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.

Schlüsselwörter

Tissue Engineering - Hepatozytensphäroide - Transplantation - Flusskultur - Bioreaktor

Key words

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

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