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CELL LINES
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Write the cell lines to be frozen in the current freeze preservation book.

Print/ hand write labels with cell line name, batch number and passage number (attached lines only).

NB: The cell line to be frozen must be in exponential log growth phase.

1. Pour off the cell culture media saving approximately 30ml in a centrifuge tube for the centrifugation step.
2. Wash cell sheet twice with PBS.
3. Add sufficient 2.5% trypsin/0.25% EDTA to cover the cell sheet i.e. 3-5 ml per 175cm² flask.
4. Pour off excess trypsin and incubate at 37°C ± 1°C until the cells detach from the flask (approximately 5 to 10 minutes).
5. Resuspend the cells in the culture media, saved in 1.1.
6. Centrifuge cultures at 150g for 5 min.

1. Aliquot the cells into centrifuge tubes.
2. Centrifuge cultures at 150g for 5 min.

1. For cell lines which are not particularly difficult to culture use 50% FCS, 40% medium, 10% DMSO; for complicated cells use 90% FCS, 10% DMSO or glycerol as appropriate. Allow 1ml for each ampoule.

1. Pour off supernatant from cell pellet following centrifugation. Resuspend cell pellet in cryoprotectant.
2. Aliquot 1 ml of cells per cryotube, tighten tops.

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