Dynamics of amino acid metabolism of primary human liver cells in 3D bioreactors

Guthke, Reinhard; Zeilinger, Katrin; Sickinger, Stephan; Schmidt‐Heck, Wolfgang; Buentemeyer, H.; Iding, Kai; Lehmann, J.; Pfaff, Michael; Pless, Gesine; Gerlach, Jörg C.

doi:10.1007/s00449-005-0040-1

Cited by 14 publications

(9 citation statements)

References 15 publications

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“…Recently, several types of co-culture systems have been performed to improve hepatocyte morphology and function by culturing with different cells, including liver-derived or non-liver derived mesenchymal cells (Ijima et al, 2008;Lu et al, 2005;Nishikawa et al, 2008). Besides, threedimensional culture system is expected to represent an artificial equivalent of the hepatic structure at the lobular level (Guthke et al, 2006). In order to mimic native sinusoidal structure, in which hepatocytes interact with hepatic stellate cells, kupffer cells or pit cells through the walls composed of endothelial cells, we adopted microsphere-based co-culture to support C3A cells in a bioreactor.…”

Section: Introductionmentioning

confidence: 99%

Co‐culture with mesenchymal stem cells enhances metabolic functions of liver cells in bioartificial liver system

Yang

Pan

et al. 2012

Biotech & Bioengineering

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Bioartificial liver provides a combination of three-dimensional support, matrix interactions, and extracellular cues to create a bio-mimic microenvironment for maintaining hepatic-specific functions of liver cells in vitro. However, its transformal and metabolic functions are not yet satisfactory for clinic application. In this study, hepatoma-derived C3A cells were co-cultured with human placental mesenchymal stem cells (hPMSC) in microspheres placed in a fluidized bioreactor. The secretion of albumin and urea, the expression of metabolizing enzymes at both transcriptional and translational levels and the drug metabolism functions of co-cultured C3A cells were determined. With the three-dimensional culture system, when C3A cells were co-cultured with hPMSCs in separate microspheres, the secretion of albumin and activity of CYP1A2 were significantly improved although the enhancement of urea synthesis and CYP3A4 activity was less prominent. Combining co-culture system with fluidization significantly increased the secretion of urea and the activities of CYP1A2, CYP3A4 but not the albumin synthesis. Interestingly, the levels of phospho-PKA (Thr 197), phospho-PKC, phospho-ERK1/2 (Thr 202/Tyr 204) and CaMKII were all found to decrease in co-cultured C3A cells, implicating suppressed signaling pathways in those cells. Taken together, our results suggest that co-culturing of liver cells with hPMSC cells in three-dimensional fluidized bioreactor significantly improved the preservation of liver cells' metabolic functions which may greatly enhance the efficacy of bioartificial liver treatment.

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Section: Introductionmentioning

confidence: 99%

Co‐culture with mesenchymal stem cells enhances metabolic functions of liver cells in bioartificial liver system

Yang

Pan

et al. 2012

Biotech & Bioengineering

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“…Additionally, the block in glycolysis was released, and inhibitions of LDH, the Krebs cycle, and fatty acid b-oxidation were demonstrated with acetaminophen toxicity. Clearly, transplanted human livers display normal biochemistry and physiology, and some 3D human bioartificial liver systems have been shown to consume glucose and glutamine (Guthke et al 2006;Seagle et al 2008). Therefore, the dynamic changes in substrates, paracrine (soluble or insoluble), and/or endocrine factors must be missing from the monolayer culture system used in this study.…”

Section: Discussionmentioning

confidence: 98%

“…Although some metabolomic footprint studies have been performed with 3D human bioartificial liver devices (Guthke et al 2006;Seagle et al 2008), to our knowledge such 14 C/ 13 C studies have not utilized primary human hepatocytes. To gain a better understanding of the inherent differences between human and rodent hepatocyte energy metabolism, this study compared rat and human hepatocyte monolayer cultures using stable isotope resolved metabolomics (SIRM) (Fan et al 2009).…”

