Extracorporeal Plasma Perfusion of Cultured Hepatocytes: Effect of Intermittent Perfusion on Hepatocyte Function and Morphology

Stefanovich, Peter; Matthew, Howard W.T.; Toner, Mehmet; Tompkins, Ronald G.; Yarmush, Martin L.

doi:10.1006/jsre.1996.0372

Cited by 30 publications

(20 citation statements)

References 4 publications

(4 reference statements)

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“…Our data show that gene transcription of HepaRG-BAL cultures can be restored after 16 hours of h plasma exposure by recirculating culture medium through the device, indicating that alternated treatment-and restoration runs are also a viable strategy to increase the life-span of BALs, as proposed by others previously [7].…”

Section: Discussionsupporting

confidence: 62%

“…Healthy-donor human plasma ( h plasma) is known to induce intracellular lipid accumulation, stress and a decrease in hepatic functionality of primary hepatocytes and hepatic cell lines, through unclarified mechanisms [7,8]. In addition, plasma from liver failure patients contains not only detrimental compounds which are normally detoxified by the liver, such as ammonia, bile acids, and lactate, but also compounds associated with inflammation and infection, such as endogenous damage-associated molecular patterns released from necrotic cells, cytokines and chemokines, as well as lipopolysaccharides and other stimulators of innate immune response due to bacterial translocation [9][10][11].…”

Section: Introductionmentioning

confidence: 99%

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HepaRG-Progenitor Cell Derived Hepatocytes Cultured in Bioartificial Livers Are Protected from Healthy- and Acute Liver Failure-Plasma Induced Toxicity

Wenum¹,

Treskes²,

Adam³

et al. 2018

Cell Physiol Biochem

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Background/Aims: For applicability of cell-based therapies aimed at the treatment of liver failure, such as bioartificial livers (BALs) and hepatocyte transplantation, it is essential that the applied hepatocytes tolerate exposure to the patient plasma. However, plasma from both healthy donors and acute liver failure (ALF) patients is detrimental to hepatocytes and hepatic cell lines, such as HepaRG. We aimed to elucidate the underlying mechanisms of plasma-induced toxicity against HepaRG cells in order to ultimately develop methods to reduce this toxicity and render HepaRG-BAL treatment more effective. Methods: Differentiated HepaRG cells cultured in monolayers and laboratory-scale BALs were exposed to culture medium, healthy human plasma, healthy porcine plasma and ALF porcine plasma. Healthy human plasma was fractionated based on size- and polarity, albumin depleted and heat treated to characterize the toxic fraction. The cells were assessed for viability by total protein content and trypan blue staining. Their hepatic differentiation was assessed on transcript level through qRT-PCR and microarray analysis, and on functional level for Cytochrome P450 3A4 activity and ammonia elimination. Mitochondrial damage was assessed by JC-1 staining and mitochondrial gene transcription. Results: Sixteen hours of healthy human plasma exposure did not affect viability, however, hepatic gene-transcript levels decreased dramatically and dose-dependently within four hours of exposure. These changes were associated with early NF-kB signaling and a shift from mitochondrial energy metabolism towards glycolysis. Healthy human plasma-toxicity was associated with the dose-dependent presence of heat-resistant, albumin-bound and (partly) hydrophobic toxic compound(s). HepaRG cells cultured in BALs were partially protected from plasma-toxicity, which was mainly attributable to medium perfusion and/or 3D configuration applied during BAL culturing. The detrimental human plasma effects were reversible in BAL-cultured cells. Porcine ALF-plasma elicited mitotoxicity additional to the basal detrimental effect of porcine healthy plasma, which were only partially reversible. Conclusion: A specific fraction of human plasma reduces hepatic differentiation of HepaRG cultures, in association with early NF-κB activation. In addition, ALF-plasma elicits mitotoxic effects. These findings allow for a targeted approach in preventing plasma-induced cell damage.

