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(Click here to download a text version of this information)
INTRODUCTION
To ensure that we have sufficient and continuous supplies of cells, e.g. in case of infections of breakdown of equipment leading to loss of growing cultures, stocks of cells are stored as cryopreserved cells. Normal, during freezing, particularly when rapid, ice crystals form within cells that affect membrane lipids & lipoproteins leading to splitting of cell membranes, resulting in loss of internal architecture and release of enzymes and processes that destroy the cell when it is thawed (process of frost-bite!). Osmotic damage also occurs as water is lost from the cells due to increased salt concentrations in the extracellular medium and the cells therefore shrink. However, if cells are cooled slowly and at a constant rate cooling occurs with the complete loss of water, meaning that no intracellular ice crystals will be present in the cells and they can then be preserved dehydrated. To allow this to happen the freezing is performed in a -70°C freezer at a rate of 1°C/min until the temperature reaches -70°C, after which the cells are transferred into liquid nitrogen (-196°C). The rate at 1°C/min freezing allows for sufficient time at -20°C to -30°C for cell shrinkage. Cells may be stored for many years At -196°C without substantial loss of cell viability. It is important to note that cells have to be stored sterile in special plastic or (less often used today) glass containers called cryovials that can withstand the rate of freezing and thawing.
To achieve the correct rate of cooling it is necessary to use either an active or passive controlled rate freezer. The former electronically controls cooling at a programmable preset rate using liquid nitrogen. Passive freezers either rely on holding cells at a predetermined temperature e.g. -25°C for a certain time period prior to plunging them into liquid nitrogen or using a precooled block or container and solvent (e.g. "Mr Frosty") which cools the cells at -1°C/minute to around -150°C. Of these the Mr Frosty is by far the cheaper option, and uses the cooling of cells (in special cryovials) placed in the inner chamber of plastic container, the outer, sealed chamber of which is filled with isopropanol. Upon placing this container in a -70°C freezer the isopropanol cools at rate of 1°C/min, thus allowing the cells to cool at this rate. An alternative is to seal the cells in a polystyrene container of 2-4 cm thick walls, which is then placed in a 70°C freezer However, although this has often been used in the past, ECACC found cell viability rarely to exceed 75% and advise against this method. Other methods of freezing mentioned above that act at controlled rate will normally preserve cells at their original viability.CRYOPRESERVATIVES
DMSO (Dimethyl sulphoxide): DMSO penetrates the cells and exerts a protective effect on them during the freezing process by binding water and reducing the availability for the formation of intracellular ice crystals, thus reducing the critical temperature range in which cell damage takes place.
Glycerol: his is another commonly used protective agent, which functions in the same way. It tends to be sued when the cells are sensitive to DMSO (which can be toxic to some cells),. However, care should be taken as glycerol penetrates some cells slowly.
SAFETY
(note: the following is for advice only. Instiutional and/or local safety rules should be followed at all times. Advice should be sought from your local safety or technical officer at all times before starting work)
In addition to normal cell culture safety issues the following should be noted:
- LIQUID NITROGEN is extremely dangersous and can cause freeze-burns very easily. Only trained operators, wearing appropriate safety clothes are allowed to handle it.
- When placing or removing cells from liquid nitrogen it is recommend that two operators be present.
- Wear a full (HOWIE) laboratory coat, the special shoes/insulated gloves/etc. provided for use with liquid N2 and a full protective face mask. NOTE: Ampoules in liquid N2 can EXPLODE when removing them from the dewar!!!
- Only approved equipment and containers must be used with liquid nitrogen at all times.
- USE ONLY PLASTIC AMPOULES DESIGNED FOR STORAGE IN LIQUID NITROGEN!!! Screw-top Cryovials are OK but push-fit eppendorfs are NOT! If you are not sure then ask the supplier.
- Handle frozen ampoules with care at all times! IT IS RECOMMENDED THAT SAFETY GLASSES SHOULD BE WORN UNTIL CELLS HAVE BEEN THAWED!
PROCEDURE FOR FREEZING & RESUSCITATING CELLS
(last updated Nov. 10th, 2003)
FREEZING OF CELLS
1. Cells should be harvested in the recommended manner for that cell line, i.e. that used for routine subculture. Cells should be in an active growing (log) phase, near confluency but not at it, when selected for freezing. Ideally:
- Cells should be grown in antibiotic-free medium, as antibiotics can interfere with cell growth (however, this requires confidence in your own techniques and should not be done until the operator has confidence that can freeze and resuscitate cells without risk of infection)
- Mycoplasma and microbial contaminants should be tested for.
