Spinner flasks

The conventional culture media can be used (DMEM, IMDM, etc.) but additional components can be added to increase the success of the culture if considered necessary. Pluronic F-68 (polyglycol) (BASF, Wyandot) can be added at 0.1% to protect the cells against mechanical damage, especially at reduced serum concentrations. Carboxymethyl cellulose (CMC) (15-20 cP) can also be used at 0.1% for this purpose. Antifoam (6 p.p.m.) (Dow Chemical Co.) is recommended if foaming occurs which usually happens if the serum concentration is above 5%. The use of Hepes buffer can also be considered to stabilize pH during set up and if gassing facilities are not available. 

Add 200 ml medium to a 1 litre spinner flask, gas with 5% COj, and warm to 37 °C. Add 2-4 X 10 cells (1-2 x 10 /ml) harvested from a growing culture of cells (i.e. not a stationary or dying culture). NB this is a higher inoculum than used for stationary cultures. 

Place spinner vessel on magnetic stirrer at 37 C and set at 100-200 r.p.m. (this is variable depending upon the vessel size, geometry, fluid volume, and individual cell. Set a speed which visually shows complete homogeneous mixing of the cells throughout the medium—150 r.p.m. is usually a safe choice). 

Monitor the growth daily by removing a small sample through the side-arm in a Class II cabinet and carrying out a cell count and visual inspection for cell morphology and lack of microbial contamination. 

Monitor the pH (colour change), especially in closed (non-gassed) systems, and should the culture become acidic (under pH 6.8) add sodium bicarbonate (5.5X stock solution) or re-gas with CO2. After three days a better option would be to allow the cells to settle out, remove 40-709S of the medium, add fresh pre-warmed medium to the culture, and continue stirring. 

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Fig. 17. Response of CRL-8018 hybridoma cells to increasing levels of well-defined laminar shear in the concentric cylinder viscometer for 10 min. Spinner flask cultures were seeded with cells from routine T-flask cultures that were 3 days old. Cell samples were taken from the spinner flask cultures during late-exponential growth and sheared in the viscometer [17]...

Table 17.13 Growth of MRC5 Cells Cell Density versus Time for MRC5 Cells Grown on Cytodex I Microcarriers in 250 mL Spinner Flasks Inoculated at 2.26 and 4.76 cells/bead...

Hawboldt et al. (1994) have also reported data on anchorage dependent Vero cells grown on Cytodex I microcarriers in 250 mL spinner flasks and they are reproduced here in Table 17.14. 

Andersen et al. (1996) and Andersen (1995) have studied the effect of temperature on the recombinant protein production using a baulovinis/insect cell expression system. In Tables 17.15, 17.16, 17.17, 17.18 and 17.19 we reproduce the growth data obtained in spinner flasks (batch cultures) using Bombyx mori (Bm5) cells adapted to serum-free media (Ex-Cell 400). The working volume was 125 ml and samples were taken twice daily. The cultures were carried out at six different incubation temperatures (22, 26,28, 30 and 32 TT). 

Spinnerets, for olefin fiber extrusion, 22 627, 630-631, 11 231-232 Spinner flasks, 15 691-692 Spinning. See also Solution spinning Yarn spinning technologies air gap, 11 209 of continuous-filament yarns,... 

Sterile Spinner flasks (200 ml and 1 litre), spinner apparatus... 

Seed the cells into a new tissue culture or spinner flask at a density of 250,000 cells/ml using the medium in Step 17 (some fresh medium may be added if required). 

Pipette Sf9 cell culture containing 2x10 cells/ml into a spinner flask. 

Transfer the spinner flask to the hood and adjust cell density to 1x10 cells/ml by adding the medium prepared in Step 8. 

In cancer research, the multicellular tumor spheroid model is the most widely used. Spheroids are three-dimensional, often heterogeneous organoids containing gradients of cell proliferation, oxygen, and nutrients. Spheroids may be developed in suspension or in 3-D matrices. The former method has been adapted in various ways to include the use of spinner flasks (86), rotating cell... 

