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Intracellular nucleotide pools and ratios as tools for monitoring dedifferentiation of primary porcine hepatocytes in culture

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Abstract

The effect of two culture configurations (single collagen gel and double collagen gel) and of two hormones (insulin and glucagon) on the differentiated status and the intracellular nucleotide pools of primary porcine hepatocytes was investigated. The objective was to analyze and monitor the current state of differentiation supported by the two culture modes using intracellular nucleotide analysis. Specific intracellular nucleotide ratios, namely the nucleoside triphosphate (NTP) and the uridine (U) ratio were shown to consistently reflect the state of dedifferentiation status of the primary cells in culture affected by the presence of the two hormones insulin and glucagon. Continuous dedifferentiation of the cells was monitored in parallel by the reduction of the secretion of albumin, and changes in UDP-activated hexoses and UDP-glucuronate. The presence of insulin maintained the differentiated status of hepatocytes for more than 12 days when cultivated under double gel conditions whereas glucagon was less effective. In contrast, cells cultivated in a single gel matrix immediately started to dedifferentiate upon seeding. NTP and U ratios were shown to be more sensitive for monitoring dedifferentiation in culture than the albumin secretion. Their use allowed the generation of an easily applicable NTP–U plot in order to give a direct graphical representation of the current differentiation status of the cultured cells. Moreover, the transition from functional and differentiated hepatocytes to dedifferentiated fibroblasts could be determined earlier by the nucleotide ratios compared to the conventional method of monitoring the albumin secretion rate.

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Abbreviations

ADP:

adenosine-5′-diphosphate

AEC:

adenylate energy charge

AMP:

adenosine-5′-monophosphate

ATP:

adenosine-5′-triphosphate

CTP:

cytidine-5′-triphosphate

DG:

double gel

EGTA:

ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid

FALGPA:

furylarcraloyl-Leu-Gly-Pro-Ala

GlcUA:

glucuronate

GTP:

guanosine-5′-triphosphate

ELISA:

enzyme linked immunosorbent assay

GPDH:

glucose-6-phosphate dehydrogenase

HEPES:

N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)

Hex:

hexose

HK:

hexokinase

IP-RP-HPLC:

ion pair reversed phase high performance liquid chromatography

LDH:

lactate dehydrogenase

NTP:

nucleoside triphosphate

PFK:

phosphofructokinase

PK:

pyruvate kinase

OPA:

o-phthaldialdehyde

PCA:

perchloric acid

SG:

single gel

U:

uridine

UDP:

uridine-5′-diphosphate

UDP-Gal:

UDP-galactose

UDP-GalNAc:

UDP-N-acetylgalactosamine

UDP-Glc:

UDP-glucose

UDP-GlcNAc:

UDP-N-acetylglucosamine

UDPGNAc:

sum of UDP-GalNAc and UDP-GlcNAc

UDP-Hex:

UDP-Hexose (UDP-Gal, UDP-Glc)

UDP-GlcUA:

UDP-glucuronate

UTP:

uridine-5′-triphosphate

References

  • Atkinson DA (1977) Cellular energy metabolism and its regulation. Academic Press, New York

    Google Scholar 

  • Andus T, Geiger T, Hirano T, Kishimoto T, Tran-thi TA, Decker K, Heinrich PC (1988) Regulation of synthesis and secretion of major acute-phase proteins by recombinant human interleukin-6 (BSF-2/IL-6) in hepatocyte primary culture. Eur J Biochem 173:287–293

    Article  CAS  Google Scholar 

  • Annoni G, Weiner FR, Colombo M, Czaja MJ, Zern MA (1990) Albumin and collagen gene regulation in alcohol- and virus-induced human liver. Gastroenterology 98:197–202

    CAS  Google Scholar 

  • Bader A, De Bartolo L, Haverich A (2000) High level benzodiazepine and ammonia clearance by flat membrane bioreactors with porcine liver cells. J Biotechnol 81:95–105

    Article  CAS  Google Scholar 

  • Bader A, De Bartolo L, Haverich A (1997) Initial evaluation of the performance of a scaled-up flat membrane bioreactor (FMB) with pig liver cells. In: Crepaldi G, Demetrioou AA, Muraca M (eds) Bioartificial liver: the critical issues. CIC International Editions, Rome, pp 36–41

    Google Scholar 

  • Bader A, Knop E, Frühauf N, Crome O, Boker K, Christians U, Oldhafer K, Ringe B, Pichlmayr R, Sewing KF (1995) Reconstruction of liver tissue in vitro: geometry and characteristic flat bed, holllow fiber, and spouted bed bioreactors with reference to the in vivo liver. Artif Organs 19:941–950

