Cryopreservation of Cell Lines

OPS Diagnostics has several products used for cryogenically storing cell lines, such as Cryogenic Vials and Cryogenic Storage Boxes. Please visit our website to see these and other cryogenic products.

Introduction

The preservation of cells is an extremely important aspect of cell culture.  The only effective means of preservation of animal cells is by freezing, which can be accomplished with either liquid nitrogen or by employing cryogenic freezers.  The freezing process involves slowly reducing the temperature of prepared cells to -30 to -60°C followed by a transfer to temperatures less than -130°C.  Once at ultralow temperatures, the cells are biologically inert and can be preserved for years.

Preparation

Cryopreserving cultured cells differs from preserving bacteria and fungi in that higher viability is required.  Where a 1% survival rate of a microbial culture can be practical, such low viability is unacceptable with cultured cells.  High survival rates are clearly important for cell lines due to the expense and difficulty in preparation , slow relative rate of growth, and tendency to change with repeated passage in culture.  Consequently, methods used for cell culture cryopreservation must ensure high viability (i.e., >90%).

Factors that can affect the viability of cryopreserved cells include growth conditions prior to harvesting, the physiological state of the cells, the cell density, choice of cyroprotectant, and handling techniques.  Actively growing cells harvested from late-logarithmic to early-stationary phase cells usually yield the highest number of viable cells following freezing.  Once harvested, the desirable final concentration of cells should be between 10⁶ to 10⁷ cells/ml.  Higher densities are often useful with adherent cells since thawed cells can be diluted and plated at a desired density.  Cryoprotectants such as DMSO and glycerol are valuable to prevent cell dehydration during the freezing process.  The cell suspension is generally prepared at a concentration twice that desired for freezing so that an equal volume of 2X cyroprotectant can be added.  Gentle handling techniques during harvest and concentration will improve viability of the recovered cells.  Excessive enzymatic treatment, vigorous pipetting, and high-speed centrifugation should be avoided.

Cryoprotectants

The diffusion of cryoprotective agents such as glycerol or dimethylsulfoxide (DMSO) into a cell will result in a partial replacement of intracellular water and help to prevent dehydration (from ice formation) during freezing.  Glycerol is also known to stabilize proteins in their native states and to assist in the maintenance of critical macromolecular interactions at subzero temperatures.  The cryoprotectant should be prepared separately by combining the cryoprotective agent and the growth medium for the cells.  Cryoprotective agents are usually used individually in concentrations ranging from 5-15% (v/v) with the optimum varying with the cell type.   It is important that the cryoprotective agents be of highest possible quality and sterilized prior to use.   Glycerol may be sterilized by autoclaving for 15 minutes and should be stored in small aliquots to prevent introduction of contaminants.  DMSO should be sterilized by filtration with a 0.2 µm nylon syringe filter and stored at -20°C in small, single-use sealed aliquots.  Air oxidation of DMSO is relatively rapid and these products are toxic to cells.  DMSO should not be allowed to come into contact with the skin as it is rapidly absorbed and is a reported neurotoxin.   Preformulated cryoprotective media can also be purchased.

Equilibration

Cells mixed with the cryoprotectant require an equilibration time at room temperature prior to the onset of the cooling process.  This time generally ranges from 15 to 45 minutes and allows penetration of the cell by the cryoprotectant for maximum protective effect.

Cooling:

The rate of cooling controls the size of the ice crystals and the rate at which they are formed, both of which may affect cell recovery.  In most cases a slow, uniform cooling rate of -1°C per minute from ambient is effective.  Since programmable-rate freezing units are not generally available to the cell culturist, alternative methods have been developed.  Placement of the cryovials in a Styrofoam rack (from 15 ml centrifuge tubes) on the shelf of a -80°C freezer for 2-3 hours will result in a non-uniform cooling rate but is close to  -1°C per minute and satisfactory for a range of cell types.  Transfer should then be made to the storage temperature.

Storage

The temperature at which frozen cells are stored will affect their viability.  Storage at -80°C may permit slow chemical reactions (due to small amounts of unfrozen water), which will eventually result in cell death.  A temperature of less than  -130°C is required to completely stabilize cell preparations.  This is usually achieved by storage in liquid nitrogen (-196°C), liquid nitrogen vapor, or in an cryogenic freezer (-150°C).  All three methods are used with each presenting its own strengths.

Liquid nitrogen is a non-mechanical method of cryopreserving cells.  A large thermos-like container is used to house either racks or sleeves that hold cryogenic vials.  Cells stored in nitrogen can be placed above the liquid in a cold vapor phase or in the liquid nitrogen itself (-196°C).  Simple systems rely on a cycle of filling the tank and allowing the nitrogen to evaporate followed by refilling.  Liquid nitrogen storage systems do not require electricity, but rather a ready source of liquid nitrogen.  A small 50 liter tank will require filling every 5-6 weeks at a yearly cost of $900 to $1000.  Though liquid nitrogen is widely used for cell preservation, two problems exist with this storage method.  Cells stored in vapor phase can experience wide temperature fluctuations (i.e., -120 to -195°C), which can be potentially damaging to cells.  Secondly, capped vials submersed in the liquid can leak and pick up contaminants and also pose a risk of exploding when removed from the liquid (see Recoverybelow).

