed toward lipogenesis, because the contribution of pentose phosphate pathway in nonproliferating cells is minor (31). Finally, we assumed that excess lactate was produced by glutaminolysis, and confirmed our assumption using offline measurement of glutamine uptake (Fig.…”

Transcriptomic and Metabolomics Profiling of Phage–Host Interactions between Phage PaP1 and Pseudomonas aeruginosa

“…Metabolic profiling and peak identification were performed by Anachro Technologies Inc. (Wuhan, China) using described methods (Weljie et al, 2006; Ouattara et al, 2012). All of the NMR experiments were performed on an Agilent DD2 600 MHz spectrometer equipped with a triple-resonance cryogenic probe at 298.15 K. Metabolites were identified and quantified using Chenomx NMR Suit (version 8.1, Chenomx, Edmonton, AB, Canada).…”

Microfluidics meets metabolomics to reveal the impact of Campylobacter jejuni infection on biochemical pathways

“…Microfluidic cultures of different organs have been reported in the literature including lung (Sellgren et al, 2014, Huh et al, 2010), kidney (Jang et al, 2013, Baudoin et al, 2007), liver (Ouattara et al, 2012, Prot et al, 2011, Kang et al, 2015, Zhang et al, 2008), intestinal tract (Kim et al, 2012, Kim and Ingber, 2013, Mahler et al, 2009), placenta (Lee et al, 2015) and combinations of multiple organs (Esch et al, 2014, Maschmeyer et al, 2015). Studies have shown that fluid dynamics in µF impacts cell biology compared to traditional stagnant culture conditions.…”

“…Metabolomics analysis is a powerful discovery tool to identify metabolites that are important for differentiating, for example, non-infected and infected cell systems, and can provide unique insight into host-pathogen interaction and progression of infection. Metabolomics analyses of cell media have been reported previously on microfluidic culture of HepG2-C3A and MDCK cells using a polydimethylsiloxane (PDMS) µF comprised of multiple chambers and channels (Ouattara et al, 2012, Shintu et al, 2012). In the study analyzing media from HepG2/C3A cells cultured in µF or petri dishes, metabolomics showed a clear differentiation of the samples in a multivariate analysis using orthogonal partial least squares discriminant analysis (OPLS-DA); indicating that cells cultured under fluid flow had higher glucose, glutamine and ethanol consumptions compared to cells grown in stagnant cultures, while stagnant culture conditions caused higher cellular consumption of lactate (Shintu et al, 2012).…”

“…In the study analyzing media from HepG2/C3A cells cultured in µF or petri dishes, metabolomics showed a clear differentiation of the samples in a multivariate analysis using orthogonal partial least squares discriminant analysis (OPLS-DA); indicating that cells cultured under fluid flow had higher glucose, glutamine and ethanol consumptions compared to cells grown in stagnant cultures, while stagnant culture conditions caused higher cellular consumption of lactate (Shintu et al, 2012). Another study of HepG2/C3A cells combining metabolomics profiling and metabolic flux analysis showed that cells cultured in µF had similar glycolytic activity as cells cultured in petri dishes, while exhibiting higher TCA cycle activity which were supported by transcriptional activity of hypoxia-regulating factor-1 (Ouattara et al, 2012). Metabolomics analysis in the field of infectious diseases have been used to uncover host-pathogen interactions (Milner et al, 2015, Jung et al, 2015, Sanchez and Lagunoff, 2015, Villar et al, 2015), antibiotic susceptibility (Lobritz et al, 2015), and find potential strategies and targets for prevention, control, and therapeutics (McGarvey et al, 2009).…”