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Zentralblatt für Kinderchirurgie 2002; 11(4): 188-194
DOI: 10.1055/s-2002-36617
Originalarbeit

© Georg Thieme Verlag Stuttgart · New York

Präimplantations-Optimierung der Kultur von Hepatozyten im Flussbioreaktor für das Tissue Engineering von Lebergewebe auf 3-dimensionalen biologisch abbaubaren Polymeren

Pre-implantation Optimization of Culture Conditions for Hepatocytes in a Flow Bioreactor for Hepatic Tissue Engineering on 3-dimensional Biodegradable PolymersÉ. Török1,3 , J.-M. Pollok1 , P. X. Ma2 , P. M. Kaufmann1 , M. Dandri4 , J. Petersen4,5 , M.-R. Burda4 , D. Kluth1 , F. Perner3 , X. Rogiers1
  • 1Klinik und Poliklinik für Chirurgie, Abteilung für Hepatobiliäre Chirurgie und Abteilung für Kinderchirurgie, Universitätsklinikum Hamburg-Eppendorf, Hamburg
  • 2Dental School, University of Michigan, Ann Arbor; MI, USA
  • 3Klinik für Transplantation und Chirurgie, Semmelweis Medizinische Universität, Budapest, Ungarn
  • 4Heinrich-Pette-Insititut, Universität Hamburg, Hamburg
  • 5Klinik und Poliklinik für Innere Medizin, Medizinische Kernklinik, Universitätsklinikum Hamburg-Eppendorf, Hamburg
Further Information

Publication History

Publication Date:
14 January 2003 (online)

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Zusammenfassung

Ziel: Unsere Hypothese ist, dass die in vitro Konditionierung von Hepatozyten innerhalb einer biologisch abbaubaren Poly-L-Lactit (PLLA) Polymer-Matrix vor der Implantation das Überleben und die Funktion der Hepatozyten nach der Transplantation verbessert. Das Ziel unserer Studie war es, die Kulturbedingungen für Hepatozyten in einem pulsatilen Flussbioreaktor zu optimieren.
Material und Methoden: PLLA-Scheiben wurden mit Ratten-Hepatozyten in einer Konzentration von 2,5, 5, 10, 20 und 40 × 106 Zellen/ml besiedelt. Die besiedelten Polymer-Scheiben wurden einem rezirkulierten vertikalen Fluss von 0, 7, 15, 24, 32 und 52 ml/min von angereichertem Williams' Medium E ausgesetzt und nach 6 Tagen aus der Flusskultur entnommen.
Ergebnisse: Hepatozyten formten nur unter Flussbedingungen sphäroidale Aggregate von 50 bis 260 µm im Durchmesser. Diese Aggregate hatten leberähnliche Morphologie und eine spezifische aktive metabolische Funktion. Die Zahl der Sphäroide wurde mittels Phasenkontrastmikroskopie untersucht. Die Dehydrogenase Enzymfunktion der Hepatozyten wurde mittels MTT-Test untersucht. Die H & E-Histologie zeigte vitale Hepatozyten innerhalb von Sphäroiden kleiner als 200 µm im Durchmesser. Nur die wenigen Sphäroide mit einem größeren Durchmesser zeigten eine zentrale Nekrose. Die immunhistochemische Färbung konnte die Albuminproduktion der Hepatozyten innerhalb der Sphäroide darstellen. Die optimale Zellaussiedelungskonzentration betrug 10 × 106 Zellen/ml mit einer Flussgeschwindigkeit von 24 ml/min.
Schlussfolgerung: Hepatozytensphäroide, die mit dieser Flussbioreaktor Kulturmethode erzeugt wurden, könnten sich als funktionelle Einheit für das Tissue Engineering eines in vivo Leberersatzes nützlich erweisen.