    CAS  Google Scholar 

  • Boudreau N, Sympson CJ, Werb Z, Bissell MJ (1995) Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix. Science 267:891–893

    Article  CAS  Google Scholar 

  • Bucher NL, Robinson GS, Farmer SR (1990) Effects of extracellular matrix on hepatocyte growth and gene expression: implications for hepatic regeneration and the repair of liver injury. Sem Liver Dis 10:11–19

    CAS  Google Scholar 

  • Carabaza A, Guinovart JJ, Ciudad CJ (1986) Activation of hepatocyte glycogen synthase by metabolic inhibitors. Arch Biochem Biophys 250:469–475

    Article  CAS  Google Scholar 

  • Castell JV, Gomez-Lechon MJ, David M, Fabra R, Trullenque R, Heinrich PC (1991) Acute-phase response of human hepatocytes: Regulation of acute-phase protein synthesis by interleukin-6. Hepatology 12:1179–1186

    Google Scholar 

  • Chaney AL, Marbach EP (1962) Modified reagents for the determination of urea and ammonia. Clin Chem 8:130–132

    CAS  Google Scholar 

  • Chou IN, Zeiger J, Rapaport E (1984) Imbalance of total cellular nucleotide pools and mechanism of the colchicine-induced cell activation. Proc Natl Acad Sci USA 81:2401–2405

    Article  CAS  Google Scholar 

  • De Bartolo L, Jarosch von Schweder G, Haverich A, Bader A (2000) A novel full-scale flat membrane bioreactor utilizing porcine hepatocytes: Cell viability and tissue-specific functions. Biotechnol Prog 16:102–108

    Article  CAS  Google Scholar 

  • Demetriou AA, Whiting J, Levenson SM, Chowdhury NR, Schechner R, Michalski S, Feldman D, Chowdhury JR (1986) New method of hepatocyte transplantation and extracorporeal liver support. Ann Surg 204:259–270

    Article  CAS  Google Scholar 

  • Deutschmann SM, Valley U, Jäger V, Wagner R (1994) Cell cycle analysis as a tool for control and regulation of mammalian cell cultures in bioreactors. In: Spier RE, Griffiths JB, Berthold W (eds) Animal cell technology: products of today, prospects of tomorrow. Butterworths-Heinemann, Oxford, UK, pp 354–356

    Google Scholar 

  • Dunn JCY, Tompkins RG, Yarmoush ML (1991) Long-term in vitro function of adult hepatocytes in a collagen sandwich configuration. Biotechnol Prog 7:237–245

    Article  CAS  Google Scholar 

  • Dunn JCY, Yarmush ML, Koebe HG, Tompkins RG (1989) Hepatocyte function and extracellular matrix geometry: long-term culture in a sandwich configuration. FASEB J 3:174–177

    CAS  Google Scholar 

  • Ekberg K, Chandramouli V, Kumaran K, Schumann WC, Wahren J, Landau BR (1995) Gluconeogenesis and glucuronidation in liver in vivo and the heterogeneity of hepatocyte function. J Biol Chem 270:21715–21717

    Article  CAS  Google Scholar 

  • Elsdale T, Bard J (1972) Collagen substrata for studies on cell behaviour. J Cell Biol 54:626–637

    Article  CAS  Google Scholar 

  • Fang F, Orend G, Watanabe N, Hunter T, Ruoslahti E (1996) Dependence of cyclin E-CDK2 kinase activity on cell anchorage. Science 271:499–502

    Article  CAS  Google Scholar 

  • Flendrig LM, La Soe JW, Joerning GGA, Steenbeck A, Karlsen OT, Bovee WM, Ladiges NC, te-Velde AA, Chamuleau RA (1997) In vitro evaluation of a novel bioreactor based on an integral oxygenator and a spirally wound nonwoven polyester matrix for hepatocyte culture as small aggregates. J Hepatol 26:1379–1392

    Article  CAS  Google Scholar 

  • Frisch SM, Francis H (1994) Disruption of epithelial cell-matrix interactions induces apoptosis. J Cell Biol 124:619–626

    Article  CAS  Google Scholar 

  • Gawlitzek M, Valley U, Wagner R (1998) Ammonium ion/glucosamine dependent increase of oligosaccharide complexity in recombinant glycoproteins secreted from cultivated BHK-21 cells. Biotechnol Bioeng 57:518–528

