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Primary rat hepatocytes were cultured in a perfusion bioreactor system in an attempt to develop a bioartificial liver support device. The bioreactor is based on immobilisation of cells attached to microporous carriers packed into a stationary bed reactor. The bioreactor is perfused with media and oxygenated independently thereby allowing better homogeneity through optimum control of nutrient and oxygen transfer. Hepatocytes which are essentially non-dividing cells when cultured in vitro, are required to be maintained for prolonged periods with minimum loss of cell viability and function. Attachment to microcarriers was optimised by investigating the influence of various parameters including temperature, serum levels, pH and cell density.

Dielectrophoresis is a well established and effective means for the manipulation of viable cells. Various applications have been found, ranging from electrofusion, to individual cell manipulation, and to differential separation from cell mixtures. Its effectiveness, however, greatly depends upon the utilization of very low electrical conductivity media. High conductivity media, as in the case with cell culture media result only in negative dielectrophoresis (i.e., induction of weaker repulsive forces) and excessive medium heating. Recently, a dielectrophoresis-based cell separation device (DEP-filter) has been developed for perfusion cultures that successfully overcomes these problems and provides a very high degree of viable cell separation while most of the nonviable cells are removed from the bioreactor by the effluent stream. The latter results in high viabilities throughout the culture period and minimization of lysed cell proteases in the bioreactor. However, an important question that remains to be answered is whether we have any adverse effects by exposing the cultured cells to high frequency dielectrophoretic fields for extended periods of time. A special chamber was constructed to quantitate the effect under several operational conditions. Cell growth, glucose, lactate and monoclonal antibody production data suggest that there is no appreciable effect and hence, operation over long periods of time of our DEP-filter should not have any adverse effect on the cultured cells.

In this study, we investigated culture conditions of primary porcine hepatocytes for development of a hybrid artificial liver using polyurethane foam (PUF)/spheroid culture. Porcine hepatocytes in the pores of hydrophilic PUF only formed spheroids spontaneously within 1.5 days of culture, and maintained ammonia detoxification activity in Williams’ E medium supplemented with 10∼20% fetal bovine serum (FBS) for a week. The porcine hepatocyte spheroids in multi-channel (MC) PUF packed-bed module also expressed the highest activities of ammonia detoxification and albumin production at an immobilized cell density of 1.0×107 cells/cm3 module and at an intrachannel medium velocity range from 60 to 80 cm/min.

We cultivated the hybridoma producing vWF antibody in a 10L bioreactor in order to investigate the characteristics oftwo different cultures, intermittent and continuous bleeding. To compare with bleeding culture non-bleeding culture was also performed. Non-bleeding culture achieved a cell density of 2.1 x 107 cells/ml at 16th day with 110 mg/l ofvwF mAb titer. However, the culture was stopped after the 18 days, due to membrane clogging. Intermittent and continuous bleeding cultures were maintained the cell density around 1 x10 cell/ml. The vWF mAb titer was 45mg/l and 53mg/l for intermittent and continuous bleeding culture. As for volumetric productivity, the continuous bleeding culture was higher than intermittent bleeding culture by 4 1%. For glucose consumption rate (GCR), lactate production rate (LPR), glutamine consumption rate (GnCR), and ammoniaproduction rate (APR), continuous bleeding culture showed 28%, 70%, 31% and 40% higher than intermittent bleeding culture. These results suggest that physiological activity ofthe continuous bleeding culture was higher than that of intermittent bleeding culture.

Factor Xa is a serine protease, whose high selectivity can be used to cleave protein tags from recombinant proteins. A fusion protein comprised of a self-activating form of factor X linked to a cellulose-binding module, saCBMFX, was produced in a stable transformed Sf9 insect cell line. The activity of the insect cell produced saCBMFX was higher than the equivalent mammalian cell produced material. A 1.5 l batch fermentation reached a maximum cell concentration of 1.6 x 10(7) cells ml(-1) and a final saCBMFX concentration of 4 mg l(-1). The production of saCBMFX by this cell line was also analyzed in a 1.5 l perfusion system using an ultrasonic filter as a cell-retention device for flow rates up to 3.5 l day(-1). The cell-retention efficiency of an air backflush mode of acoustic filter operation was greater than 95% and eliminated the need to pump the relatively shear sensitive insect cells. In the perfusion system over 4 x 10(7) Sf9 cells ml(-1) were obtained with a viability greater than 80%. With a doubling of viable cell concentration from 1.5 to 3 x 10(7) cells ml(-1) the saCBMFX production rate was doubled to 6 mg l(-1) day(-1). The saCBMFX volumetric productivity of the perfusion system was higher than the batch fermentations (0.6 mg l(-1) day(-1)) by an order of magnitude.