Cryopreservation enables investigators to store cell cultures and to prevent the need to have all cell lines in culture at all times. Storing cells is invaluable when working with primary cells due to their limited life span. MOREOVER, CRYOPRESERVATION DECREASES RISK OF:
- microbial contamination (bacteria, yeast, fungi, viruses)
- mycoplasma contamination
- cross contamination with other cell lines
- genetic drift due to genomic instability resulting in physiological and morphological consequences)
IMPORTANTLY, cryopreservation allows investigators to conduct collaborative studies by using cells at a consistent passage number Centralized cell storage decreases costs (consumables and staff time)
To ensure successful cryopreservation and resuscitation of a wide variety of primary and established cell lines, essential steps should be followed. The precise requirement may vary among cell lines and as a general rule, cells must be cooled at a rate of –1°C to –3°C per minute and thawed quickly by incubation at 37°C (for 3-5 minutes). General considerations:
- Cell cultures must have a viability of >95%
- There should be no signs of contamination by microbes (mycoplasma and other infectious agents (see mycoplasma detection service).
- Cell population should be in the log phase of growth; this can be achieved by using pre-confluent cultures (consult TCCF director).
- Use a cryoprotectant such as dimethyl sulphoxide (DMSO) or glycerol and/or a high concentration of serum/protein (>20%) or combination thereof to protect the cells from rupture by the formation of ice crystals. The most commonly used cryoprotectant is DMSO at a final concentration of 5 to 10%, it cannot always be used due its toxicity or its impact on cell differentiation.
- The TCCF distributes ready-made cell freezing media for cryopreservation (DMSO, glycerol and serum-free formulations)
NOTE: The use of DMSO may not be appropriate for all cell lines (e.g. where DMSO is used to induce differentiation). In such cases an alternative such as glycerol should be used.
Following controlled-rate freezing (see above) in the presence of cryoprotectants, cell lines can be cryopreserved in a suspended state for indefinite periods provided a temperature of less than -165°C is maintained. TCCF uses ultra-low temperatures, attained by use of specialized containers (usually by immersion in liquid or vapor-phase nitrogen). The advantages and disadvantages can be discussed with the TCCF director.
Our liquid nitrogen storage vessels are equipped with alarms system. TCCF personnel ensure liquid nitrogen delivery in a timely fashion. It is highly recommended that valuable cell stocks should be backed up by storage at a second site.
The TCCF storage vessels should include a racking/ inventory system designed to organize the contents for ease of location and retrieval. This should be supported by accurate record keeping and inventory control incorporating the following:
- Ampules are individually labeled, using low temperature-resistant labels.
- The location of each ampule is recorded in an electronic database, and on a spreadsheet (back-up). A paper record is also maintained.
- There is a system to ensure that no ampule can be deposited or withdrawn without updating the records.