We have developed a flow-through device which uses high frequency, low energy ultrasonic resonance fields to transiently aggregate hybridoma cells and return them by sedimentation to a perfusion bioreactor. The system retained up to 99 percent of the inflowing viable cells with no measurable effect on viability. Viable cells were selectively retained at up to 3 percent higher efficiency than nonviable cells. A stirred tank bioreactor was operated for 700 hours with acoustic cell recycle. Concentrations greater than 5 x 10(7) cells/ml were attained with a 5-fold increase in antibody concentration and a 70-fold increase in volumetric productivity compared with batch culture.

Ultrasound was used to enhance the sedimentation of hybridoma cells from medium in a 75 mL resonator chamber. Forces in the acoustic standing waves aggregated the cells, and the aggregates were then rapidly sedimented by gravity. Cell separation increased with acoustic treatment time and cell concentration. The separation efficiency was over 97% for cell concentrations between 10(6) and 10(7) cells/mL. During acoustic treatment at 180 W/L, the medium temperature increased at a rate of 1.3 degrees C/min. Ultrasonic exposures up to 220 W/L did not influence the viability or subsequent growth and antibody production of the cells. A decrease in cell viability was observed at a power level of 260 W/L. Batch separation efficiencies were as high as 98%. Acoustic separation was tested under semicontinuous operation, and above 90% separation efficiency was achieved at a flow rate of 0.7 L/h.

Analysis of cellular ATP as a means of measuring viable biomass loading was investigated in hybridoma cell culture. ATP analysis by the luciferin-luciferase assay was compared with trypan blue-stained hemocytometer counts. The cell-specific ATP content varied between 2 and 6 fmol per viable cell over a batch culture. ATP levels were highest during exponential growth, and decreased during the stationary and decline phases. Electronic counting and volume measurements were performed to assay the viable cell biomass. Cell sorting, using fluorescein diacetate, was used to separate viable and nonviable cells in cultures with between 35% and 90% viable cells. Viable cells contained over 2 orders of magnitude greater cell-specific ATP than nonviable cells. Cell-specific ATP correlated directly with the viable cell volume rather than viable cell numbers. Over the range of batch culture conditions, ATP analysis should provide a more accurate measurement of hybridoma viable biomass than hemocytometer counts.

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