SUBCULTURING ATTACHED AND SUSPENSION CELLS

LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

REFERENCE NO: AHC/1998/3/2.1/2

TITLE: SUBCULTURING ATTACHED AND SUSPENSION CELLS

MATERIALS

The cell line
Appropriate tissue culture medium (pre-warmed to 37°C) and tested for microbial contaminants
Centrifuge tubes and Bench top centrifuge
Tissue culture flasks
Trypan blue stain (0.4%)
Haemacytometer
Pipettes
Ca²⁺, Mg²⁺- free PBS for attached cells (pre-warmed to 37°C)
0.25% Trypsin/EDTA for attached cells (pre-warmed to 37°C)
Inverted microscope

PROCEDURE

For Attached Cell Lines

Check the cells for microbial contamination by microscopic examination.

Check cells for attachment, then decant medium and wash the cell layer twice with PBS (i.e. approximately 1ml per 10cm² surface area).

Add trypsin/EDTA (i.e. approximately 0,5 to 2ml per 25cm² surface area). Spread the trypsin over cell layer.

Incubate at 37 ± 1°C until the cells are detached (approx. 5 to 10 min). Once cells start to detach, tap flask gently and check that they are all loose using microscope.

Wash cells into corner of flask with fresh medium (2 to 3ml/25cm²).

Pipette gently to break-up the clumps.

Perform a viable cell count where necessary, e.g. when preparing cells for cryopreservation, by taking up a sample and counting by the trypan blue exclusion method (AHC/1998/3/3.1/2.1).

Where specific seeding densities are required centrifuge cell suspension at 200g for 5 minutes and discard supernatant.

Resuspend the cells or cell pellet at required seeding density in fresh media in new flasks or roller bottles.

For Suspension Lines

Follow the protocol above except omit steps 2 to 6.

Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
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