alcohol (and therefore the samples) will cool down at an
approximate rate of 1
C/min. After 100 min, the samples
are at À80
C and can be directly transferred to liquid nitrogen
for storage.
5. Nannochloropsis gaditana and Rhodomonas lens can also be
cryopreserved using a passive freezer, but in our experience,
survival and fitness of these species are improved when cryopreserved using a controlled rate freezer (see Note 4) with the
following cooling ramp: 1
C/min down to À10
C, hold at
À10
C for 1 min and cool down at 3
C/min to À40
C,
holding this temperature for a couple of minutes before transferring the straws to liquid nitrogen for storage.
6. Storage in liquid nitrogen requires handling of samples while
immersed in liquid nitrogen, and there are therefore several
security precautions that should be applied (see Note 5). Cryotubes (and/or straws) can then safely be transferred using the
large forceps or tweezers into the storage containers. Storage
containers can be cardboard or plastic boxes or canisters
(depending on whether cryo-tubes or straws are used).
7. For thawing, transfer the cryo-tubes/straws from the liquid
nitrogen to a warm water bath (35
C) and leave them until all
ice melts, avoiding swirling the sample too much to prevent
mechanical damage. This step can either be conducted with
sterile water inside the flow cabinet or outside, in which case
each tube/straw should be wiped with 70% ethanol prior to
further manipulation. Empty one tube (or four to eight straws)
into sterile 10 mL test tubes and dilute the cryoprotective
agent by adding fresh culture media in ten steps of 200 μL,
one min apart. After this 1:1 stepwise dilution, top the vial up
to 5 mL for incubation.
8. For the first 24 h, it is recommended to incubate under low
light intensity to allow for a gradual cell reactivation of photosynthetic mechanisms. Step 7 is also recommended to be performed under low light.
9. Viability assays: success can be assessed simply by examining
cells under the inverted microscope, by measuring cell density
(manual cell counting, flow cytometry quantification, measurement of in vivo fluorescence) over time (typically a few weeks),
or by using vital stain. Cryopreserved cultures typically exhibit
a quite long lag phase before reaching exponential growth and
providing an active culture than can be scaled up to larger
volumes.
614
Estefania Paredes et al.
approximate rate of 1
C/min. After 100 min, the samples
are at À80
C and can be directly transferred to liquid nitrogen
for storage.
5. Nannochloropsis gaditana and Rhodomonas lens can also be
cryopreserved using a passive freezer, but in our experience,
survival and fitness of these species are improved when cryopreserved using a controlled rate freezer (see Note 4) with the
following cooling ramp: 1
C/min down to À10
C, hold at
À10
C for 1 min and cool down at 3
C/min to À40
C,
holding this temperature for a couple of minutes before transferring the straws to liquid nitrogen for storage.
6. Storage in liquid nitrogen requires handling of samples while
immersed in liquid nitrogen, and there are therefore several
security precautions that should be applied (see Note 5). Cryotubes (and/or straws) can then safely be transferred using the
large forceps or tweezers into the storage containers. Storage
containers can be cardboard or plastic boxes or canisters
(depending on whether cryo-tubes or straws are used).
7. For thawing, transfer the cryo-tubes/straws from the liquid
nitrogen to a warm water bath (35
C) and leave them until all
ice melts, avoiding swirling the sample too much to prevent
mechanical damage. This step can either be conducted with
sterile water inside the flow cabinet or outside, in which case
each tube/straw should be wiped with 70% ethanol prior to
further manipulation. Empty one tube (or four to eight straws)
into sterile 10 mL test tubes and dilute the cryoprotective
agent by adding fresh culture media in ten steps of 200 μL,
one min apart. After this 1:1 stepwise dilution, top the vial up
to 5 mL for incubation.
8. For the first 24 h, it is recommended to incubate under low
light intensity to allow for a gradual cell reactivation of photosynthetic mechanisms. Step 7 is also recommended to be performed under low light.
9. Viability assays: success can be assessed simply by examining
cells under the inverted microscope, by measuring cell density
(manual cell counting, flow cytometry quantification, measurement of in vivo fluorescence) over time (typically a few weeks),
or by using vital stain. Cryopreserved cultures typically exhibit
a quite long lag phase before reaching exponential growth and
providing an active culture than can be scaled up to larger
volumes.
614
Estefania Paredes et al.