Continuous cell suspension

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

One of the most important bioreactors is the stirrer bioreactor, in which a continuous culture of cell suspensions can be maintained. This kind of bioreactor is constructed in three parts bioreactor vessel with stirrer and both medium and effluent vessels with pump systems (Figure 6.9). In a continuous cell suspension culture reactor, the following points are noted ... 

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). 

FIGURE 7.5 7,8-BPQ increases intracellular Ca2+ in murine spleen cells (A) and in both B and T cells (B). Single cell suspensions were prepared from murine spleens. Splenocytes were loaded with Fluo-3/AM dye for one hour and then treated with 7,8-BPQ, 1,6-BPQ, 3,6-BPQ, or DMSO (control). Surface-marker-defined T cells and B cells were treated with 7,8-BPQ or DMSO. Following treatment, the immediate intracellular Ca2+ response was continuously monitored for 15 minutes. Results are shown as the change in Mean Channel Fluorescence SEM. The numbers shown in this figure were the averages of triplicate determinants. Adapted from Gao et al., 2005. 

Pipet 100 pL of the cell suspension onto treated slides, spread using the edge of a clean slide, and allow to dry. Alternatively, cells can be attached to the slide by cytospin preparation. Continue with step 2 immediately below (Subheading 3.2.2.). 

Dilute 2 X 10 cells in 17 ml Soln. A and warm up to 37 °C. Prepare a solution of H-PN 200-110 (about 1 600 000dpm) in 3 ml. Mix both solutions and continue incubation at 37 °C. Take 1-ml probes in duplicate at timet = 0,l,2,4>6,8,10,12,15,and20 min after mixing (- Bt ). Precipitate the probes with 2 ml of ice-cold Soln. D immediately after sampling and filter on Whatman GF/C glass fiber filters. Prepare a blank ( Bo ) by incubation of a mixture of 850-pl cell suspension and 150 pi Soln. C at 37 °C for 20 min. Wash the filters with Soln. D. Dry the filters at air and count for radioactivity. 

The main advantages of continuous cell lines are (i) faster cell growth, achieving high cell densities in culture, particularly in bioreactors (ii) the possible use of defined culture media available in the market, mainly serum-free and protein-free media and (iii) the potential to be cultured in suspension, in large-scale bioreactors. 

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. 

Continuous cultivation is used mainly in research and development activities, at small scales. It is characterized by a continuous feed of fresh medium and a continuous removal of cell suspension, both at the same flow rate, and at constant bioreactor volume. 

These methods are, however, largely unnecessary when it is desired to clone continuous cell lines. Thus a dilute suspension of HeLa cells (containing about 100 cells in 5 ml) will grow up to form colonies, each derived from a single cell. It is important to prevent the movement of these cells (and hence mixing of clones) and sometimes they are overlayered with soft agar. 

Culture of PPLO. It is important to use both a cell suspension and the culture supernatant from cells grown for 3 days. Take up the sample in a Pasteur pipette. Pierce duplicate PPLO agar plates with the pipette about 10 times each or simply cover the agar with the culture supernatant. Incubate in 5% C02 in either N2 or air in a sealed jar, i.e. anaerobically or aerobically. Colonies appear in 3-7 days but incubation should be continued for 3-5 weeks when the colonies assume a typical fried egg appearance (Fig. 9.3). 

Alternatively, some subunit viral vaccines can be generated by rDNA techniques and expressed in a continuous cell line or insect cells. Recent advances in bio reactor design and operation have improved the successful production of IPV in large-scale bioreactors. However, roller bottles or flasks are still used for most current vaccine production. Development of insect cell culture will allow for very large-scale liquid suspension culture (143). Several vaccine candidates such as gpl60 for HIV and gD protein for herpes have been demonstrated in the insect cell culture system. However, no vaccine has... 

---