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Medium: Standard serum-free F12"CDME; supplement with:
“/ 2"C10% fetal bovine serum (see Chapter 8 for a detailed discussion of serum reduction or elimination)
“/ Insulin, 5 ^g/ml
Y/Pluronic F-68 (Gibco/BRL #24040-016), 0.1%
Y/HEPES buffer, 15 mM
Spinners: Use well-siliconized (see note) spinners. If the cells cake around the spinner shaft and on the sides of the spinner at the medium surface, the spinner is not properly siliconized. We prefer 250-ml spinners with 50- to 100-ml volume of medium. Spinner bases are set for a speed of 50"C80 rpm. This should be just sufficient to keep cells in suspension and allow for good mixing of gases without undue mechanical damage to the cells. Set up two spinners in parallel. Carry both spinners, subculturing on different days, to prevent loss of the culture due to contamination. Suspension adaptation of most established (transformed) cell lines usually takes from 21 to 90 days. However, some normal cells will not suspension adapt without transformation.
Culture: Remove cells from the starting culture with trypsin and neutralize the trypsin with serum (or STI if using serum-free culture). Set up the spinners at 3"C5 x 105 cells/ml. Check cells daily for growth and viability. More sodium bicarbonate solution may be added if the pH in the spinner drops below 6.8. After the first day or so, the caps on the spinner should be loosened to allow for increased oxygen and carbon dioxide exchange. On day 3 or 4, the cells should be counted and passaged. Initially, cells should be centrifuged and sufficient fresh medium added to the total cell pellet to bring the cell number to between 3 and 5 x 105/ml. As the cells start growing logarithmically to densities over 1 x 106/ml, they may be passaged by dilution of the suspended cells with fresh medium. If direct passage by dilution is to be used, the cell density should be such as to allow at least a 1:5 split. If cells cannot be split at this ratio or higher, the cells should be centrifuged, the old medium discarded, and sufficient cells seeded in fresh medium to obtain the desired inoculum density. Carryover of too much exhausted medium can progressively inhibit cell growth through the presence of toxic cell waste products or through nutrient depletion. If cells clump in the spinner, excessively large clumps should be allowed to settle so that they are not passaged.
After 2"C10 weeks, the cells should be capable of logarithmic growth in suspen-
Suspension adapting a CHO cell line. Cells should be carried at high density until they start to grow vigorously. When cells can grow from 104 to over 106 cells/ml, they can be considered adapted.
sion to reach densities of >106 cells/ml when inoculated at densities of 5"C10 x 104 cells/ml. Cell viability should remain at >90% throughout the growth period. At this point the cells are termed suspension adapted, even though they may still attach to tissue culture plastic, especially in medium containing serum or attachment factors. The cells may exhibit some clumping during prolonged growth in suspension, but this is not necessarily a disadvantage as long as the clumps are not so large as to cause necrosis of cells in the center due to nutrient limitation or to fall out of suspension.
The cell counts and passage times (arrows) are shown in Fig. 11.2 for the course of adapting a CHO recombinant clone. The cells were considered suspension adapted after 5 weeks.
Scaling-Up Suspension-Adapted Cells
Larger (1 to 3 liters, and greater quantities) spinners can easily be set up using the same cells and medium as used in the smaller (e.g., 250 ml) spinners (Fig. 11.3). If the cells have been suspension adapted, or grow easily in suspension (e.g., hybridoma cells), then obtaining increased volumes of harvest medium should be straightforward. Make sure all spinners are well sialated to prevent sticking of both the cells and the protein produced by the cells. Oxygen diffusion rapidly becomes limiting for many cells when grown in the larger spinners. If the cells are growing less rapidly or to lower density in large spinners than in the smaller spinners, decrease the volume of medium in the spinners to about one third the designated volume (e.g., 300 ml of medium in a 1-liter spinner). This increases the surfaceto-volume ratio and allows improved oxygenation. Controlling pH may also be a problem, particularly if longer culture times are desired in order to allow accumulation of product in the medium. A bicarbonate solution can be added back to the culture as it becomes acid, to increase buffering capacity and keep the pH in the desired range of 7.0 to 7.5. It is also possible to purchase "instrumented spinners," which allow for aeration to increase oxygenation and for pH control. These are, however, more complicated, more labor intensive, and
A spinner set up for growing large volumes of cells in suspension. This spinner allows for aeration through the pump at right. (Courtesy of E. Braun Biotech International)