Cryogenic freezers are an alternative to the traditional methods of cryopreserving animal cells.  Cryogenic freezers use high efficiency compressors to reach temperatures as low as -150°C.  No filling is necessary with freezers although back-up non-mechanical refrigerants are available for added security.  Additionally, freezers are generally easier to catalog than many liquid nitrogen systems.

Recovery

Unlike the freezing process, rapid thawing of frozen cells is necessary to maintain viability.  Certain precautions should be exercised when thawing cells.  Vials stored in liquid nitrogen, especially screw capped tubes, often fill with liquid nitrogen while submersed.  When these tubes are removed from the tank, the tubes may pressurize and burst.  Thus, a face shield or goggles should be worn while thawing cells.  Vials stored in cryogenic freezers are a reduced risk of bursting.  Directly after removal from storage, vials should be thawed with agitation (but not for fragile hybridoma cells) in a 37°C water bath.  As the last ice crystals are melting, the vial is removed from the water.  Wipe, spray, or submerse the vial with 70% ethanol before opening it in a biosafety hood.

It is prudent when working with an unfamiliar cell line to determine the percentage of viable cells recovered by Trypan Blue staining.  This may serve to uncover any deficiencies in the cryopreservation process.  Note that safety precautions must be taken when recovering vials from the liquid nitrogen.  Insulated gloves should be worn to protect against burns from the low temperatures.  Though specially-designed cryovials are used to store cells, a face shield and laboratory coat serve to protect against fragments of exploding vials caused by introduction of liquid nitrogen (an all too common occurrence with leaky vials).

Cryopreservation Protocol

Materials

• Cultured cells
• Hemocytometer and cover slip
• 2X cryoprotective medium (e.g., DMEM with serum and 15% DMSO or glycerol)
• Cryovials or glass ampoules
• Propane torch (for glass ampoules)
• Cryogenic freezer (-150°C)
• -80°C freezer

Protocol (Freezing)

1. If cells are a monolayer culture, gently trypsinize to detach.
2. Count an aliquot of cells in hemocytometer.
3. Adjust the density of cells in culture medium to 1 X 10⁷ cells/ml.  Add an equal volume of 2X cryoprotective medium.  Allow the cells to sit at room temperature 15 min. so that the cryoprotectant can diffuse into the cell.
4. Transfer 1 ml of cells to a plastic or glass vial. 
   Note:For plastic vials it is necessary to use caps with O-rings that will allow for a tight fit.  Most plastic cryogenic tubes placed in liquid nitrogen do not form tight seals and will allow liquid nitrogen to seep into the tube if submersed.  This can be a concern since cultured cells, viruses, and bacteria, which may be present in the liquid phase of the nitrogen, can potentially contaminate a culture stored in a screw capped tube.  This is not a concern if cells are stored in only the vapor phase or in a cryogenic freezer.  To completely enclose the cells, glass ampoules can be used and sealed with a flame by rolling the neck of the ampoule in a flame until it becomes soft and pliable.  Using forceps, slowly pull the neck of the ampoule while continuing to roll the tube.  As the neck separates from the vial, roll the end of the vial in the flame to seal.  Once the vial is cooled, it can be submersed in a solution of Methylene Blue or Trypan Blue in order to ensure the vials are closed.  Wash the vials to remove the stain and examine the cell suspension for the dye.  Any dye on the inside of the vial means the vial was not sealed and should be discarded.
5. Clearly label the vials using permanent ink.  Include information on cell type and date.  This should be cross referenced to additional information on the cell line.  Cool the vials at a rate of 1°/min until they have reached -80°C.  Glass vials should be slanted while freezing so that  the liquid can expand without cracking the glass.  This can be done by placing the vials in a Styrofoam box and placing in a -80°C freezer overnight.
6. Transfer to a cryogenic freezer for permanent storage.

Protocol (Thawing)

1. Remove vials from the cryogenic freezer.  If cells are stored in liquid nitrogen, use tongs and insulated gloves, keeping in mind that pressure will build up inside the vials as the nitrogen expands into a gas.  The vial may shatter, thus wear goggles and a lab coat.
2. Thaw in a 37°C water bath with constant gentle shaking until completely thawed (<1 minute).  Carefully observe whether the glass vials have cracked during freezing or thawing.
3. Wash the vial with 70% ethanol.  Open the cryogenic tube or snap the ampoule aseptically.  Plate cells immediately into pre-warmed medium.
4. After attachment of monolayer cells, usually 1 to 10 hours, change the medium to remove the cryoprotectant.  If the cells are non-adherent, allow a sufficient recovery time (about 6 hours), then gently pellet (5 minutes at 400 X g) and resuspend in fresh medium.

References

American Type Culture Collection.  1992.  Quality Control Methods for Cell Lines, 2nd Ed.

Coriell, L.L.  1979.  Preservation, storage, and shipment.  In: Methods in Enzymology, vol. 58:29-36

Morris, C.B.  1995  Cryopreservation of animal and human cell lines.  In: Methods in Molecular Biology, Vol. 38:179-187.