    Article  CAS  Google Scholar 

  • Gawlitzek M, Papac DI, Sliwkowski MB, Ryll T (1999) Incorporation of 15N from ammonium into the N-linked oligosaccharides of an immunoadhesin glycoprotein expressed in Chinese hamster ovary cells. Glycobiology 9:125–131

    Article  CAS  Google Scholar 

  • Gauldie J, Richards C, Harnish D, Landsorp P, Baumann H (1987) Interferon β2/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the majur acute-phase protein response in liver cells. Proc Natl Acad Sci USA 84:7251–7255

    Article  CAS  Google Scholar 

  • Gerlach J, Schnoy N, Smith MD, Neuhaus P (1994) Hepatocyte culture between woven capillary network-A micros-copy study. Artif Organs 18:1–5

    Article  Google Scholar 

  • Grammatikos SI, Tobien K, Noë W, Werner RG (1999) Monitoring of intracellular ribonucleotide pools is a powerful tool in the development and characterization of mammalian cell culture processes. Biotechnol Bioeng 64:357–367

    Article  CAS  Google Scholar 

  • Grummt F, Paul D, Grummt I (1977) Regulation of ATP pools, rRNA and DNA synthesis in 3T3 cells in response to serum or hypoxanthine. Eur J Biochem 76:7–12

    Article  CAS  Google Scholar 

  • Inaba T, Tait A, Nakano M, Mahon WA, Kalow W (1988) Metabolism of diazepam in vitro by human liver. Drug Metab Depos 16:605–608

    CAS  Google Scholar 

  • Itoh Y, Okanoue T, Enjo F, Sakamoto S, Takami S, Yasui K, Kagawa K, Kashima K (1994) Regulation of hepatocyte albumin and α1-acid glycoprotein secretion by monokines, dexamethasone, and nitric oxide synthase pathway: Significance of activated liver nonparenchymal cells. Dig Dis Sci 39:851–859

    Article  CAS  Google Scholar 

  • Kim BH, Sung SR, Choi EH, Kim YI, Kim KJ, Dong SH, Kim HJ, Chang YW, Lee JI, Chang R (2000) Dedifferentation of conditionally immortalized hepatocytes with long-term in vitro passage. Exp Mol Med 32:29–37

    Google Scholar 

  • Kobayashi A, Goto M, Kobayashi K, Akaike T (1994) Receptor-mediated regulation of differentiation and proliferation of hepatocytes by synthetic polymer model of asialoglycoprotein. J Biomater Sci Polymer Edn 6:325–342

    CAS  Google Scholar 

  • Koj A, Gauldie J, Regoeczi E, Sauder DN, Sweeney GD (1984) The acute-phase response of cultured rat hepatocytes. Biochem J 224:505–514

    CAS  Google Scholar 

  • Larsen BR, West FG (1981) A method for quantitative amino acid analysis using precolumn o-phthaldialdehyd derivatization and high performance liquid chromatography. J Chromatogr Sci 19:259–265

    CAS  Google Scholar 

  • Meredith JE Jr, Fazeli B, Schwartz MA (1993) The extracellular matrix as a cell survival factor. Mol Biol Cell 4:953–961

    CAS  Google Scholar 

  • Miners JO, Mackenzie PI (1991) Drug glucuronidation in humans. Pharmacol Ther 5:347–369

    Article  Google Scholar 

  • Murphree S, Moore EC, Peterson D (1974) Temporal variation of adenine ribonucleotides during the cell cycle of Chinese hamster fibroblasts in culture. Exp Cell Res 83:189–190

    Article  Google Scholar 

  • Oude Elferink RP, Jansen PL (1994) The role of the canalicular multispecific organic anion transporter in the disposal of endo- and xenobiotics. Pharmacol Ther 64:77–97

    Article  CAS  Google Scholar 

  • Ranucci CS, Kumar A, Surenda PB, Moghe PV (2000) Control of hepatocyte function on collagen foams: sizing matrix pores toward selective induction of 2-D and 3-D cellular morphogenesis. Biomaterials 21:783–793

    Article  CAS  Google Scholar 

  • Rapaport E, Garcia-Blanco MA, Zamecnik PC (1979) Regulation of DNA replication in S phase nuclei by ATP and ADP pools. Proc Natl Acad Sci USA 76:1643–1647

    Article  CAS  Google Scholar 

  • Re F, Zanetti A, Sironi M, Polentarutti N, Lanfrancone L, Dejana E, Colotta F (1994) Inhibition of anchorage-dependent cell spreading triggers apoptosis in cultured human endothelial cells. J Cell Biol 127:537–546

    Article  CAS  Google Scholar 

  • Rothschild MA, Oratz M, Schreiber SS (1988) Serum albumin. Hepatology 8:385–401

