Culture systems stationary cultures

Batch fermentation is the most widely used method of amino add production. Here the fermentation is a dosed culture system which contains an initial, limited amount of nutrient. After the seed inoculum has been introduced the cells start to grow at the expense of the nutrients that are available. A short adaptation time is usually necessary (lag phase) before cells enter the logarithmic growth phase (exponential phase). Nutrients soon become limited and they enter the stationary phase in which growth has (almost) ceased. In amino add fermentations, production of the amino add normally starts in the early logarithmic phase and continues through the stationary phase. 

Typical commercial cell culture systems include batch or fed-batch suspension reactors and perfused immobilized-cell reactors. However, the transient nature of batch culture causes difficulties in studying the effects of external stimuli on growth, metabolism and product formation. Due to metabolite concentration gradients, and the difficulty of obtaining representative cell samples, immobilized-cell reactors are also poorly suited for the analysis of cell growth and metabolism. As a result it is desirable to use well-defined model systems. Continuous-flow suspension reactors allow metabolic parameters to be measured at steady state, after cells have adapted to new (and possibly inhibitory) conditions. Perfusion reactors (with cells immobilized on suspended or stationary supports) extend these benefits to anchorage-dependent cells, and provide model systems for cell responses in vivo. However, while it is instructive to study the behaviour of cells under well-defined conditions, the results obtained must be verified in the culture system selected for commercial production. 

Althou hybridoma cells are spherical and well adapted to suspension culture they do grow well in stationary culture resting on the substrate, sometimes with very light attachment. This means that a wide selection of culture vessels and systems are available for their culture ranging from a simple tissue culture plate or flask to highly sophisticated bioreactors with full instrumentation to control the physiological environment (1-3). Commercially hybridoma cell lines are grown at scales up to 2000 litres and beyond in culture units scaled-up from laboratory size vessels. Laboratory, pilot, and production scale is ill-defined so in this chapter laboratoiy scale will be taken as 10 litre volume cultures, and below. 

The roller bottle technique is a well-established and successful culture method widely used for the production of cells and products. One reason for this is that a single contamination event does not mean that the whole batch is lost, as with a single unit process. However, roller culture needs a considerable financial investment both in the apparatus itself and in incubator/hot-room facilities. Also, some cell lines (particularly epithehal) may not be as successfully grown in roller bottles as in stationary bottles. Common problems are streaking, clumping or inadequate spreading over the total surface (e.g. non-locomotory cell lines). An alternative scale-up route is to use multisurface stationary systems such as the Nunclon Cell Factory (Nunc) (see section 5.7) or the CellCube (Costar). 

Cultures in the stationary phase were harvested by centrifugation at 10,000 g for 10 min. Aliquots of 1.2 ml were washed with different concentrations of sucrose and trehalose. After this, they were dehydrated for 30 min in a vacuum centrifuge (Integrated Speed Back System ISS 100 (Savant Knowledge Storm, Inc. 2520 Northwinds Parkway Suite 300 Alpharetta, GA 30004) at 70°C. Then they were immediately resuspended in distilled... 

---