Continuous cell suspension processing

Terranova, B. E., and Bums, M. A., Continuous Cell Suspension Processing Using Magnetically Stabilized Fluidized BedsBiotechnol. Bioeng., 37 110... 

The alternative to batch mode operation is continuous operation. In the continuous mode there is a continuous flow of medium into the fermentor and of product stream out of the fermentor. Continuous bioprocesses often use homogenously mixed whole cell suspensions. However, immobilised cell or enzyme processes generally operate in continuous plug flow reactors, without mixing (see Figure 2.1, packed-bed reactors). 

For cells growing continuously in suspension, the subculture process can be performed similarly to the method used for microbial cultures. Trypsin treatment is not required and subculture is faster and less traumatic for the cells. Total medium exchange is not generally performed for these cultures since it would require a centrifugation step. Culture maintenance can be performed by dilution with fresh medium after adequate cell growth. 

Cells are thawed into culture medium and continuously processed from T-flasks to the NLF 22 bioreactor. At each level the parameters measured and the cumulative cultivation time required are shown. The volume of cell suspension required to proceed to the next step is given. 

Compared to cell suspensions, immobilized cells have several advantages the process can develop continuously, while the product is released from the cells and separated from the culture medium the culture medium can be changed, allowing several compounds to be added and removed easily and quickly the biomass can be rejuvenated in situ by perfusion of the growth medium to the cells at different time intervals and relatively low amounts of biomass can be used efficiently [16, 17]. 

Suspension systems can be operated in different modes batch, fed-batch, chemostat, and perfusion (Fig. 1). These operation modes differ basically in the way nutrient supply and metabolite removal are accomplished, which in turn determines cell concentration, product titer and volumetric productivity that can be achieved [8]. The intrinsic limitation of batch processes, where cells are exposed to a constantly changing environment, limits full expression of growth and metabolic potentials. This aspect is partially overcome in fed-batch cultures, where a special feeding strategy prolonges the culture and allows an increase in cell concentration to be achieved. In perfusion and chemostat processes nutrients are continuously fed to the bioreactor, while the same amount of spent medium is withdrawn. However, in perfusion cultures the cells are retained within the bioreactor, as opposed to continuous-flow culture (chemostat), which washes cells out with the withdrawn medium [9]. 

Fermentation is typically conducted in dilute suspension culture. The low concentration in such systems limits reaction efficiency, and the presence of particulate and colloidal solids poses problems for product recovery and purification. By circulating the fermentation broth through an ultrafiltration system, it is possible to recover product continuously as they are generated while minimizing loss of enzyme or cells and keeping product concentration in the bioreactor below the self-inhibition level for the biocatalyst. This process is referred to as perfusion. As the ultrafiltration unit is part of the production process, the entire system is often considered a membrane reactor. 

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