Subculture of Suspension Cell Lines

Aim
In general terms cultures derived from blood (e.g. lymphocytes) grow in suspension. Cells may grow as single cells or in clumps (e.g. EBV transformed lymphoblastoid cell lines). For these types of lines subculture by dilution is relatively easy. But for lines that grow in clumps it may be necessary to bring the cells into a single cell suspension by centrifugation and resuspension by pipetting in a smaller volume before counting.

Materials

• Media– pre-warmed to 37ºC (refer to the ECACC Cell Line Data Sheet for the correct medium)

• 70% Ethanol in water (Prod. No. R8382)

Equipment

• Personal protective equipment (sterile gloves, laboratory coat, safety visor)

• Waterbath set to 37ºC

• Microbiological safety cabinet at appropriate containment level

• Centrifuge

• CO₂ incubator

• Inverted phase contrast microscope

• Haemocytometer (Bright-line, Prod. No. Z359629, Improved Neubauer, Camlab CCH.AC1)

• Pre-labeled flasks

Procedure

1. View cultures using an inverted phase contrast microscope. Cells growing in exponential growth phase should be bright, round and refractile. Hybridomas may be very sticky and require a gentle knock to the flask to detach the cells. EBV transformed cells can grow in very large clumps that are very difficult to count and the center of the large clumps may be non-viable.

2. Do not centrifuge to subculture unless the pH of the medium is acidic (phenol red = yellow) which indicates the cells have overgrown and may not recover. If this is so, centrifuge at 150g for 5 minutes, re-seed at a slightly higher cell density and add 10- 20% of conditioned medium (supernatant) to the fresh media.

3. Take a small sample of the cells from the cell suspension (100-200uL - Protocol 6 - Cell Quantification). Calculate cells/ml and re-seed the desired number of cells into freshly prepared flasks without centrifugation just by diluting the cells. The data sheet will give the recommended seeding densities.

4. Repeat this every 2-3 days.

Key Points

1. If the cell line is a hybridoma or other cell line that produces a substance (e.g. recombinant protein or growth factor) of interest retain the spent media for analysis.