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

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.

Tissue Engineering - Hepatozytensphäroide - Transplantation - Flusskultur - Bioreaktor

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
• 2 American Liver Foundation .Fact sheet - Hepatitis, liver and gallbladder diseases in the United States. http://gi.ucsf.edu/alf/info/facsheet.html 1997
• 3 Eurotransplant International Foundation .Annual Report. 1999
• 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
• 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
• 6 Folkman J, Hochberg M. Self-regulation of growth in three dimensions.  J Exp Med. 1973;  138 745-753
• 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
• 8 Gupta S, Chowdhury R. Hepatocyte transplantation: Back to the future.  Hepatology. 1992;  15 156-162
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 16 Langer R, Vacanti J P. Tissue Engineering.  Science. 1993;  260 920-926
• 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
• 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
• 19 Ma P X, Zhang R. Synthetic nano-scale fibrous extracellular matrix.  J Biomed Mat Res. 1999;  46 60-72
• 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
• 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
• 22 Mooney D J, Vacanti J P. Tissue engineering using cells and synthetic polymers.  Transplant Rev. 1993;  7 153-162
• 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
• 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
• 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
• 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
• 27 Pollok J M, Vacanti J P. Tissue engineering.  Semin Ped Surg. 1996;  5 191-196
• 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
• 29 Seglen P O. Preparation of isolated rat liver cells.  Methods Cell Biol. 1976;  13 29-83
• 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
• 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
• 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
• 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
• 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
• 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
• 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); 
• 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
• 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
• 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
• 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
• 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
• 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