    CAS  Google Scholar 

  • Ryll T, Valley U, Wagner R (1994) Biochemistry of growth inhibition by ammonium ions in mammalian cells. Biotechnol Bioeng 44:184–119

    Article  CAS  Google Scholar 

  • Ryll T, Wagner R (1991) An improved ion-pair HPLC method for the quantification of a wide variety of nucleotides and sugar-nucleotides in animal cells. J Chromatogr 570:77–88

    CAS  Google Scholar 

  • Ryll T, Wagner R (1992) Intracellular ribonucleotide pools as a tool for monitoring the physiological state of in vitro cultivated mammalian cells during production processes. Biotechnol Bioeng 40:934–946

    Article  CAS  Google Scholar 

  • Soars MG, Buchell B, Riley RJ (2002) In vitro analysis of human drug glucuronidation and prediction of in vivo metabolic clearance. J Pharm Exp Ther 301:382–390

    Article  CAS  Google Scholar 

  • Soars MG, Riley RJ, Findlay KAB, Coffey MJ, Burchell B (2001) Evidence for significant differences in microsomal drug glucuronidation by canine and human liver and kidney. Drug Metab Dispos 29:121–126

    CAS  Google Scholar 

  • Tilles AW, Baskaran H, Roy P, Yarmush ML, Toner M (2001) Effects of oxygenaton and flow on the viability and function of rat hepatocytes cocultured in a microchannel flat-plate bioreactor. Biotechnol Bioeng 73:379–389

    Article  CAS  Google Scholar 

  • Tobe S, Takei Y, Kobayashi K, Akaide T (1992) Receptor-mediated formation of multilayer aggregates of primary cultured adult rat hepatocytes on lactose-substituted polysterene. Biophys Biochem Res Commun 184:225–230

    Article  CAS  Google Scholar 

  • Valley U, Ryll T, Wagner R (1993) Process control of BHK cells by nucleotide pool measurement. In: Kaminogawa S, Ametani A, Hachimura S (eds) Animal cell technology: basic and applied aspects, vol 5, Kluwer Acad Pub, Dordrecht, pp 93–97

    Google Scholar 

  • Valley U, Nimtz M, Conradt HS, Wagner R (1999) Incorporation of ammonium into intracellular UDP-activated N-acetylhexosamines and into carbohydrate structures in glycoproteins. Biotechnol Bioeng 64:401–417

    Article  CAS  Google Scholar 

  • Yagi K, Sumiyoshi N, Nakashima Y, Michibayashi N, Kawase N, Miura Y, Mizoguchi T (1998) Stimulation of liver functions in hierachical co-culture of bone marrow cells and hepatocytes. Cytotechnology 26:5–12

    Article  CAS  Google Scholar 

  • Zhu X, Ohtsubo M, Boehmer RM, Assoian RK (1996) Adhesion-dependent cell cycle progression linked to the expression of cyclin D1, activation of cyclin E-cdk2 and phosphorylation of the retinoblastoma protein. J Cell Biol 133:392–403

    Article  Google Scholar 

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Acknowledgements

We are grateful to Dr. Mark Smith as a native speaker for helpful assistance in proofreading the manuscript.

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Author notes

  1. Dirk Rocker

    Present address: Altana Pharma Deutschland, Byk-Gulden-Straße 2, D-78467, Konstanz, Germany

  2. Friedemann Hesse

    Present address: Austrian Center of Biopharmaceutical Technology, Muthgasse 18, A-1190, Vienna, Austria

  3. Roland Wagner

    Present address: Miltenyi Biotec GmbH, Bioprocess Sciences, Robert-Koch-Straße 1, D-17166, Teterow, Germany

Authors and Affiliations

  1. Cell Culture Technology Department, German Reseach Centre for Biotechnology (GBF), Mascheroder Weg 1, D-38124, Braunschweig, Germany

    Dirk Rocker, Friedemann Hesse, Augustinus Bader & Roland Wagner

  2. Universität Leipzig, Biotechnologisch-Biomedizinisches Zentrum, Zelltechniken und angewandte Stammzellbiologie, Deutscher Platz 5, D-04103, Leipzig, Germany

    Augustinus Bader

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  1. Dirk Rocker
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Correspondence to Roland Wagner.

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Rocker, D., Hesse, F., Bader, A. et al. Intracellular nucleotide pools and ratios as tools for monitoring dedifferentiation of primary porcine hepatocytes in culture. Cytotechnology 51, 119–132 (2006). https://doi.org/10.1007/s10616-006-9019-2

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