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confidence: 99%

Stable isotope resolved metabolomics of primary human hepatocytes reveals a stressed phenotype

et al. 2011

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The development of techniques allowing the culturing of primary mammalian hepatocytes has provided great insights into liver physiology. For most applications, it is desirable for hepatocytes in culture to mimic hepatocytes in vivo. We used stable isotope resolved metabolomics (SIRM) to assess glucose and glutamine utilization in primary rat and human hepatocytes maintained in standard culture media. Primary rat hepatocytes readily metabolized 13 C-glucose and 13 C-glutamine made evident by 13 C incorporation into glycogen, glycolytic end products, and Krebs cycle intermediates and responded to insulin and glucagon appropriately. In contrast, no glucose or glutamine consumption was detected in primary human hepatocytes over 4 h of exposure to high media concentrations of 13 C-glucose or 13 C-glutamine even in the presence of insulin. Nonetheless, cultured human hepatocytes were metabolically active and viable, as demonstrated by incorporation of media 13 C-octanoic acid into Krebs cycle intermediates and ketone bodies. The failure to utilize glucose was not due to inhibition of glucokinase (hexokinase IV) since the human hepatocytes could readily incorporate 13 C-glucose into glucuronic acid, as demonstrated by the production of 13 C-glucuronide conjugates after addition of acetaminophen to the media. These novel observations support inhibition of phosphofructokinase-1, the other regulatory enzyme in glycolysis. Parts of this phenotype could be reproduced in the rat hepatocytes by replacing insulin with glucagon to the media. We conclude that under standard culture conditions human hepatocytes are in an extreme starved state. We believe this may result from prolonged fasting in the human liver donors combined with exposure to stress hormones such as, epinephrine, glucagon, and cortisol. Efforts should now be exerted to find culture conditions that will reverse this state to achieve more metabolically relevant cultures of human hepatocytes.

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“…Without these measurements a large fraction of the fluxes cannot be determined. Therefore, a literature value for the oxygen uptake (∼ 54 mM) was used [3]. The results can be summarized as follows (for the following considerations, the fluxes are based on the carbohydrate uptake flux, representing 100%): Carbohydrate uptake: Glucose and galactose are taken up and the main part of these carbohydrates is metabolized via glycolysis.…”

Section: B Summary Flux Analysismentioning

confidence: 99%

“…This allows to support the liver function until liver transplantation or until organ recovery in case of acute hepatic failure. For this study a cell culture bioreactor for extracorporeal liver support is used that was introduced previously [2], [3], [4]. The bioreactor is composed of three perfused interwoven capillary systems within a polyurethane housing ( Figure 1).…”

Section: Introductionmentioning

confidence: 99%

A reduced stoichiometric model to describe metabolism in hepatocytes

Kremling

Zeilinger

Gerlach

et al. 2006

2006 IEEE Conference on Computer Aided Control System Design, 2006 IEEE International Conference on Control Applications, 2006

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Stoichiometric models are widely used in Metabolic Engineering and Systems Biology to estimate the flux distribution in cellular systems during steady-state operation. For a number of applications it is desirable to have a model with a small number of state variables. This is of particular interest, if static metabolic models are combined with dynamical models of gene expression. Such integrated models can be used to analyze, monitor and control cell culture bioreactors working as extracorporeal liver support systems. Such systems provide the option for bridging the liver function in case of acute hepatic failure until liver transplantation or until regeneration of the patient's own organ. In this contribution a reduced stoichiometric model of the central metabolism of hepatocytes, i.e. the most important cell type in the liver, is presented.

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Dynamics of amino acid metabolism of primary human liver cells in 3D bioreactors

Cited by 14 publications

References 15 publications

Co‐culture with mesenchymal stem cells enhances metabolic functions of liver cells in bioartificial liver system

Co‐culture with mesenchymal stem cells enhances metabolic functions of liver cells in bioartificial liver system

Stable isotope resolved metabolomics of primary human hepatocytes reveals a stressed phenotype

A reduced stoichiometric model to describe metabolism in hepatocytes

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