- Viability should be assessed to ensure that cells are at the appropriate (usually >80%) viable.
2. Prepare freeze medium by allowing 1 ml for each ampoule (normally a 25 cm2 flask full of cells is frozen per ampoule). ECACC recommend the use of 90% serum + 10% cryoprotectant for all of its cells unless otherwise specified on the data sheet. Most cell lines can be frozen in the appropriate culture medium supplemented with 20% serum and 10% cryoprotectant. This is usually DMSO but in certain instances (refer to datasheet or other recommendations for the particular cell line) glycerol is recommended. The chosen cryopreservative should be tested on each cell line before use. Do not include antibiotics as these can inhibit growth of cells on resuscitation and also suppress any existing bacterial or fungal contamination upon resuscitation.
3. Resuspend the cell pellets in the freeze medium to give a final cell concentration of between 2 and 4 x 106 cells/ml and aliquot 1ml into each ampoule. The ampoules should be clearly marked using a permanent marker pen (one that has ink that can withstand freezing in liquid nitrogen!!). If there is any doubt about rapid exposure of the cryoprotectant causing toxic shock to the cells, resuspend the pellets in half the required volume of freeze medium without cryoprotectant, mix and then add equal volume of medium to which double the volume of cryoprotectant has been added. Normally a flask full of cells should be frozen per ampule.
4. SAFETY NOTICE: USE ONLY PLASTIC AMPOULES DESIGNED FOR STORAGE IN LIQUID NITROGEN!!! Screw-top Cryovials are OK but push-fit eppendorfs are NOT! If you are not sure then ask the supplier.
5. Freeze the cells at a cooling rate between 1-3°C/min using a polystyrene box or a 'Mr Frosty' (BDH cat no. 215/0884/00) or similar in a -80°C freezer for 24 hours prior to transfer to gaseous phase liquid Nitrogen. When the temperature has reached -150°C , transfer the ampoule to a gas phase liquid Nitrogen storage vessel. (NOTE: a programmable freezer is suitable alternative to a box/Frosty)
6. It is advisable to test the cell viability by thawing one ampoule after freezing. Viability's below 70% may cause problems in starting new cultures, due to low cell numbers and the presence of debris.
RESUSCITATION (THAWING & CULTURE) OF CELLS
1). Handle ampoules with care! Wear a full (HOWIE) laboratory coat, the special insulated gloves provided for use with liquid N2 and a full protective face mask (ampoules in liquid N2 can EXPLODE when removing them from the dewar!!!
2). Leave the ampoule at room temp for approx. 1 minute and then transfer to a water bath at 37°C for 1-2 minutes until fully thawed (it is important to thaw quickly to minimise any membrane damage that can occur on thawing). DO NOT immerse ampoule fully in the water as this can lead to contamination.
3). NOTE: The next stages all take place in a class II cabinet
4). Wipe the ampoules all over with a tissue soaked in 70% alcohol. prior to opening
5). Slowly pipette the contents of the ampoule directly into a flask of pre-warmed (37°C) media in order to achieve the recommended optimum cell density for growth (refer to date sheet or other source of recommendation for the particular cell type in you are not user what this value is).
6). NOTE: centrifugation, and washing, of the cells prior to culture to remove DMSO is no longer recommended (by ECACC) as the centrifugation itself can damage the cells. The DMSO should be gradually diluted out during subsequent sub-culture. If in any doubt then it recommended that the medium is changed the next day, or when the cells have adhered, etc.
7). Culture, and maintain, the cells in the flask as normal, i.e. 5% CO2, 37°C, etc. Don't forget to regularly freeze down fresh stocks, so that you end up with stocks of different passage number. This is useful in case of infections or genetic changes (loss of desired attributes) in the immortal cells being grown
NOTES:
i). ECACC recommends that a stock of the cell line(s) should be frozen as soon as possible after receipt at between 2 to 4 x 106 cells/ml as a precaution
BIBLIOGRAPHY
i). Cryopreservation of Animal Cells in Advances in Biotechnology Processes (1988), 7, A.R. Liss