Cryopreservation PDF

Cryopreservation SBP 3410 – Cell & Tissue Culture Prof. Daud Israf Cell Signaling Laboratory Faculty of Medicine & Health Sciences Universiti Putra Malaysia [email protected] SBP 3410– Cryopreservation – Sli...

Cryopreservation SBP 3410 – Cell & Tissue Culture Prof. Daud Israf Cell Signaling Laboratory Faculty of Medicine & Health Sciences Universiti Putra Malaysia [email protected] SBP 3410– Cryopreservation – Slide 1/25  Learning Outcome At the end of this class the student is able to: Define cryopreservation. Describe the various equipment and apparatus used in the cryopreservation of cell lines. Explain basic principles and concepts in the cryogenic preservation of cell lines. SBP 3410– Cryopreservation – Slide 2/25  Rationale for cell freezing Genotypic drift due to genetic instability. Senescence and extinction of the cell line. Transformation of growth characteristics and acquisition of malignancy-associated properties. Phenotypic instability due to selection and dedifferentiation. Contamination by microorganisms or cross- contamination by other cell lines. Misidentification due to careless handling. Incubator failure. Saving time and materials by not maintaining lines other than those in current use. Need for distribution to other users. SBP 3410– Cryopreservation – Slide 3/25  Theoretical background to cell freezing Slow freezing allows water to leave the cell but not so slowly that ice crystal growth is encouraged. Use hydrophilic cryoprotectants to sequester water. Store cells at lowest possible temperature to minimize effects of high salt concentrations on protein denaturation in micelles within the ice. Thawing rapidly to minimize ice crystal growth and generation of solute gradients formed as the residual intracellular ice melts. SBP 3410– Cryopreservation – Slide 4/25  Cell concentration Cells appear to survive freezing best when frozen at a high cell concentration. This is due to reduced viability on thawing requiring a higher seeding concentration. Survival is improved at a high cell concentration if cells are leaky because of cryogenic damage. A high concentration at freezing also allows sufficient dilution of the cryoprotectant at reseeding after thawing, so that centrifugation is unnecessary (at least for most cells). SBP 3410– Cryopreservation – Slide 5/25  Freezing medium The medium in which cells are frozen is comprised of media, serum and a cryoprotectant. Most common cryoprotectants: glycerol or dimethyl sulfoxide (DMSO). DMSO more effective, penetrates the cell better. Concentrations of between 5% and 15% have been used, but 7.5% or 10% is more usual. In some cells (haematopoietic) DMSO may be toxic or induce cells to differentiate after thawing. In these cases glycerol is preferable. Many laboratories also increase the serum concentration in freezing medium to 40%, 50%, or even 100%. SBP 3410– Cryopreservation – Slide 6/25  Cooling rate Cooled at 1oC/min. The cooling curve is governed by : → ambient temperature → any insulation incl. the ampoule → specific heat and volume of the ampoule contents → latent heat absorption during freezing. SBP 3410– Cryopreservation – Slide 7/25  Ways to control cooling rate Cotton wool and polystyrene foam boxes. Ampoule canes in tubular foam pipe insulation. Freezer neck plug. Nunc freezing container. Controlled-rate programmable freezer. If recovery is low, adjust cooling rate (i.e., by use of more or less insulation). SBP 3410– Cryopreservation – Slide 8/25  Containers for slowing the cooling rate FIGURE 19.3 Neck Plug Cooler. Modified neck plug for narrow necked freezers, allowing controlled cooling at different rates. Shown is the section of the freezer neck with the modified neck plug in place. The retaining ring is used to set the height of the ampoules within the neck of the freezer. The lower the height, the faster the cooling. SBP 3410– Cryopreservation – Slide 9/25  Considerations when cooling Cooling rate is proportional to the difference in temperature between ampoules and ambient air. At −70oC, cells will cool rapidly to around −50oC, but the cooling rate falls off significantly after that. Do not estimate at the projected 1oC/min cooling rate, as the bottom of the curve is asymptotic. To be safe leave the ampoules at −70oC overnight before transferring them to liquid nitrogen. Upon removal, cells will heat up at a rate of about 10oC/min. It is critical that they do not warm up above −50oC, so the transfer to the liquid nitrogen freezer must take significantly less than two minutes. SBP 3410– Cryopreservation – Slide 10/25  Ampoules Plastic ampoules (polypropylene) are safer than glass. Use alcohol and low temperature resistant markers/labels. Labelling and different colored caps help identification. A reference record must be kept. Plastic ampoules will leak if too slack or too tight (due to distortion of the o-ring). Inexperienced users should avoid glass ampoules as they have a serious risk of explosion when thawed. If glass ampoules are used, make sure perfectly sealed and use vapour phase cooling instead of -70oC freezer. SBP 3410– Cryopreservation – Slide 11/25  Cryofreezers (liquid nitrogen containers) Transfer cells rapidly to a cryofreezer following cooling. Storage systems include canisters or trays. Either submerge cells in liquid phase (- 190oC) or suspend in vapour phase (- 110oC). Weekly monitoring with a dipstick. Liquid-phase storage has an explosion hazard so wear face shield/goggles. If liquid nitrogen storage is not available, the cells may be stored in a conventional freezer (-70oC). Deterioration (5-10% per annum) may occur at -70oC. SBP 3410– Cryoprservation – Slide 12/25  Cryofreezer design SBP 3410– Cryopreservation – Slide 13/25  Freezing cells SBP 3410– Cryopreservation– Slide 14/25  Freezer record SBP 3410– Cryopreservation – Slide 15/25  Thawing frozen ampoules SBP 3410– Cryopreservation – Slide 16/25  Freezing flasks/plates Flasks are frozen by growing cells to late log phase, adding 5%-10% DMSO to the smallest volume of medium that will effectively cover the monolayer, and placing the flask in an expanded polystyrene container of 15-mm wall thickness. The insulated container is placed in a −70◦C freezer, and it will freeze at approximately 1◦C/min. Survival is good for several months, as long as the flask in its container is not removed from the freezer. Twenty-four-well plates may also be frozen in the same manner with about 150 μL freezing medium per well and can be used to store large numbers of clones during evaluation procedures. SBP 3410– Cryopreservation – Slide 17/25  Vitrification Although the slow cooling method is suitable for most cultured cell lines, some cells, such as preimplantation embryos and human embryonal stem cells, require rapid cooling and vitrification. It is possible that their three dimensional structure, although very small, still presents diffusion limitations during the slow cooling process. Vitrification is the transformation of a liquid into a glass (a supercooled liquid) and is achieved by plunging the cells in a plastic capillary tube, or straw , into liquid nitrogen. The cryoprotectants DMSO and ethylene glycol are used with sucrose in stepwise increasing concentrations. SBP 3410– Cryopreservation – Slide 18/25  Design and control of freezer stocks SBP 3410– Cryopreservation – Slide 19/25  Serial replacement of culture stock Replace stock cultures from the freezer at regular intervals to minimize genetic drift and phenotypic variation. After 2 1/2 months, thaw out another vial, check and expand it to replace existing stocks. Discard existing stocks after 3 months, and move to new stock. Repeat every 3 months with cells that have a population- doubling time (PDT) of approximately 24 h; cell lines with shorter or longer PDTs may need shorter or longer replacement intervals, respectively. SBP 3410– Cryopreservation – Slide 20/25  Cell banking Several cell banks exist for the secure storage and distribution of validated cell lines. Because many cell lines may come under patent restrictions, particularly hybridomas and other genetically modified cell lines, it has also been necessary to provide secure patent repositories with restricted access. As a rule, it is preferable to obtain your initial seed stock from a reputable cell bank, where the necessary characterization and quality control will have been done. Furthermore it is highly recommended that you submit valuable cultures to a cell bank as this will protect you against loss and allow distribution of the cells to others unless you wish distribution restricted, which you can specify. SBP 3410– Cryopreservation – Slide 21/25  Commercial cell banks SBP 3410– Cryopreservation – Slide 22/25  Cell transport Transport as frozen ampoules or as living cultures. Always provide detailed instructions. Ship frozen ampoules in solid CO2. For growing cultures the cells should be at the mid- to late-log phase. Flask is filled with medium, taped securely around the neck and sealed in a small polythene bag. Further wrapping for protection is recommended and sealed in an envelope with cushioned inflatable outer jacket. Label ‘‘fragile’’ and, in large letters: DO NOT FREEZE! On receipt, remove medium leaving the normal amount for culture. Wean onto new medium, but keep the original shipping medium in case of adaptation problems. Use a courier service for shipping and follow custom control regulations. SBP 3410– Cryopreservation – Slide 23/25  Transportation containers for cells SBP 3410– Cryopreservation – Slide 24/25  Summary Cryopreservation involves the validation and freezing of stock cultures for safe use in research. Cryoprotectants are used with suitable cooling apparatus and procedures with cryofreezers to maintain cell viability for long-term storage. Freezer records are important for tracing a cell lines history. Thawing should be done rapidly in an aseptic environment to ensure cell viability and sterility. Stock maintenance should follow SOPs to prolong usage. Cell banks maintain and sell cell lines to researchers. Cells can be transported either frozen or at mid-log phase growth and require strict adherence to proper packaging. SBP 3410– Cryopreservation – Slide 25/25