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REFERENCE NO: AHC/1998/4/2.2.1 Appendix 2



Cell lines are provided either as frozen cultures (in plastic cryo-ampoules delivered on dry ice) or as active (growing) cultures in plastic flasks or tubes. Frozen cells should not be stored at -80 C for extended periods of time, but in liquid nitrogen (-196 C).

Frozen Cells

  1. Thaw the ampoule in a waterbath (25-37 C).
  2. Transfer the cells to a 10-15 ml centrifuge tube.
  3. Add 6-10 ml prewarmed medium to the cells, mix gently (some scientists recommend dropwise addition of medium).
  4. Centrifuge the cells at 200 x g for 5 min.
  5. Decant supernatant, resuspend the cells in a defined volume of medium, remove a small sample in order to determine cell number and viability, and centrifuge again.
  6. Decant supernatant and adjust cell suspension to the recommended concentration in culture medium containing all supplements listed on the cell line information sheet.

Active Culture

  1. The culture vessel is completely filled with medium to prevent loss of cells in transit. Remove the medium and add freshly prepared, prewarmed medium sufficient to cover the bottom of the flask.
  2. Incubate and subdivide the culture as outlined in the cell line information sheet. If the cells became detached from the plastic surface in transit: remove the entire contents of the flask and centrifuge at 200 x g for 5 min.
  3. Decant the supernatant and resuspend the cells in fresh culture medium (remove a small sample in order to determine cell number and viability).
  4. Seed the cell suspension in a flask of suitable size according to the instructions given for the respective cell line.

Suspension culture

  1. For shipment the flask(s) or tube(s) was completely filled with medium. Remove the entire volume and centrifuge at 200 x g for 5 min.
  2. Resuspend the cells in a defined volume of medium and determine cell number and viability; possibly, wash the cells again.
  3. Finally, resuspend the cells in freshly prepared culture medium containing the required supplements and incubate as recommended in the cell line information sheet.
  4. For the first few days we suggest using a higher concentration of FBS than usually required; we strongly recommend the omission of any antibiotics for long term culturing. If not otherwise stated: incubate and propagate the cells at 37 C in a humidified atmosphere containing 5% CO2.
  5. Do not forget to freeze one or more ampoules as soon as possible (i.e. when you split the cells for the first time).

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