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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

REFERENCE NO: AHC/1998/3/2.4/2


TITLE: CRYOPRESERVATION OF CELL LINES


PROCEDURE

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.

Attached Cell Lines

  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 37C 1C 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.

Suspension Cell Lines

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

Preparation of Cryoprotectant

  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.

Aliquoting of Cells

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

NB: The number of cells in an ampoule should be approximately 2 - 4 x 106 cells/ampoule.

Controlled Rate Freezing

 Load samples into appropriate controlled rate freezer and follow manufacturers instructions.


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

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