Stirred bioreactors are common in animal cell culture, as they offer a homogenous enviroiunent, representative sampling, better access to process control and an increased oxygen transfer. Several of these techniques (spinner flasks and stirred vessel bioreactors) have been tested successfully for the cultivation of hematopoietic cells [58,64-67]. 

For the cultivation of animal cells, inoculum expansions have traditionally been conducted in T-flasks, shake flasks, spinner flasks, or roller bottles. Typi-... 

Whatever the scale or method of culture (T-flask, Schott bottle, spinner flask, or bioreactor), the temperature of the culture medium with which the cells are in contact is always a fundamental state variable, because it interferes with growth and the production process. However, it is a process variable that is easy to monitor and control. On a small scale the culture flask is usually put in a thermostatically controlled incubator, where the measured value of a thermometer sends a sign to turn the heating on or off ( on-off control ). In bioreactors, there are equivalent systems, as will be seen later. Usually, however, a resistance thermometer sensor type is used (resistance temperature detector or RTD), the electric... 

Fig. 3.6. Spinner flasks. The flask on the stirrer is protected from excessive heat by a layer of foam insulation. The vessel on the right is wrapped in aluminium foil for sterilisation. The model with the solid spindle which is allowed to swivel top and bottom is preferable to the one with metal swivel (on the left) as the latter tends to stir...

Fig. 3.7. Techne spinner flasks and stirrers. This type of stirrer produces no heat and can be controlled at low speeds. The suspended ball combined with the raised centre of the flask readily keeps cells or microcarriers in suspension. (Courtesy of Techne.)...

The magnet is driven by a magnetic stirrer motor on which the vessel stands. Traditional magnetic stirrers when running for long periods create excessive heat and the vessel requires to be insulated by having a sheet of expanded polystyrene foam interposed between it and the motor. Bellco spinner flasks (Fig 3.6) are available from Arnold Horwell Ltd. (Appendix 3). 

Transient Transfection of Suspension Cells in Spinner Flasks... 

Approximately 18-20 h prior to transfection, seed cells at a cell density of 5 x 105 cells per mL in serum-tree growth medium. Seed 25 x 106 cells in 50 mL of culture media in a 125 mL Coming Spinner Flask to reach approximately double the density overnight. 

Use 0.4% Trypan Blue to stain cells and count viable cells at seeding. Viability should be at least 95% at this stage. Culture cells in 50 mL media in Coming Spinner Flasks at 8% CO2, 37°C, in a humidified incubator (see Note 1). 

The cell line has been adapted for growth in serum-free media using spinner flasks and stirred tanks and cultures can be scaled up readily to produce high yields of recombinant protein (Jain et at, 1991). In batch cultures, infection of the cells with baculovirus can be monitored easily by measuring the increase in cell volume and decrease in cell viability. Several days after infection the cells lyse, releasing the recombinant protein product. Product titre is influenced by the oxygen requirement of the insect cells. 

Cells are cultured in FIO supplemented with 15% horse serum and 2.5% FBS. The cells are adaptable to growth in suspension culture in spinner flasks using Eagle s MEM. 

The growth vessel and some supplements are determined by whether the cells are grown attached or in suspension. When the cells are anchorage dependent, they can be grown in plates or flasks. As serum is reduced, it may be necessary to add attachment factors to the culture. For cells grown in suspension in spinner flasks or fermenters, non-ionic surfactants (F68) that increase viscosity may be needed to minimize shear stress caused by agitation. 

For suspension cultures, spinner flasks (Techne, Wheaton, Bellco, Cellon, etc.) and bottles are available, which provide sufficient capacity for most small-scale preparation of cells. Sizes range from 25 to 10 000 ml, and the stirring rate can be adapted for each cell type. 

Figure 4.7.1 Cumulative concentrations of non-attached CHO cells in a serum-free medium from confluent microcarriers in spinner flasks at ( ) 80, (A) 115 and ( ) 150 rpm. Cumulative values are the sum of suspended cells by nuclei counting in the supernatant. Microcarrier concentration was 5 g H. Based on data of Borys and Papoutsakis (1992) by permission of Cytotechnology.

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