new freezing protocol

Elizabeth Harris chlamy at ACPUB.DUKE.EDU
Fri Jun 28 12:32:41 EST 1996

Dick Sayre just sent me this to pass along to all of you:

      Using a modification (a range of cryoprotectants and concentrations
for different strains) of the ATCC protocol we have tried freezing cells
three times using CC-2137 (WT) and once using a cell wall-less, Arg (-)
strain, CC-425. The results have been quite encouraging. The protocol is as

1. Grow cells to approx. 1,000,000 per ml in TAP (CC-2137) or TAP plus Arg
(CC-425). Pellet cells and resuspend in 1/10 volume fresh growth media.
2. To a 1.8 ml Nunc style cryotube add 250 ul of appropriate growth media
containing 2 - 10% (v/v) methanol or DSMO. We find 6% methanol (3% final
concentration) works best for CC-2137 and CC-425 (see below).
3. Add 250 ul (equal volume) of 10X cells to the tube. This gives a final
cryoprotectant concentration range of between 1-5% (v/v).
4. Place the tubes in a Nalgene Cryo 1C Freezing Container (esssentially an
isopropanol bath, costs about $35, catalogue # 5100-0001) and place the
Cryo-container in a minus 80 C freezer. The cryo-container slowly freezes
the cells at about 0.9-1.0 C/min. Leave Cryo-container in freezer until
isopropanol reaches - 40 C; about 65-72 min.
5. Remove tubes and immediately freeze in liquid nitrogen. Store at liquid
nitrogen temperatures.
6. Thaw cells by placing in a 35 C water bath for two minutes with gentle
7. Transfer cells to 10 mls of appropriate growth medium and grow 6 -18
hours before plating.

We have screened various cryoprotectant concentrations using either methanol
or DMSO. Here are the averages of three trials for methanol. The lowest
level of survival we have obtained using 3% (v/v) methanol is 15% and the
highest is 31%.

Strain                      Cryoprotectant          %volume         %survival
 CC-2137                  Methanol                       1

        No cells (strain CC-2137) survived when frozen without
cryoprotectant or when using DMSO. We have frozen strain CC-425 only once
and the survival numbers are similar to CC-2137 with methanol, however,
unlike CC-2137, there is a low level of survival of CC-425 when using DMSO.
One caveat, we have only thawed cells after 1 day in storage, however,
according to the ATCC representative there should not be a substantial loss
in viability over time.
*   Richard T. Sayre                                              *
*   Associate Professor                                         *
*   Depts. of Biochemistry and Plant Biology      *
*   Ohio State University                                      *
*   310F Kottman Hall, 2021 Coffey Rd.             *
*   Columbus, OH 43210 USA                             *
*                                                                            *
*   (614) 292-9030 office                                      *
*   (614) 292-8379 lab                                          *
*   (614) 292-7162 fax                                          *
*    email: sayre.2 at                                  *

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