13. Fill up the Erlenmeyer flask containing the beads to a total
volume of 100 mL with growth medium containing 0.9 M
sorbitol (see Note 5).
14. Cultivate cells under routine conditions for 2 more days.
3.2 Cryoprotection
1. Cool the Erlenmeyer flask containing cells immobilized in
alginate beads to 4
C.
2. Add 5 mL 100% Me 2 SO to the Erlenmeyer flask containing
100 mL cell/beads suspension, resulting in a final concentration of 5% Me 2 SO in the suspension (see Note 6). Work under
sterile conditions (see Note 7).
3. Incubate for a total duration of 60 min at 4
C (see Note 8).
3.3 Freezing
1. Separate the beads and medium by filtering through a Buchner
funnel (without Nylon net filter), and drain off the medium (see
Note 2).
2. Transfer five beads into a 2 mL cryovial, using a sterilized/
flamed forceps (see Note 9).
3. Place the cryovials into the cooling device and cool from 4
C
down to À40
C, using a cooling rate of À0.25
C/min (see
Note 10).
4. Hold the vials at a temperature of À40
C for 15 min (see Note
10).
5. Transfer the vials immediately into liquid nitrogen.
6. Store samples in liquid nitrogen.
3.4 Recovery
1. For thawing, transfer vials from liquid nitrogen immediately
into a water bath of 40
C (see Note 11).
2. Keep the samples at 40
C until completely thawed (~3 min).
3. Pour the thawed beads onto a sterile Petri dish (60 mm diameter) containing normal solid growth medium (see Note 12).
4. Press the beads gently onto the medium. Make sure that not
more than 1/3 of the beads are submersed in the medium.
5. After 4 h, transfer the beads to another Petri dish with fresh
solid growth medium.
6. Within 2–6 weeks, cells grow out of the beads and can be used
to reinitiate a new suspension culture.
4 Notes
1. In the initial and logarithmic phase of a cell culture, the cellular
water content is generally low and increases at the end of the
growth curve. Furthermore, cells are most vigorous in the
logarithmic growth phase and best suited for freezing.
642
Heinz Martin Schumacher et al.
volume of 100 mL with growth medium containing 0.9 M
sorbitol (see Note 5).
14. Cultivate cells under routine conditions for 2 more days.
3.2 Cryoprotection
1. Cool the Erlenmeyer flask containing cells immobilized in
alginate beads to 4
C.
2. Add 5 mL 100% Me 2 SO to the Erlenmeyer flask containing
100 mL cell/beads suspension, resulting in a final concentration of 5% Me 2 SO in the suspension (see Note 6). Work under
sterile conditions (see Note 7).
3. Incubate for a total duration of 60 min at 4
C (see Note 8).
3.3 Freezing
1. Separate the beads and medium by filtering through a Buchner
funnel (without Nylon net filter), and drain off the medium (see
Note 2).
2. Transfer five beads into a 2 mL cryovial, using a sterilized/
flamed forceps (see Note 9).
3. Place the cryovials into the cooling device and cool from 4
C
down to À40
C, using a cooling rate of À0.25
C/min (see
Note 10).
4. Hold the vials at a temperature of À40
C for 15 min (see Note
10).
5. Transfer the vials immediately into liquid nitrogen.
6. Store samples in liquid nitrogen.
3.4 Recovery
1. For thawing, transfer vials from liquid nitrogen immediately
into a water bath of 40
C (see Note 11).
2. Keep the samples at 40
C until completely thawed (~3 min).
3. Pour the thawed beads onto a sterile Petri dish (60 mm diameter) containing normal solid growth medium (see Note 12).
4. Press the beads gently onto the medium. Make sure that not
more than 1/3 of the beads are submersed in the medium.
5. After 4 h, transfer the beads to another Petri dish with fresh
solid growth medium.
6. Within 2–6 weeks, cells grow out of the beads and can be used
to reinitiate a new suspension culture.
4 Notes
1. In the initial and logarithmic phase of a cell culture, the cellular
water content is generally low and increases at the end of the
growth curve. Furthermore, cells are most vigorous in the
logarithmic growth phase and best suited for freezing.
642
Heinz Martin Schumacher et al.