398
Related Techniques
10. Rinse the gel beads in 30-ml aliquots each of 0.1 % CHES and l.l %
CaCI 2 , and two washes of 30-ml aliquots of 0.85% KCI solution.
11. React the cell-containing gel beads with 0.05% PPL (Mv = 2.7 x 10 5 )
solution for 6 min.
12. Rinse the beads in 30 ml each 0.1 % CHES, 1.1 % CaCI 2 , and 0.85% KCI
solution.
l3. Wash the beads twice with 0.85% KCI solution.
14. Incubate the beads in 0.05 M sodium citrate solution for 6 min to
reliquify the interior of the capsules.
15. Place the beads in a mixture of equal volumes of 0.85% KCI solution
and double-strength culture medium in a tube, and mix by rocking
end to end for 20-30 min to allow the lower molecular weight sodium alginate to diffuse out of the capsules and to allow the capsules
to swell toward equilibrium.
16. Wash the beads three times in 0.85% KCI solution.
17. React the beads with 30 ml of 0.02%-0.15% PPL (Mv = 2.2 x 10 4 ) for
6 min.
18. Wash the beads three times with 30 ml 0.85% KCI solution.
19. React the beads with 30 ml of 0.03% sodium alginate solution for 4
min.
20. Wash the beads twice with 30 ml KCI solution to remove unbound
alginate.
21. Transfer the beads into complete culture medium.
Results
Cells are encapsulated and multiply in the microcapsules.The cells tend
to settle on the bottom of the capsules. The cells are liberated into culture medium by passing the capsule suspension through a 26-G needle
to rupture the capsules. King et al. (1989) were able to grow cell up to a
density of 8 x 10 7 cells/ml using microcapsules.
Comments
In addition to the microcapsule method, several other methods can be
used to immobilize the cells.
Microcarrier Method
The common primary matrix materials for micro carriers are dextran,
cellulose, gelatin, and plastic (polystyrene). Of these, dextran-based
micro carriers (Cytodex 1,2,3; Pharmacia Fine Chemicals) are most
widely used. The important features of micro carriers are surface charge,
density and size. In particular, surface charge is crucial for cell attachment to and cell growth on micro carriers. With regard to size, particles
with a diameter in the range 100-230 flm are most commonly used. Cells
on the surface of the micro carriers may suffer mechanical damage due
Related Techniques
10. Rinse the gel beads in 30-ml aliquots each of 0.1 % CHES and l.l %
CaCI 2 , and two washes of 30-ml aliquots of 0.85% KCI solution.
11. React the cell-containing gel beads with 0.05% PPL (Mv = 2.7 x 10 5 )
solution for 6 min.
12. Rinse the beads in 30 ml each 0.1 % CHES, 1.1 % CaCI 2 , and 0.85% KCI
solution.
l3. Wash the beads twice with 0.85% KCI solution.
14. Incubate the beads in 0.05 M sodium citrate solution for 6 min to
reliquify the interior of the capsules.
15. Place the beads in a mixture of equal volumes of 0.85% KCI solution
and double-strength culture medium in a tube, and mix by rocking
end to end for 20-30 min to allow the lower molecular weight sodium alginate to diffuse out of the capsules and to allow the capsules
to swell toward equilibrium.
16. Wash the beads three times in 0.85% KCI solution.
17. React the beads with 30 ml of 0.02%-0.15% PPL (Mv = 2.2 x 10 4 ) for
6 min.
18. Wash the beads three times with 30 ml 0.85% KCI solution.
19. React the beads with 30 ml of 0.03% sodium alginate solution for 4
min.
20. Wash the beads twice with 30 ml KCI solution to remove unbound
alginate.
21. Transfer the beads into complete culture medium.
Results
Cells are encapsulated and multiply in the microcapsules.The cells tend
to settle on the bottom of the capsules. The cells are liberated into culture medium by passing the capsule suspension through a 26-G needle
to rupture the capsules. King et al. (1989) were able to grow cell up to a
density of 8 x 10 7 cells/ml using microcapsules.
Comments
In addition to the microcapsule method, several other methods can be
used to immobilize the cells.
Microcarrier Method
The common primary matrix materials for micro carriers are dextran,
cellulose, gelatin, and plastic (polystyrene). Of these, dextran-based
micro carriers (Cytodex 1,2,3; Pharmacia Fine Chemicals) are most
widely used. The important features of micro carriers are surface charge,
density and size. In particular, surface charge is crucial for cell attachment to and cell growth on micro carriers. With regard to size, particles
with a diameter in the range 100-230 flm are most commonly used. Cells
on the surface of the micro carriers may suffer mechanical damage due