Abstract

Purpose: We hypothesize that in vitro conditioning of hepatocytes within biodegradable poly-L-lactic acid (PLLA) polymer matrices prior to implantation may increase hepatocyte survival and function after transplantation. The purpose of this study was to optimize the culture conditions of hepatocytes in a pulsatile flow bioreactor.
Material and Methods: PLLA discs were seeded with rat hepatocytes in a concentration of 2.5, 5, 10, 20 and 40 × 106 cells/ml. Seeded discs were exposed to recirculated perpendicular flow of 0, 7, 15, 24, 32, 52 ml/min of supplemented Williams' Medium E and harvested after six days in flow culture.
Results: Only under flow conditions the hepatocytes formed spheroidal aggregates of 50 to 260 µm in diameter with a liver like morphology and active metabolic function. The number of spheroids was examined by phasecontrast microscopy and the dehydrogenase enzyme function of the hepatocytes was tested using MTT. H & E histology showed vital hepatocytes within the spheroids less than 200 µm in diameter but central necrosis in the spheroids exceeding this size. Immunohistochemical staining confirmed albumin production of hepatocytes within the spheroids. The optimal cell seeding concentration was 10 × 106 cells/ml with a flow speed of 24 ml/min.
Conclusion: Spheroids of hepatocytes cultured with this flow bioreactor method may prove useful as a functional unit for tissue engineering of an in vivo liver substitute.

Schlüsselwörter

Tissue Engineering - Hepatozytensphäroide - Transplantation - Flusskultur - Bioreaktor

Key words

Tissue engineering - hepatocyte spheroids - transplantation - flow-culture - bioreactor

