Efficient Isolation of Viable High Producing Mammalian Cell Lines with the Fluorescence Activated Cell Sorter

The establishment of cell lines producing human antibodies for therapy of cancer or viral and bacterial infections is a very labour and cost-intensive process when using current methodology. The proposed project will optimize a technology that measures the secretion rate of individual cells, which should reduce the expenditure required by a factor of at least 10 by directly measuring the specific production rate of single cells, so that they can be isolated using a flow cytometer and cell sorter. Two cell lines will be used for the demonstration of the potential of the technique: recombinant CHO-cells producing a human antibody and human-mouse heterohybridoma cells. With the recombinant CHO cells the goals will be to demonstrate the possibility of combining different selection criteria. For model purposes the production kinetics of subclones will be changed from growth-associated to maintenance-associated. select and characterise stable producers in the absence of pressure analyse cellular properties together with cell specific secretion rates for better understanding of the physiology of product formation in mammalian cells. Human-mouse heterohybridoma cells will be used to optimise the technique for selection of lymphoid cells (problems are expected due to possible surface expression of antibodies) modify the technique to allow the selection of hybridoma cells producing antigen specific antibodies directly after cell fusion. This will be achieved by using a fluorescently labelled antigen for staining. analyse expression of lymphocyte surface markers in combination with secretion rates on primary lymphocytes as well as on heterohybridoma cells. Identification of surface markers that correlate with production rates will allow the pre-enrichment of likely high-producers by a fast bulk sorting method such as magnetic cell separation.