The use of a conventional tissue culture plate as an optically accessible perfusion chamber for in situ assays and for long-term cultivation of mammalian cells

摘要

An alternative culture system has been developed based on a conventional tissue culture plate (3.5 cm diameter) which is changed into a closed perfusion chamber. The system can easily be scaled up from one to several chambers. The shape and the size of the area of cell growth may be designed to individual experimental demands. The whole culture chamber is optically accessible, so cell growth and morphology can be evaluated by light microscopy. Furthermore the cellular physiology can be characterised by any fluorimetric assay using a bottom type fluorescence reader. A peristaltic pump sustains a constant medium flow through the chamber thus creating true homeostasis. The use of HPLC-valves and connectors allows the switching between different media or assay solutions. Thus it is possible to perform in situ assays also measuring transient effects. A protocol for vitality tests using calcein-AM is worked out for an adherent cell line and for a suspension cell line. The lower detection limits are 7 × 102 cells cm-2 for the adherent cells and 5 × 104 cells mL-1 for the suspension cells. The upper limits are 1–2 × 105 cells cm-2 respectively 8 × 106 cells mL-1.