Literatur

  • 1 Aiken J, Cima L, Schloo B, Mooney D, Johnson L, Langer R, Vacanti J P. Studies in rat liver perfusion for optimum harvest of hepatocytes.  J Pediatr Surg. 1990;  25 140-145
    PubMedGoogle Scholar
  • 2 American Liver Foundation .Fact sheet - Hepatitis, liver and gallbladder diseases in the United States. http://gi.ucsf.edu/alf/info/facsheet.html 1997
    Google Scholar
  • 3 Eurotransplant International Foundation .Annual Report. 1999
    Google Scholar
  • 4 Asano K, Koide N, Tsuji T. Ultrastructure of multicellular spheroids formed in the primary culture of adult rat hepatocytes.  J Clin Electron Microsc. 1989;  22 243-252
    PubMedGoogle Scholar
  • 5 Asonuma K, Gilbert J C, Stein J E, Takeda T, Vacanti J P. Quantification of transplanted hepatic mass necessary to cure the Gunn rat model of hyperbilirubinemia.  J Pediatr Surg. 1992;  27 298-301
    CrossrefPubMedGoogle Scholar
  • 6 Folkman J, Hochberg M. Self-regulation of growth in three dimensions.  J Exp Med. 1973;  138 745-753
    CrossrefPubMedGoogle Scholar
  • 7 Gerlach J C, Schnoy N, Encke J, Smith M D, Müller C, Neuhaus P. Improved Hepatocyte in vitro maintenance in a culture model with woven multicompartment capillary systems: Electron microscopy studies.  Hepatology. 1995;  22 546-552
    CrossrefPubMedGoogle Scholar
  • 8 Gupta S, Chowdhury R. Hepatocyte transplantation: Back to the future.  Hepatology. 1992;  15 156-162
    PubMedGoogle Scholar
  • 9 Kaufmann P M, Kneser U, Fiegel H, Pollok J M, Kluth D, Izbicki J R, Herbst H, Rogiers X. Is there an optimal concentration of cotransplanted islets of Langerhans for stimulation of hepatocytes in three dimensional matrices?.  Transplantation. 1999;  68 272-279
    PubMedGoogle Scholar
  • 10 Kawaguchi M, Koide N, Sakaguchi K, Shinji T, Tsuji T. Combination of epidermal growth factor and insulin is required for multicellular spheroid formation of rat hepatocytes in primary culture.  Acta Med Okayama. 1992;  46 195-201
    PubMedGoogle Scholar
  • 11 Kim S S, Utsunomija H, Koski J A, Wu B M, Cima M J, Sohn J, Mukai K, Griffith L G, Vacanti J P. Survival and function of hepatocytes on a novel three-dimensional synthetic biodegradable polymer scaffold with an intrinsic network of channels.  Ann Surg. 1998;  228 8-13
    CrossrefPubMedGoogle Scholar
  • 12 Kneser U, Kaufmann P M, Fiegel H C, Pollok J M, Kluth D, Izbicki J R, Herbst H, Rogiers X. Long term differentiated function of heterotopically transplanted hepatocytes on three dimensional polymer matrices.  J Biomed Mater Res. 1999;  47 494-503
    CrossrefPubMedGoogle Scholar
  • 13 Koide N, Sakaguchi K, Koide Y, Asano K, Kawaguchi M, Matsushima H, Takenami T, Shinji T, Mori M, Tsuji T. Formation of multicellular spheroids composed of adult rat hepatocytes in dishes with positively charged surfaces and other nonadherent environments.  Exp Cell Res. 1990;  186 227-235
    CrossrefPubMedGoogle Scholar
  • 14 Koide N, Shinji T, Tanabe T, Asano K, Kawaguchi M, Sakaguchi K, Koide Y, Mori M, Tsuji T. Continued high albumin production by multicellular spheroids of adult rat hepatocytes formed in the presence of liver-derived proteoglycans.  Biochem Biophys Res Commun. 1989;  161 385-391
    CrossrefPubMedGoogle Scholar
  • 15 Landry B, Bernier D, Ouellet C, Gayety R, Marcia N. Spheroidal aggregate of rat liver cells: histotypic reorganization, biomatrix deposition, and maintenance of functional activities.  J Cell Biol. 1985;  101 914-923
    CrossrefPubMedGoogle Scholar
  • 16 Langer R, Vacanti J P. Tissue Engineering.  Science. 1993;  260 920-926
    CrossrefPubMedGoogle Scholar
  • 17 Lazar A, Peshwa M V, Wu F J, Chi C M, Cerra F B, Hu W S. Formation of porcine hepatocyte spheroids for use in a bioartificial liver.  Cell Transplant. 1995;  4 259-268
    PubMedGoogle Scholar
  • 18 Ma P X, Langer R. Fabrication of biodegradable polymer foams for cell transplantation and tissue engineering. In: Yarmush M, Morgan J (eds.) Tissue Engineering Methods and Protocols. Huma
    Google Scholar
  • 19 Ma P X, Zhang R. Synthetic nano-scale fibrous extracellular matrix.  J Biomed Mat Res. 1999;  46 60-72
    PubMedGoogle Scholar
  • 20 Marshall N J, Goodwin C J, Holt S J. A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function.  Growth Regul. 1995;  5 69-84
    PubMedGoogle Scholar
  • 21 Matsushita T, Ijima H, Koide N, Funatsu K. High albumin production by multicellular spheroids of adult rat hepatocytes formed in the pores of polyurethane foam.  Appl Microbiol Biotechnol. 1991;  36 324-326
    PubMedGoogle Scholar
  • 22 Mooney D J, Vacanti J P. Tissue engineering using cells and synthetic polymers.  Transplant Rev. 1993;  7 153-162
    PubMedGoogle Scholar
