Zusammenfassung
Hintergrund
Perfusionskultursysteme werden heute überwiegend für die Untersuchung zellphysiologischer Fragestellungen und für die Züchtung dreidimensionaler Gewebekonstrukte verwendet. In der Regel sind diese Systeme relativ teuer und ermöglichen keine kontinuierliche mikroskopische Kontrolle der wachsenden Zellen. Einfache und kostengünstige Perfusionskultursysteme stehen bisher nicht zur Verfügung.
Material und Methoden
Es wurde ein neuartiges Perfusionskultursystem entwickelt, dessen Module aus einer Halterung zum Einsatz unterschiedlicher Medienversorgungssysteme, Mikrodosierpumpen und laminar durchströmter Kulturkammern mit je 8 cm3 Kulturfläche bestehen. Die Perfusionskammern wurden mit humanen osteoblastären Zellen aus der Gewebekultur besiedelt (5000/cm2) und nach Adhärenz der Zellen über 10 Tage perfundiert (0,5 ml/min). Als Kontrollgruppe wurden osteoblastäre Zellen in baugleichen Kulturkammern ohne Mediumperfusion mitgeführt. Nach 10 Tagen wurden die Zellzahlen nach dem Coulterprinzip bestimmt. Als Differenzierungsmerkmal wurde die alkalische Phosphatase photometrisch gemessen.
Ergebnisse
In den Perfusionskulturen konnte in 10 Tagen die 3- bis 4fache Zellmenge im Vergleich zur Kontrollgruppe gezüchtet werden. Die Werte der alkalischen Phosphatase lagen gleich hoch bzw. nur gering niedriger, was auf eine erhaltene osteoblastäre Differenzierung der Zellen bei höherer Proliferation hinweist.
Fazit
Eine möglichst hohe Zahl in vitro vermehrter Zellen ist die Grundvoraussetzung für die klinische Anwendung des Tissue-Engineering. Das erprobte Perfusionskultursystem ermöglicht durch eine kontinuierliche Medienversorgung eine höhere Proliferationsrate osteoblastärer Zellen bei erhaltener Differenzierung. Durch die Verwendung handelsüblicher Petri-Schalen ist die permanente mikroskopische Kontrolle der Kulturen möglich.
Abstract
Background
Today, perfusion culture systems are mainly used to investigate cellular physiology and to cultivate three-dimensional tissue complexes. As a rule, these systems are relatively expensive and do not enable continuous microscopic monitoring of the growing cells. Simple and inexpensive perfusion culture systems have not been available up to now.
Methods
A novel perfusion culture system was developed in which the modular components consist of a mounting apparatus for inserting various media supply systems, microdispenser pumps, and laminar-flow culture chambers, each with a culture volume of 8 cm3. The perfusion chambers were inoculated with human osteoblast cells from the tissue culture (5,000/cm2) and were perfused for 10 days after adherence of the cells (0.5 ml/min). As a control group, osteoblast-like cells were cultured in identically constructed culture chambers without medium perfusion. After 10 days, the cell counts were determined in accordance with the Coulter principle. Alkaline phosphatase was measured photometrically as a characteristic for differentiation.
Results
Compared with the control group, three to four times the quantity of cells were produced within 10 days in the perfusion cultures. The alkaline phosphatase values were equally high or only slightly lower, indicating that osteoblast differentiation of the cells was maintained with a higher proliferation.
Conclusions
As large a number of in vitro proliferated cells as possible is a prerequisite for clinical application of tissue engineering. By continuously supplying medium, the tested perfusion culture system enables a higher rate of proliferation of osteoblast-like cells with maintenance of differentiation. Continuous microscopic monitoring of the cultures is possible using commercially available Petri dishes.
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Bücheler, M., Bücheler, BM., Hagenau, K. et al. Vermehrung und Differenzierung humaner Nasenseptum-Osteoblasten . HNO 56, 301–305 (2008). https://doi.org/10.1007/s00106-007-1653-2
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DOI: https://doi.org/10.1007/s00106-007-1653-2