(250–300 mL), one Buchner funnel (60 mm diameter) with
nylon net (100 μm pore size), two Buchner funnels (60 mm
diameter) without nylon net, and flammable metal spatula or
spoon.
9. The following materials have to be purchased sterilized: 50 mL
Falcon tubes and 2 mL cryovials.
10. Pipette tips with widened opening. To widen the opening of a
pipette tip, cut using scissors or a scalpel. A self-made positioning device helps to standardize this (see Note 9).
11. Technical equipment: gyratory shaker, programmable cooling
device (e.g., a Planer K10 from Planer plc or for cooling rates
of 1
C/min a Mr. Frosty from Nalgene).
3 Methods
3.1 Immobilization
and Pre-culture
1. Use cultures in the logarithmic growth phase (see Note 1).
Harvest cells by filtration through a nylon net filter in a Buchner funnel. Let the medium gently drain off and do not apply
vacuum (see Note 2).
2. Wash cells twice in the Buchner funnel with calcium-free
growth medium.
3. Fill a volume of 10 mL cells in a sterile 50 mL Falcon tube using
a sterile metal spoon or spatula. Fill up to 30 mL (see Note 3)
with growth medium containing 3% alginate (see Note 4).
4. Close the Falcon tube and mix gently to suspend the cells.
5. Transfer the suspension carefully and dropwise into 250 mL of
a solution of growth medium containing 100 mM calcium
chloride in a 500 mL beaker. This should be done drop-bydrop using a pipette tip with a widened opening while stirring
gently.
6. For polymerization, stir gently for 20 min.
7. Let the alginate beads settle down, pour off the medium containing calcium, and wash the beads twice with standard
growth medium.
8. After washing, separate the beads from the medium by filtering
through a sterile Buchner funnel (without a Nylon net filter)
(see Note 2).
9. Transfer the beads into a 300 mL Erlenmeyer flask.
10. Fill up to a total volume of 100 mL normal liquid growth
medium (see Note 3).
11. Cultivate under normal growth conditions for 3 days.
12. Let the beads settle down and pour off the medium.
Cryopreservation of Plant Cell Lines
641
nylon net (100 μm pore size), two Buchner funnels (60 mm
diameter) without nylon net, and flammable metal spatula or
spoon.
9. The following materials have to be purchased sterilized: 50 mL
Falcon tubes and 2 mL cryovials.
10. Pipette tips with widened opening. To widen the opening of a
pipette tip, cut using scissors or a scalpel. A self-made positioning device helps to standardize this (see Note 9).
11. Technical equipment: gyratory shaker, programmable cooling
device (e.g., a Planer K10 from Planer plc or for cooling rates
of 1
C/min a Mr. Frosty from Nalgene).
3 Methods
3.1 Immobilization
and Pre-culture
1. Use cultures in the logarithmic growth phase (see Note 1).
Harvest cells by filtration through a nylon net filter in a Buchner funnel. Let the medium gently drain off and do not apply
vacuum (see Note 2).
2. Wash cells twice in the Buchner funnel with calcium-free
growth medium.
3. Fill a volume of 10 mL cells in a sterile 50 mL Falcon tube using
a sterile metal spoon or spatula. Fill up to 30 mL (see Note 3)
with growth medium containing 3% alginate (see Note 4).
4. Close the Falcon tube and mix gently to suspend the cells.
5. Transfer the suspension carefully and dropwise into 250 mL of
a solution of growth medium containing 100 mM calcium
chloride in a 500 mL beaker. This should be done drop-bydrop using a pipette tip with a widened opening while stirring
gently.
6. For polymerization, stir gently for 20 min.
7. Let the alginate beads settle down, pour off the medium containing calcium, and wash the beads twice with standard
growth medium.
8. After washing, separate the beads from the medium by filtering
through a sterile Buchner funnel (without a Nylon net filter)
(see Note 2).
9. Transfer the beads into a 300 mL Erlenmeyer flask.
10. Fill up to a total volume of 100 mL normal liquid growth
medium (see Note 3).
11. Cultivate under normal growth conditions for 3 days.
12. Let the beads settle down and pour off the medium.
Cryopreservation of Plant Cell Lines
641