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Introduction and Overview

Mammalian cells can be cryopreserved (frozen) and maintained for many years (typically ten years or more) by careful preparation and strict attention to the details of the following guidelines. The two approaches outlined under this protocol can be used to successfully cryopreserve primary, normal, or continuous (immortal) cell types from either anchorage dependent or suspension growth systems. (This includes B and T lymphocytes separated from blood, transformed or not.) The basic tenant to follow in cryopreserving and subsequently recovering cells is to freeze slowly and thaw rapidly. Two of the most commonly used approaches to accomplish this are outlined here.

Cryopreservation Medium

The medium used to cryopreserve cells should always be the same formulation as that used to propagate the cells with the addition of fetal bovine serum (FBS) (in the case of serum-free cultures) or the increase in concentration of FBS, but to a final concentration of no more than 20%. The FBS will tend to bind toxic materials that may be released if some cells are lysed during the freezing or thawing process. A cryopreservative MUST also be used. Either of two (2) may be used. The primary choice is sterile dimethyl sulfoxide (DMSO) at a final concentration of between 7% and 10%, with ten percent being optimum in most cases. The other choice is sterile glycerol, also at a final concentration of 10% in the medium. Glycerol should be used if the cell type to be cryopreserved may be adversely or unwantedly affected by exposure to strong bi-polar compounds such as DMSO. An example of this is HL-60 cells which could become differentiated upon exposure to DMSO. Where this would be of concern or could negatively affect the subsequent culture, glycerol would be the preferred cryopreservative. Important: The cryopreservative, especially DMSO, must be used fresh each time. Do not purchase large volumes and continue to open and close the reagent bottle. Both cryopreservatives will either rapidly oxidize and/or absorb potentially toxic materials from the air. Cryopreservation medium should be made fresh each time in only the volumes required for that day. It can be kept at +4°C for up to one week and used usually without problem. However, extremely sensitive cultures, especially cultures that have been propagated in serum free medium may require very fresh, pure cryopreservation medium.

Preparation of Cells

Anchorage dependent cells should be harvested by trypsinization (or other appropriate method) when newly confluent or before (70-80% confluency is ideal). Suspension cells should be harvested in mid log phase growth, between 4 x 105 and 8 x 105 cells/ml. Count the cells using a hemacytometer and resuspend the cells in fresh cryopreservation medium at a final concentration of 2-5 x 106 cells/ml. Aliquot 1 ml of cell suspension into each of either 1.8 ml or 2.0 ml cryovials. Label the vials with a marker or special laser printed cryo labels resistant to liquid nitrogen temperatures, vapor phase = between –150°C to –180°C and liquid phase = 196°C.

Freezing the Cells

Method 1. Programmable rate freezer

If a programmable rate freezer is available, this approach is by far the best. The program should be set to begin at the temperature of the cells when you start, either room temperature or if the cells have to be held for longer than about an hour after being aliquoted but before the freezing process is started, put the cells at +4°C and set the program to start at that temperature. Then, the program should lower the temperature at a rate of -1°C per minute down to –30°C. Between –30°C and –50°C, set the rate to cool at -0.5°C per minute. After –50°C to below -100°C, the rate can go back to –1°C per minute. Once the cells are below –100°C, they can be placed directly into liquid nitrogen storage.

Method 2. Insulated container

In the absence of a programmable rate freezer, alternatively, cells can be frozen by placing the vials in an insulated rack or similar insulated container that has both wall insulation and insulation between the vials. A common item used is a Styrofoam rack that 15 ml centrifuge tubes are often packaged in. The vials are placed in the holes where the tubes were and another rack is placed over the top of the tubes, effectively “sandwiching” the vials between the Styrofoam racks. That way, the vials are also insulated from each other individually. This is an important arrangement. The attempt is to accomplish the controlled, slow freezing process outlined above. By placing the vials in the rack arrangement described and then putting the assembly in a –70°C or –80°C freezer, the vials will freeze slowly due to the time it takes to cool the surrounding insulation. After leaving the vials in the rack arrangement overnight, or even up to several days, they can be transferred directly to liquid nitrogen storage.

Thawing cryopreserved cells

Cells that have been appropriately cryopreserved should be recovered as follows. Cells are thawed rapidly by submersing the cryovial containing the cells in pre-warmed sterile double distilled, deionized water (37° C, 1-2 min.) and the cell suspension then plated into a 25 cm2 tissue culture flask in 10 ml of the appropriate growth medium. If cells have to be transported from liquid nitrogen over any lengthy distance or time, they must be transported either in liquid nitrogen or dry ice. Never transport cryopreserved cells on regular ice. This will begin slow thawing which is the opposite of what is desired for good viable recovery. After 24 to 48 hours, the medium can be changed to remove the cryopreservative. However, the initial dilution, when first plating the cells, will bring the concentration of cryopreserevative down to below 1%, a concentration that will typically not be toxic or otherwise problematic to the culture.

Notice of disclaimer.
All methods discussed under this protocol have been used at the TCF for over 17 years with freeze and subsequent recovery rates of over 99%. This protocol is not copyrighted nor is it proprietary. It may be used and distributed without prior consent of the TCF. However, the TCF makes no guarantee, expressed or implied, that similar results will be achieved by using this protocol. For further information regarding any aspect of this protocol or services provided by the TCF, please contact Steve Oglesbee, Director, at 919-966-4324.

TCF General Protocol EBV.v10 2/7/2007