BUCHI Encapsulator B-395 Pro - Manual (Page 64)

6 Operation
64 B-395 Pro Operation Manual, Version C
Procedure
1. Prepare the reaction vessel and autoclave it as described in section 6.10 and 6.11.
2. Prepare all solutions and labware.
3. Fill the autoclaved reaction vessel with 225 mL polymerization solution.
4. A cell culture with ca. 6×10
6 cells (or according to personal need) is centrifuged and the pellet is
re-suspended in 2 mL sterile MOPS washing buffer and mixed with 10 mL 1.5 % sodium-alginate
solution. Give care, that no or only few air bubbles are introduced during mixing.
5. Fill a 20 mL syringe with the cell-alginate suspension and attach the syringe to the reaction vessel
in a laminar air hood.
6. Fix the reaction vessel to the Encapsulator control unit, which is placed on the bench.
7. Start bead formation with previously established parameters.
8. Allow for bead hardening for 5 minutes, then stop stirrer and drain off polymerization solution.
NOTE
The beads and later the capsules should always be covered by a small amount of liquid to prevent
clumping, otherwise re-suspension of the beads and capsules would become difficult and the
membrane might be damaged.
9. Pump in 75 mL 0.05 % PLL solution and let form the PLL-alginate membrane for 10 minutes.
10. Drain of the 0.05 % PLL-solution.
11. Pump in 150 mL MOPS washing buffer, stir for 1 min and then drain off.
12. Pump in another 150 mL MOPS washing buffer, stir for 5 min and then drain off the buffer.
13. Pump in 100 mL 0.03 % alginate solution and allow 5 minutes stirring for the formation of the
outer alginate membrane, then drain off the alginate solution.
14. Pump in 150 mL MOPS washing buffer, stir for 1 min and then drain off the buffer.
15. Pump in 150 mL depolymerization solution and stir approx. 10 minutes to dissolve the alginate of
the bead core. Appropriate dissolution time is dependent on the molecular weight and purity of the
alginate, and the susceptibility of the encapsulation product to the depolymerization solution.
16. Drain off the depolymerization solution.
17. Pump in 150 mL MOPS washing buffer, resuspend the capsules and transfer them into the bead
collecting flask.
18. Transfer the capsules in culture medium and cultivate them.
NOTE
Dissolved alginate diffuses out slowly. Depending on the alginate in use and the thickness of the
capsule membrane, it can take up to 2 h for substantial amounts to leave the capsule. To maximize
removal of the core you can:
• Extend the extraction time in MOPS or in a culture medium without bivalent ions.
• Cultivate the cells in a medium containing < 50 mg/l of Ca ions.
• Cultivate the cells in a medium with a ratio of monovalent ions (Na + , K + ) to bivalent ions
(Ca 2+ , Mg 2+ ) between 20:1 and 50:1.