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Section: Discussionsupporting

confidence: 62%

Section: Introductionmentioning

confidence: 99%

HepaRG-Progenitor Cell Derived Hepatocytes Cultured in Bioartificial Livers Are Protected from Healthy- and Acute Liver Failure-Plasma Induced Toxicity

Wenum¹,

Treskes²,

Adam³

et al. 2018

Cell Physiol Biochem

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“…Otherwise, the oxygen consumption could lead to local domains where a significant fraction of cells are either hypometabolic or dead from low oxygen tension (Foy et al, 1994;Gerlach et al, 1990;Hu et al, 1997;Jauregui et al, 1996;McGuire et al, 1995;Nyberg et al, 1992a, 1994Stefanovich et al, 1996;Yarmush et al, 1992). This is particularly true of newer high-density cell packing topologies or multicellular systems (Bader et al, 1995;Dixit, 1994;Gerlach, 1996;Gerlach et al, 1994;Hu et al, 1997;Hughes and Williams, 1996).…”

Section: Introductionmentioning

confidence: 93%

Oxygen Consumption Characteristics of Porcine Hepatocytes

Balis

Behnia

Dwarakanath

et al. 1999

Metabolic Engineering

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120

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“…We previously reported that primary rat hepatocytes cultured in standard hepatocyte culture medium become severely steatotic and exhibit decreased hepatocellular functions within a short time when exposed to plasma, as would be the case during clinical operation of a bioartificial liver device Stefanovich et al, 1996). More recently, we noted that preconditioning primary rat hepatocytes in culture medium containing low levels of insulin reduced intracellular lipid accumulation during subsequent plasma exposure (Chan et al, 2002), and that amino acid supplementation to the plasma restored albumin and urea secretion, as well as ammonia removal (Chan et al, 2002;Washizu et al, 2000).…”

Section: Introductionmentioning

confidence: 97%

Metabolic flux analysis of cultured hepatocytes exposed to plasma

Chan

Berthiaume

Lee

et al. 2002

Biotech & Bioengineering

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Hepatic metabolism can be investigated using metabolic flux analysis (MFA), which provides a comprehensive overview of the intracellular metabolic flux distribution. The characterization of intermediary metabolism in hepatocytes is important for all biotechnological applications involving liver cells, including the development of bioartificial liver (BAL) devices. During BAL operation, hepatocytes are exposed to plasma or blood from the patient, at which time they are prone to accumulate intracellular lipids and exhibit poor liver-specific functions. In a prior study, we found that preconditioning the primary rat hepatocytes in culture medium containing physiological levels of insulin, as opposed to the typical supraphysiological levels found in standard hepatocyte culture media, reduced lipid accumulation during subsequent plasma exposure. Furthermore, supplementing the plasma with amino acids restored hepatospecific functions. In the current study, we used MFA to quantify the changes in intracellular pathway fluxes of primary rat hepatocytes in response to low-insulin preconditioning and amino acid supplementation. We found that culturing hepatocytes in medium containing lower physiological levels of insulin decreased the clearance of glucose and glycerol with a concomitant decrease in glycolysis. These findings are consistent with the general notion that low insulin, especially in the presence of high glucagon levels, downregulates glycolysis in favor of gluconeogenesis in hepatocytes. The MFA model shows that, during subsequent plasma exposure, low-insulin preconditioning upregulated gluconeogenesis, with lactate as the primary precursor in unsupplemented plasma, with a greater contribution from deaminated amino acids in amino acid-supplemented plasma. Concomitantly, low-insulin preconditioning increased fatty acid oxidation, an effect that was further enhanced by amino acid supplementation to the plasma. The increase in fatty acid oxidation reduced intracellular triglyceride accumulation. Overall, these findings are consistent with the notion that the insulin level in medium culture presets the metabolic machinery of hepatocytes such that it directly impacts on their metabolic behavior during subsequent plasma culture.

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Extracorporeal Plasma Perfusion of Cultured Hepatocytes: Effect of Intermittent Perfusion on Hepatocyte Function and Morphology

Cited by 30 publications

References 4 publications

HepaRG-Progenitor Cell Derived Hepatocytes Cultured in Bioartificial Livers Are Protected from Healthy- and Acute Liver Failure-Plasma Induced Toxicity

HepaRG-Progenitor Cell Derived Hepatocytes Cultured in Bioartificial Livers Are Protected from Healthy- and Acute Liver Failure-Plasma Induced Toxicity

Oxygen Consumption Characteristics of Porcine Hepatocytes

Metabolic flux analysis of cultured hepatocytes exposed to plasma

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