42 Large-Scale Cell Culture
397
- Culture flasks (75 cm 2 )
- 22-G and 26-G needles
- 0.85% KCI
- 1.4% Sodium alginate solution (dissolve 1.4 g sodium alginate powder in 100 ml of 0.85% KCI solution. Sterilize the alginate solution by
heating in a boiling water bath at 100°C for 5 min)
- 0.03% Sodium alginate solution (add 2.1 ml of 1.4% sodium alginate
solution to 97.9 ml of 0.85% KCI solution)
- 1.1% CaCl 2 solution (dissolve 14.57 g CaCI 2 .2H 2 0 in 1,000 ml of distilled water)
- 1.5% CaCl 2 solution (dissolve 19.86 g CaCI 2 .2Hp in 1,000 ml of distilled water)
- 0.1% 2 (n-cydohexylamino) ethane sulfonic acid (CHES) solution {to
prepare 0.1 % CHES solution, first prepare CHES stick solution (dissolve 2.0 g CHES and 0.51 g KCI in 100 ml of distilled water), and then
add 5 ml CHES stock solution to 95 ml of 1.1 % CaCl 2 solution)
- 0.05M Sodium citrate solution (dissolve 2.58 g sodium citrate and 0.85 g
KCI in 200 ml of distilled water)
- Polylysine solution (dissolve appropriate amount of polY{L-lysine)
hydrobromide (Mv = 2.7 x 10 5 or Mv = 2.2 x 10 4 )(PLL) in sterile 0.85%
KCI solution)
- Culture media
Procedure
1. Discard the medium from a 75 cm 2 culture flask when cells have
reached sub confluence.
2. Add 10 ml of fresh culture medium to the flask.
3. Dislodge the cells from the substrate by gentle shaking or pipetting.
4. Centrifuge the cell suspension at 150 g for 10 min and discard the
medium.
5. Suspend the cell pellet in 3 ml of 1.4% sodium alginate solution.
6. Place 2 ml of the above alginate/cell suspension in a 10-ml plastic syringe.
7. Extrude this viscous solution through a 22-G needle with the aid of a
syringe pump operating at 39 cm 3 /h. Droplets forming at the needle
tip are pulled off by a stream of rapidly flowing air in the annular
region between the two coaxial cylinders. The air flow is controlled by
a rotameter at 4500 scm 3 /min. In this manner, extrude a 2-ml volume
of alginate/cell suspension into 30 ml of 1.5% CaCl2 solution in which
the droplets harden into calcium alginate beads.
8. Discard the supernatant when the beads settled in the suspension.
9. Use the remaining 1 ml sodium alginate solution containing cells to
determine the initial cell density and viability.
Solutions
397
- Culture flasks (75 cm 2 )
- 22-G and 26-G needles
- 0.85% KCI
- 1.4% Sodium alginate solution (dissolve 1.4 g sodium alginate powder in 100 ml of 0.85% KCI solution. Sterilize the alginate solution by
heating in a boiling water bath at 100°C for 5 min)
- 0.03% Sodium alginate solution (add 2.1 ml of 1.4% sodium alginate
solution to 97.9 ml of 0.85% KCI solution)
- 1.1% CaCl 2 solution (dissolve 14.57 g CaCI 2 .2H 2 0 in 1,000 ml of distilled water)
- 1.5% CaCl 2 solution (dissolve 19.86 g CaCI 2 .2Hp in 1,000 ml of distilled water)
- 0.1% 2 (n-cydohexylamino) ethane sulfonic acid (CHES) solution {to
prepare 0.1 % CHES solution, first prepare CHES stick solution (dissolve 2.0 g CHES and 0.51 g KCI in 100 ml of distilled water), and then
add 5 ml CHES stock solution to 95 ml of 1.1 % CaCl 2 solution)
- 0.05M Sodium citrate solution (dissolve 2.58 g sodium citrate and 0.85 g
KCI in 200 ml of distilled water)
- Polylysine solution (dissolve appropriate amount of polY{L-lysine)
hydrobromide (Mv = 2.7 x 10 5 or Mv = 2.2 x 10 4 )(PLL) in sterile 0.85%
KCI solution)
- Culture media
Procedure
1. Discard the medium from a 75 cm 2 culture flask when cells have
reached sub confluence.
2. Add 10 ml of fresh culture medium to the flask.
3. Dislodge the cells from the substrate by gentle shaking or pipetting.
4. Centrifuge the cell suspension at 150 g for 10 min and discard the
medium.
5. Suspend the cell pellet in 3 ml of 1.4% sodium alginate solution.
6. Place 2 ml of the above alginate/cell suspension in a 10-ml plastic syringe.
7. Extrude this viscous solution through a 22-G needle with the aid of a
syringe pump operating at 39 cm 3 /h. Droplets forming at the needle
tip are pulled off by a stream of rapidly flowing air in the annular
region between the two coaxial cylinders. The air flow is controlled by
a rotameter at 4500 scm 3 /min. In this manner, extrude a 2-ml volume
of alginate/cell suspension into 30 ml of 1.5% CaCl2 solution in which
the droplets harden into calcium alginate beads.
8. Discard the supernatant when the beads settled in the suspension.
9. Use the remaining 1 ml sodium alginate solution containing cells to
determine the initial cell density and viability.
Solutions