Typically, the adaptation of adherent cell lines to growth in suspension is carried out with the goal to achieve high cell densities and/ or growth in serum-deprived cell culture media.

The procedure described below is for cells grown in T-25 or T-75 flasks. About ten T-75 flasks provide enough cells (approximately 1-2 x 107) to seed a suspension culture in a spinner or shake flask.

For best results the following recommendations should be observed:

Use cells 50% to 80% confluent

Use cells with > 90% viability

Cell culture media

Several basal cell culture media have been developed and optimized for suspension cell culture growth. We recommend the use of the following in the procedure described below:

Minimum Essential Medium Eagle (MEM), Joklik’s Modification for Suspension Cultures, for example Sigma refs 56449C, M0518 (powder) and M8028 (liquid). These media do not contain Calcium chloride. These basal media were designed for suspension culture systems. Calcium chloride has been omitted to reduce cellular attachment. References 56449C and M0518 are powder formulations and need the addition of Sodium Bicarbonate. M8028 is a ready-to-use liquid medium that requires the addition of L-glutamine.

Dulbecco’s Modified Eagle’s Medium/ Ham’s F-12 Nutrient Mixture without Ca++ and Mg++, e.g. Sigma reference D9785. This powder formulation does not contain L-glutamine, L-leucine, L-lysine, L-methionine, CaCl2, MgCl2, MgSO4, sodium bicarbonate, and phenol red. Needs to be supplemented with L-glutamine, L-leucine, L-lysine, L-methionine, sodium bicarbonate, and phenol red. This formulation is enriched with vitamins and amino acids compared to Joklik’s MEM modification and contains also extra-buffering capacity (HEPES). This is the preferred formulation for the procedure described below.

To obtain the complete serum-free cell culture medium to be used in the procedure, supplement the basal cell culture medium of choice with 10% of XerumFree™ XF 201 or 2% XF205 serum replacement, animal component-free, chemically defined. The use of antibiotics is left at the discretion of the user.

PROTOCOL

Aspirate the growth medium from the flasks.

Add Phosphate Buffer Solution (PBS) without calcium and magnesium (5 ml for T-25 flask or 15 ml for T-75 flask). 

Leave for 5 minutes, then remove the washing solution.

Add trypsin-EDTA solution to the side of the flask opposite the cell monolayer. Use the same volumes and procedure as for a normal cell passaging. 

Incubate the flasks at 37°C for 5 to 10 minutes. 

Monitor the cells during this incubation under a microscope. 

When the cells round up, tap the flask to dislodge the cells.

Add growth medium (5 ml for T-25 flask or 10 ml for a T-75 flask) to the cell suspension.

Add soybean trypsin inhibitor. Typically a 0.25 mg/ml trypsin inhibitor solution is used at the same volume as the trypsin-EDTA solution above.

Aspirate the cell suspension and transfer to a 15 ml centrifuge tube. Centrifuge at 200 × g for 4 min. Remove the supernatant.

Suspend the cells in 5 ml of growth medium. Centrifuge at 200 × g for 4 min. Aspirate and discard the supernatant. 

Resuspend the cells in 5 ml of growth medium. 

Perform a cell count with a trypan blue stain and estimate of the cell viability.

Dilute the cell suspension with growth medium such as to obtain a density of approximately 5 × 10⁵ cells/ml.

Add Pluronic® F68 to a final concentration of 0.1 % in order to protect the cells against shear stress. A 10% sterile cell culture tested
solution is available from Sigma-Aldrich (reference P5556) – add 1 ml of this solution to 100 ml of cell suspension.

Transfer the cell suspension into sterile spinner or shake flasks. Leave enough headspace for adequate gas exchange (100 ml of cell suspension in a 250 ml spinner flask or 250 ml shake flask).

Loosen caps for gas exchange and place at 37°C in a humidified atmosphere of 5% CO2. For spinner flasks, use an impeller speed of 75 to 95 rpm. For shake flasks, rotate on a shaker platform at 125 to 135 rpm.

Perform a daily count of viable cells and plot growth kinetics of the suspension culture.

When the cell density reaches 1 × 10⁶ cells/ml (or on day 4 post-seeding) passage the cells at a density of 5 × 10⁵ cells/ml. To perform this step aspirate the desired amount of cell suspension (e.g. take 50 ml of a 1 × 10⁶ cells/ml cell suspension) and dilute it with the same volume of fresh cell culture medium containing 0.1% Pluronic F68 – this will lead to the suspension density of 5 × 105 cells/ml).

Note: if the cell density has not reached 1 × 10⁶ cells/ml by day 4, the cells should be pelleted by centrifugation and resuspended in fresh medium containing Pluronic F68.

Once cell density has reached 1 × 106 cells/ml with 90% viability by day 3 post-seeding for 3 passages, the cells are considered adapted to suspension culture. Seeding density can then be reduced to 2-3 × 10⁵ cells/ml.