Maintenance of plasmacytoma cells for fusion

2.3 Maintenance of plasmacytoma cells for fusion

Efficiency of fusion and recovery of hybrids is greatest when the plasmacytoma ‘parent’ cells are uniformly viable and growing exponentially. Incubation times and cell densities given below are a guide, but it is necessary to determine the growth characteristics of each plasmacytoma line.

2.3.1 Plasmacytoma cells for fusion

MATERIALS AND EQUIPMENT Plasmacytoma line e.g. SP2 (if necessary, recover from frozen state) Tissue culture medium RPMI 1640 (500 ml) supplemented with 5 ml 200 m ML -glutamine,

5000 units penicillin, 5 mg streptomycin, 10 ml 6.6 m M 8-azaguanine and 10–20% (v/v) fetal bovine serum (FBS)

Plastic culture flasks (75 cm 2 ), 10 ml culture volume Incubator, humidified and gassed with 5% CO 2 in air

METHOD

1 Add 10 5 plasmacytoma cells to 10 ml of tissue culture medium and place in a humid

incubator gassed with 5% CO 2 in air.

2 Each day, resuspend the cells and determine the number per ml using a haemocytometer.

3 Plot a growth curve of cell number versus time (see Fig. B2 in Appendix B).

4 As soon as the growth rate starts to decline, dilute the cells by transferring 0.2–1.0 ml aliquots of the resuspended culture to flasks containing 10 ml of fresh medium.

5 When the cells have again reached their exponential growth phase, select viable cultures for storage under liquid nitrogen.

TECHNICAL NOTES • To remove the dimethyl sulphoxide (DMSO), thaw cell rapidly under running warm water,

wash and resuspend in tissue culture medium. • These cell lines will reach a maximum density of approximately 10 6 cells/ml. Exponential growth should be maintained by diluting the culture 1 : 10 with fresh medium every 3–5 days. Under these conditions the cells will have a doubling time of 16–20 h.

• The plasmacytoma cells grow either in suspension or lightly adherent. Release the adherent cells by gently tapping the culture flask or by gentle pipetting. • Check by phase-contrast microscopy that the cells are ‘healthy’. They should be phase-bright and of regular shape with clear outlines. Even in cloned lines size variation is common. Cell viability should be between 90 and 95%.

• As with all cell lines in long-term culture care must be taken to avoid cross-contamination between cultures. • The rate of reversion to HAT resistance varies with cell lines and is a relatively rare event. Eliminate revertants by culturing the cells in medium containing 8-azaguanine or 6-thioguanine (2 × 10 –5 m ) every 3–6 months. Alternatively, import a ‘seed’ culture from a commercial

44 C H A P T E R 2: Monoclonal antibodies: production, purification and enzymatic fragmentation 44 C H A P T E R 2: Monoclonal antibodies: production, purification and enzymatic fragmentation

• As with all long-term maintenance of cell lines in vitro it is advisable to check periodically for

Mycoplasma infection. Commercial kits are available for the demonstration of Mycoplasma DNA using a fluorescent dye. A rapid, but non-specific, indication of potential Mycoplasma contam- ination may be obtained by culturing supernatant, obtained by centrifugation (150 g for 15

min) of a 5-ml sample of the suspected culture, with 3.7 × 10 3 Bq 3 H-thymidine overnight at

37°C. Incorporation of label into trichloroacetic acid (TCA)-precipitable material indicates con- tamination. More sensitive tests use DNA probes and can be performed within hours. Removing Mycoplasma infection is generally not worth the considerable effort involved, but antibiotics such as erythromycin and ciprofloxacin can help.

2.3.2 Preparation of cell for storage

MATERIALS AND EQUIPMENT Viable cells in growth phase

Freezing medium (90% (v/v) fetal bovine serum (FBS) and 10% (v/v) dimethyl sulphoxide (DMSO)) Vials for freezing Ice

METHOD

Steps 1–3 are performed at 4°C (step 1) or on ice (steps 2 and 3).

1 Collect 10 7 cells and centrifuge at 1000 g for 15 min at 4°C to remove the excess supernatant containing the monoclonal antibody, but leave a residue of medium.

2 Resuspend the cells in the freeze-down medium by gently flicking of the centrifuge tube.

3 Load into the vial for freezing and ensure thorough mixing of the cells with the medium.

4 Slowly freeze the vial as follows: vial at –20°C for 2–4 h; vial at –70°C overnight; transfer into a Dewar flask containing liquid nitrogen.

TECHNICAL NOTES • Hybridoma cells can be unstable; therefore, to ensure adequate storage, store in more than one

aliquot and in different banks. • Freeze the vials in a polystyrene box to allow the cells to freeze slowly.