  • 23 Niwa T, Koide N, Tsuji T, Imaoka S, Ishibashi F, Funae Y, Katagiri M. Cytochrome P450s of isolated rat hepatocytes in spheroid and monolayer cultures.  Res Comm Mol Path Pharm. 1996;  91 372-378
    PubMedGoogle Scholar
  • 24 Peshwa M V, Wu F J, Follstad B D, Cerra F B, Hu W S. Kinetics of formation of hepatocyte spheroids.  Biotechnol Prog. 1994;  10 460-466
    CrossrefPubMedGoogle Scholar
  • 25 Peshwa M V, Wu F J, Sharp H L, Cerra F B, Hu W S. Mechanistics of formation and cultural ultrastructural evaluation of hepatocyte spheroids.  In Vitro Cell Dev Biol Animal. 1996;  32 197-203
    PubMedGoogle Scholar
  • 26 Pollok J M, Kluth D, Cusick R A, Lee H, Utsunomiya H, Ma P X, Langer R, Broelsch C E, Vacanti J P. Formation of spheroidal aggregates of hepatocytes on biodegradable polymers under continuous flow bioreactor conditions.  Eur J Ped Surg. 1998;  8 195-199
    PubMedGoogle Scholar
  • 27 Pollok J M, Vacanti J P. Tissue engineering.  Semin Ped Surg. 1996;  5 191-196
    PubMedGoogle Scholar
  • 28 Sakaguchi K, Koide N, Asano K, Takabatake H, Matsushima H, Takenami T, Ono R, Sasaki S, Mori M, Koide Y, Tsuji T. Promotion of spheroid assembly of adult rat hepatocytes by some factor(s) present in the initial 6-hour conditioned medium of the primary culture.  Pathobiology. 1991;  59 351-356
    PubMedGoogle Scholar
  • 29 Seglen P O. Preparation of isolated rat liver cells.  Methods Cell Biol. 1976;  13 29-83
    PubMedGoogle Scholar
  • 30 Shinji T, Koide N, Tsuji T. Glycosaminoglycans partially substitute for proteoglycans in spheroid formation of adult rat hepatocytes in primary culture.  Cell Struct Funct. 1988;  13 179-188
    PubMedGoogle Scholar
  • 31 Slater T F, Saywer B, Sträuli U. Studies on succinate-tetrazolium reductase systems: III. Points of coupling of four different tetrazolium salts.  Biochim Biophys Acta. 1963;  77 383-393
    CrossrefPubMedGoogle Scholar
  • 32 Takeda T, Kim T H, Lee S K, Langer R, Vacanti J P. Hepatocyte transplantation in biodegradable polymer scaffolds using the Dalmatian dog model of hyperuricosuria.  Transplant Proc. 1995;  27 635-636
    PubMedGoogle Scholar
  • 33 Tong J Z, Bernard O, Alvarez F. Long-term culture of rat liver cell spheroids in hormonally defined media.  Exp Cell Res. 1990;  189 87-92
    CrossrefPubMedGoogle Scholar
  • 34 Tong J Z, De Lagausie P, Furlan V, Cresteil T, Bernard O, Alvarez F. Long-term culture of adult rat hepatocyte spheroids.  Exp Cell Res. 1992;  200 326-332
    CrossrefPubMedGoogle Scholar
  • 35 Ueno K, Miyashita A, Endoh K I, Takezawa T, Yamazaki M, Mori Y, Satoh T. Formation of multicellular Spheroids composed of rat hepatocytes.  Res Comm Chem Path Pharmacol. 1992;  77 107-120
    PubMedGoogle Scholar
  • 36 Uyama S, Kaufmann P M, Kneser U, Fiegel H C, Pollok J M, Kluth D, Vacanti J P, Rogiers X. Hepatocyte transplantation using biodegradable matrices in ascorbic acid-deficient rats: Comparison with heterotopically transplanted liver grafts.  Transplantation. (in press); 
    PubMedGoogle Scholar
  • 37 Uyama S, Kaufmann P M, Takeda T, Vacanti J P. Delivery of whole liver-equivalent hepatocyte mass using polymer devices and hepatotrophic stimulation.  Transplantation.. 1993;  55 932-935
    PubMedGoogle Scholar
  • 38 Vacanti J P, Langer R. Tissue engineering: the design and fabrication of living replacement devices for surgical reconstruction and transplantation.  Lancet. 1999;  354 32-34
    CrossrefPubMedGoogle Scholar
  • 39 Vacanti J P, Morse M A, Saltzman M, Domb A J, Perez-Atayde A, Langer R. Selective cell transplantation using bioabsorbable artificial polymers as matrices.  J Pediatr Surg. 1988;  23 3-9
    CrossrefPubMedGoogle Scholar
  • 40 Wu F J, Friend J R, Hsiao C C, Zilliox M J, Ko W J, Cerra F B, Hu W S. Efficient assembly of rat hepatocyte spheroids for tissue engineering applications.  Biotechnol Bioeng. 1996;  50 404-415
    PubMedGoogle Scholar
  • 41 Yamada K, Kamihira M, Hamamoto R, lijima S. Efficient induction of hepatocyte spheroids in a Suspension culture using a water-soluble synthetic polymer as an artificial matrix.  J Biochem. 1998;  123 1017-1023
    PubMedGoogle Scholar
  • 42 Yuasa C, Tomita Y, Shono M, Ishimura K, Ichihara A. Importance of cell aggregation for expression of liver functions and regeneration demonstrated with primary cultured hepatocytes.  J Cell Phisiol. 1993;  156 522-530
    PubMedGoogle Scholar

PD Dr. med. D. Kluth

Klinik und Poliklinik für Chirurgie · Abteilung für Kinderchirurgie · Universitätsklinikum Hamburg-Eppendorf

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20246 Hamburg

Phone: 0 40-4 28 03-24 97

Fax: 0 40-4 28 03-69 14

Email: kluth@uke.uni-hamburg.de

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