CuteChlamyFreezing Chlamydomonas Cells

Johnson and Dutcher published a procedure for freezing Chlamydomonas in Trends in Genetics 9, 194-195 (1993). Bodas et al. described general methods for freezing algal cells, including Chlamydomonas, in Cryo Letters 16, 267-274 (1995). The older literature on this topic is reviewed in The Chlamydomonas Sourcebook. Recently another protocol was posted to bionet.chlamydomonas by Dick Sayre, and is reproduced here.

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

1. Grow cells to approx. 1,000,000 per ml in TAP (strain 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 strain 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 shaking.

7. Transfer cells to 1 ml 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

2137        Methanol                      1                    4.5

                                                        2                    13

                                                        3                    24

                                                        4                    22

                                                        5                    18

No cells (strain 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 2137 with methanol, however, unlike 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 Sayre