Cryopreservation of cells
- Cells can be preserved for later use by freezing stocks in liquid nitrogen.
- This process is referred to as cryopreservation and is an efficient way of sustaining stocks.
- Indeed, it is advisable that, when good cultures are available, aliquots of cells should be stored in the frozen state.
- This provides a renewable source of cells that could be used in future without necessarily having to culture new batches from tissues.
- Freezing can, however, result in several lethal changes within the cells, including formation of ice crystals and changes in the concentration of electrolytes and in pH.
- To minimise these risks a cryoprotective agent such as DMSO is usually added to the cells prior to
freezing in order to lower the freezing point and prevent ice crystals from forming inside the cells.
- In addition, the freezing process is carried out in stages, allowing the cells initially to cool down slowly from room temperature to -80°C at a rate of 1–3°C min-1.
- This initial stage can be carried out using a freezing chamber or alternatively a cryo freezing container (‘Mr Frosty’) filled with isopropanol, which provides the critical, repeatable -1°C min-1 cooling rate required for successful cell cryopreservation.
- When this process is complete, the cryogenic vials, which are polypropylene tubes that can withstand temperatures as low as -190 °C, are removed and immediately placed in a liquid nitrogen storage tank where they can remain for an indefinite period or until required.
- The actual cryogenic procedure is itself relatively straightforward.
- It involves harvesting cells as described in Section 2.5.5 and resuspending them in 1 cm3 of freezing medium, which is basically culture medium containing 40% serum.
- The cell suspension is counted and appropriately diluted to give a final cell count of between 106 and 107 cells cm-3 .
- A 0.9-cm3 aliquot is transferred into a cryogenic vial labelled with the cell type, passage number and date harvested.
- This is then made up to 1 cm3 by adding 100 mm3 of DMSO to give a final concentration of 10%.
- The cells should then be mixed gently by rotating or inverting the vial and placed in a ‘Mr Frosty’ cryo freezing container.
- The container and cells are placed in a -80 °C freezer and allowed to freeze overnight.
- The frozen vials may then be transferred into a liquid nitrogen storage container.
- At this stage cells can be stored frozen until required for use.
- All procedures should be carried out under sterile conditions to avoid contaminating cultures as this will appear once the frozen stocks are recultured.
- As an added precaution it is advisable to replace the growth medium in the 24-h period prior to
harvesting cells for freezing.
- Moreover, cells used for freezing should be in the log phase of growth and not too confluent in case they may already be in growth arrest.
- Principles and techniques of biochemistry and molecular biology 7th edition by Wilson walker.