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To minimize the risk of contamination, follow these 5 rules:
1. Always check the cells carefully before handling (by eye and on a microscope). Become familiar with the indicators of abnormal cell growth.
2. Whenever possible, maintain cultures without antibiotics for at least part of the time, to reveal cryptic contamination.
3. Check sterility of all reagents before use.
4. Use dedicated media and reagents; do not share with other cell lines.
5. Maintain a high standard of sterility at all steps.
Mycoplasma contamination, which may slow cell growth, cannot be checked under a regular microscope. To confirm or rule out such contamination, use a mycoplasma test (e.g. Roche Applied Science Mycoplasma PCR ELISA Kit).
There should be a laminar flow hood in the room dedicated to cell culture, and this hood should be usedfor all culture manipulations and storage of all equipment. The hood must be placed away from traffic orequipment that might generate air currents (e.g., centrifuges, refrigerators and freezers).Always carefully clean the hood before and after your procedure. Remove all unneeded items.It is crucial to always keep the work surface clean and tidy. To achieve this, follow these rules:
Ø Use 80% ethanol to clean the surface before starting.
Ø Place and keep on this surface only the items required for your procedure. This will reduce the possibility of contact between sterile and non-sterile items and facilitate culture manipulations.
Ø Clear space in the center of the bench, not just the front edge.
Ø Avoid spills, if they happen immediately clean the area.
Ø Remove everything when you are done, and again clean the work surface.
Reagents and media obtained from commercial suppliers will already have undergone strict quality testing. Most of the bottles are wrapped in polyethylene. The wrapping should be removed outside the hood. Unwrapped bottles should be cleaned with 80% ethanol whenever they are removed from the refrigerator or from a water bath. Regularly clean the refrigerator, the incubator and the water bath to avoid growth of mold or fungi. Imported cell lines should always be quarantined before being incorporated into your main stock. Do not perpetually use antibiotics; they will suppress some contaminants, but will not eliminate them.
Special care should be taken with caps. Use deep screw caps in preference to stoppers. When working on an open bench, flame glasspipettes and necks of the bottles before and after each use. Always use the pipettes which are best adapted your procedure; regularly clean them and check their calibration. Use a multi-channel pipette instead of a single pipette if you are working with multiwell plates. This will reduce both the time required to perform the procedure and the probability of contamination. Prepare as many reagents and equipment as possible in advance, to reduce the time the cultures are kept out of the incubator.
Cryopreservation Cell lines in continuous culture are prone to genetic drift, finite cell lines are fated forsenescence, all cell cultures are susceptible to microbial contamination, and even thebest-run laboratories can experience equipment failure. Because an established cell lineis a valuable resource and its replacement is expensive and time consuming, it is vitallyimportant that they are frozen down and preserved for long-term storage.As soon as a small surplus of cells becomes available from subculturing, they should befrozen as a seed stock, protected, and not be made available for general laboratory use.Working stocks can be prepared and replenished from frozen seed stocks. If the seedstocks become depleted, cryopreserved working stocks can then serve as a source forpreparing a fresh seed stock with a minimum increase in generation number from theinitial freezing.The best method for cryopreserving cultured cells is storing them in liquid nitrogen incomplete medium in the presence of a cryoprotective agent such as dimethyl sulfoxide(DMSO). Cryoprotective agents reduce the freezing point of the medium and also allow aslower cooling rate, greatly reducing the risk of ice crystal formation, which can damagecells and cause cell death.
DMSO is known to facilitate the entry of organic molecules into tissues. Handle reagents containing DMSO using equipment and practices appropriate for the hazards posed by such materials. Dispose of the reagents in compliance with local regulations.
Guidelines for Cryopreservation Following the guidelines below are essential for cryopreserving your cell lines for future use. As with other cell culture procedures, we recommend that you closely follow the instructions provided with your cell line for best results.
Ø Freeze your cultured cells at a high concentration and at as low a passage number as possible. Make sure that the cells are at least 90% viable before freezing. Note that the optimal freezing conditions depend on the cell line in use.
Ø Freeze the cells slowly by reducing the temperature at approximately 1oC per minute using a controlled rate cryo-freezer or a cryo-freezing container such as “Mr. Frosty,” available from NALGENE labware (Nalgene Nunc)
Ø Always use the recommended freezing medium. The freezing medium should contain a cryoprotective agent such as DMSO or glycerol.
Ø Store the frozen cells below –70oC; frozen cells begin to deteriorate above –50oC.
Ø Always use sterile cryovials for storing frozen cells. Cryovials containing the frozen cells may be stored immersed in liquid nitrogen or in the gas phase above the liquid nitrogen.
Ø Always wear personal protective equipment.
Ø All solutions and equipment that come in contact with the cells must be sterile. Always use proper sterile technique and work in a laminar flow hood.
Always use the recommended freezing medium for cryopreserving your cells. The freezing medium should contain a cryoprotective agent such as DMSO or glycerol.
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