Cell culture spinner flasks

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. 

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]...

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). 

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. 

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]. 

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... 

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. 

PROCEDURE CULTURE OF SUSPENSION CELLS IN A SPINNER FLASK... 

Place spinner flask on magnetic stirrer and stir at the minimum speed to ensure that all cells and carriers are in suspension (usually 20-30 rpm). It is advantageous if cells and carriers are limited to the lower 60-70% of the culture for this purpose. Alternatively either ... 

With some cells, or at microcarrier densities of 3 g H or more, partial medium changes should be carried out. Allow culture to settle (5-10 min), siphon off at least 50% (usually 70%) of the medium and replace with prewarmed fresh medium (serum can be reduced or omitted at this stage). Replace spinner flask on stirrer. 

The basic principles for using microcarrier culture are described, together with many notes on how to avoid problems and get the most out of this very powerful and useful technology. The description is suited to small-scale processes based on spinner flasks (i.e. 200 ml to 51) at levels that do not need special adaptations for perfusion, etc. This does not mean that scale-up is not possible in fact, it has been volumetrically scaled up to 40001 for the production of interferon and viral vaccines. It has also been scaled up in density by the use of spin-filters (Griffiths et al, 1987) to allow continuous perfusion of the culture and thus operation at microcarrier concentrations up to 15 g and cell densities over 10 ml. ... 

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