2. The BY2 cell line grows as a very fine suspension. Therefore, for
harvesting, it has to be poured through a filter. For many more
clumpy cell cultures, it may be possible to let cells settle down
(incline the Erlenmeyer flask), after which the medium can be
poured off, and the culture flask can be filled with the fresh
medium containing pre-culture osmotic conditions.
3. In many cases, the cells-to-medium ratio is important for the
cryopreservation procedure and needs to be experimentally
determined. In any case, it is important to use the same ratio
in repeated experiments. This is typically determined by comparing/measuring the volume of settled/immobilized cells (scv
settled cell volume) versus the total volume of the suspension.
4. In the presented method, controlled rate freezing is combined
with immobilization of cells in alginate. Immobilization is not
necessary but seems to stabilize the cells. Furthermore, immobilization/encapsulation in alginate beads may slow down diffusion processes during pre-culturing with osmotic and
cryoprotective agents.
5. The best substances to be used for osmotic pre-culturing, its
optimum concentration, as well as the duration of
pre-culturing all have to be determined experimentally. The
most frequently used osmotic substances are sorbitol, mannitol, and sucrose, with concentrations ranging from 0.3 M up to
1.2 M and pre-culture durations from 2 to 6 days. For embryogenic cultures, the pre-culturing period may be even longer.
6. Me 2 SO can be used as the only cryoprotective agent, at concentrations ranging from 2.5% to even 10%. For many cultures,
we obtained good results when using 5% Me 2 SO. Originally,
Withers and King [6] used the following mixtures for cryoprotection: DGS (1 M Me 2 SO, 1 M glycerol, 2 M sucrose; in
normal growth medium) and DPG (1 M Me 2 SO, 1 M glycerol,
2 M proline; in normal growth medium). These mixtures are
filter sterilized. However, they are so viscous that moderate
warming is needed to allow proper filter sterilization. For cryopreservation, half a volume of such a cryopreservation solution
is mixed with an equal volume of pre-cultured cell suspension.
It is not recommended to use the mixtures described above in
combination with immobilized cells.
7. We use Me 2 SO under sterile conditions in the clean bench, and
do not filter sterilize this. Toxic oxidation products may accumulate with time, and therefore only small batches/bottles of
Me 2 SO should be used. When stored at colder temperatures,
the substance may solidify. Me 2 SO may dissolve certain filter
materials, which are used for filter sterilization.
Cryopreservation of Plant Cell Lines
643
harvesting, it has to be poured through a filter. For many more
clumpy cell cultures, it may be possible to let cells settle down
(incline the Erlenmeyer flask), after which the medium can be
poured off, and the culture flask can be filled with the fresh
medium containing pre-culture osmotic conditions.
3. In many cases, the cells-to-medium ratio is important for the
cryopreservation procedure and needs to be experimentally
determined. In any case, it is important to use the same ratio
in repeated experiments. This is typically determined by comparing/measuring the volume of settled/immobilized cells (scv
settled cell volume) versus the total volume of the suspension.
4. In the presented method, controlled rate freezing is combined
with immobilization of cells in alginate. Immobilization is not
necessary but seems to stabilize the cells. Furthermore, immobilization/encapsulation in alginate beads may slow down diffusion processes during pre-culturing with osmotic and
cryoprotective agents.
5. The best substances to be used for osmotic pre-culturing, its
optimum concentration, as well as the duration of
pre-culturing all have to be determined experimentally. The
most frequently used osmotic substances are sorbitol, mannitol, and sucrose, with concentrations ranging from 0.3 M up to
1.2 M and pre-culture durations from 2 to 6 days. For embryogenic cultures, the pre-culturing period may be even longer.
6. Me 2 SO can be used as the only cryoprotective agent, at concentrations ranging from 2.5% to even 10%. For many cultures,
we obtained good results when using 5% Me 2 SO. Originally,
Withers and King [6] used the following mixtures for cryoprotection: DGS (1 M Me 2 SO, 1 M glycerol, 2 M sucrose; in
normal growth medium) and DPG (1 M Me 2 SO, 1 M glycerol,
2 M proline; in normal growth medium). These mixtures are
filter sterilized. However, they are so viscous that moderate
warming is needed to allow proper filter sterilization. For cryopreservation, half a volume of such a cryopreservation solution
is mixed with an equal volume of pre-cultured cell suspension.
It is not recommended to use the mixtures described above in
combination with immobilized cells.
7. We use Me 2 SO under sterile conditions in the clean bench, and
do not filter sterilize this. Toxic oxidation products may accumulate with time, and therefore only small batches/bottles of
Me 2 SO should be used. When stored at colder temperatures,
the substance may solidify. Me 2 SO may dissolve certain filter
materials, which are used for filter sterilization.
Cryopreservation of Plant Cell Lines
643