In-vitro-Kultivierung von Zellen der respiratorischen Schleimhaut auf Matrizes aus Kollagen, Poly-L-Laktid (PLLA) und Polyhydroxybuttersäure (PHB)

Zusammenfassung

Hintergrund: Der Ersatz autologer respiratorischer Schleimhaut im Rahmen der Deckung von Defekten im HNO-Bereich (Trachea, Septum, Nasennebenhöhlen) erscheint aus therapeutischer Sicht notwendig.

Methode: Zellen der respiratorischen Schleimhaut wurden durch Auswachsen oder nach Dissoziation auf Materialien aus Kollagen, PLLA und PHB in serumhaltigen und serumfreien Medium kultiviert.

Ergebnis: Auf allen Materialien konnten Fibroblasten und auch Epithelzellen gezüchtet werden. PLLA stellte sich als besonders günstige Matrix dar. Auf PHB gelang Wachstum nur nach Aminofunktionalisierung mittels Plasmabehandlung. Besiedelung konnte bei Kollagenmaterialien nicht lichtmikroskopisch, sondern nur indirekt (Membranfärbung, REM) nachgewiesen werden. Eine Differenzierung der proliferierten Zellen bis zu aktiver Zilienbewegung konnte auch nach mehreren Wochen nicht gefunden werden.

Schlussfolgerungen: Die untersuchten Materialien sind in unterschiedlichem Ausmaß zur Besiedelung mit Zellen der respiratorischen Schleimhaut geeignet. Für die Entwicklung final differenzierter Epithelzellen müssen die Kulturbedingungen geändert werden.

Abstract

Background: The replacement of respiratory mucosa after surgical resections at different locations in the ENT-field (trachea, nasal septum, sinus maxillaris) seems to be essential or at least of great benefit for patients. Presently a satisfying solution for this challenge does not exist. Therefore we have analysed the growth of cells from respiratory mucosa on different matrices.

Methods: Cell cultures are initiated mainly by growing out cells from tissue pieces but also by seeding of cells after enzymatic dissociation (sequential trypsinization) of mucosa. Cells were cultured with serum-containing and serum-free media on 3 different foils: made of collagen, of Poly-L-lactic acid (PLLA) and Poly-hydroxybutyric acid (PHB). Culturing time was on average about 4 - 5 weeks.

Results: On all tested materials fibroblasts and epithelial cells have grown in principle. PLLA was the material with best properties concerning the aim of this study whereas culturing of cells on PHB only happens after surface modification by amino-functionalisation with plasma treatment. Visualisation of cells on materials of collagen could not realised by light-microscopy due to the 3-dimensional structure and the optical properties of this material. Here the cultures were analysed by cell membrane staining and by REM. We could not find a differentiation of epithelial cells with beating cilia 6 weeks after starting the culture. However 20 days after starting the culture on PLLA (good conditions for observation with light-microscope) cells with beating cilia could be observed, in our opinion resulting from dissociated cells from tissue pieces and not from proliferated cells.

Conclusions: In principle the analysed materials are useful for the culturing of cells from respiratory mucosa. However, for obtaining differentiated epithelial cells the culture conditions have to be modified.

Schlüsselwörter

Respiratorisches Epithel · Tissue engineering · Zellkultur · Matrizes · Kollagen · PLLA · PHB

Key words

Cell culture · respiratory mucosa · culture condition · collagen · PLLA · PHB · epithel · fibroblasts

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