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CHO-K1 and VERO cells have been grown on MicroHextrade mark, a polystyrene-based microsupport with two-dimensional geometry and the consistency of their growth kinetics were established. These cells have been detached by exposure to buffered ethylenediaminetetraacetic acid (EDTA) followed by controlled mechanical shear to yield well-isolated cells suspended in EDTA. Neutralisation of the EDTA followed by restoration of the chemical composition of the growth medium causes detached CHO-K1 cells to display unaltered growth kinetics.

Tissue culture flasks were activated by electron beam irradiation and subsequently treated with N-isopropylacrylamide to make them cryoresponsive. Leaving such 'cryoflasks' unattended for 10 minutes at room temperature sufficed to almost completely detach the anchorage-dependent cells. MicroHex((R)), a polystyrene-based tissue culture microsupport with two-dimensional geometry, was handled in the same way to obtain CryoHex, i.e. a cryoresponsive MicroHex from which anchorage-dependent cells could be detached by exposure to low temperature (4-20 degrees C). Experimental conditions were determined allowing one to detach the cells from small and large microsupport culture volumes. Cells detached from CryoHex by exposure to low temperature displayed a high cell viability and, upon subcultivation on MicroHex((R)), did not show any alteration of their growth kinetics.

The procedure used to establish in situ (without cell trypsinization) the growth kinetics characteristics of anchorage-dependent cells propagated on microcarriers by Aperture Impedance Pulse Spectroscopy can be replaced by a novel method based on the time-dependent shifts of the size distribution histograms of cell-laden microcarriers. This we have named Laser Diffraction Particle Sizing.

The lengthy and cumbersome protocol used to establish the growth kinetics characteristics of anchorage-dependent cells (ADC's)in situ (i.e. while the cells adhere on their microsupport) by Aperture Impedance Pulse Spectroscopy (AIPS) can be replaced by an accelerated procedure. This we have named Turbo AIPS whereby the same results can be obtained without actually performing the manipulations leading to the determination of the biomass.

Low-frequency dielectric spectroscopy has been used in situ, i.e. while the cells are still attached to their microsupport, to monitor the changes of biomass accompanying the growth of anchorage-dependent cells. This method, when compared to Aperture Impedance Pulse Spectroscopy (also called electronic sizing), is characterized by a somewhat lower degree of resolution. Suggestions are made on how to determine the capacitance of the spent growth medium alone, still keeping the probe inserted in the bioreactor. This will make dielectric spectroscopy the first truly in situ, on-line, in real time, non-invasive measure of the biomass.

The capacitance of suspensions of CHO and HeLa cells (0.5-3 x 10(6) cells/ml) has been measured between 0.2 and 10 MHz. As frequencies decrease, there is a continuous increase in capacitance of both the cell suspension and the spent growth medium free of cells, a phenomenon which is partially attributed to an increased polarisation of the electrodes. At a given frequency, subtraction of the capacitance of the spent medium from that of the cell suspension allows one to determine the capacitance of the cells only. The intensity of this signal varies linearly with the biomass and cell size. At low frequencies such as those used in this study (0.25 MHz), where sensitivity is the highest, concentrations as low as 0.5 x 10(6) cells/ml can be accurately measured. Suggestions are made how to make these measures on-line, non-invasive and in real time.