BUCHI Encapsulator B-395 Pro - Manual (Page 61)

6 Operation
61 B-395 Pro Operation Manual, Version C
6 .13 Encapsulation procedure for immobilization of micro-organisms in
Ca-alginate beads
In this section, a simple but well established method is described for the immobilization of microorgan-
isms in Ca-alginate beads. The stability of these beads depends not only on the alginate type used,
but on the future culture conditions. The Ca ions in the hardening solution substitute for the Na ions in
the droplets causing the alginate beads to harden (this is a reversible reaction). If more resistant beads
are required, because some culture media have ingredients that will slowly dissolve the beads, Ca ions
may be replaced by Ba ions, which have a stronger affinity to alginate than Ca ions and the resultant
Ba-alginate beads will be more stable.
Sterilization of alginate solutions is best accomplished by sterile membrane filtration (0.2 µm). Heat
sterilization tends to partially degrade the alginate and unpredictably changes the viscosity and the
polymerization capacity.
For the encapsulation of animal cells, it is recommended to use another protocol because the three
dimensional structure of the Ca-alginate hinders the formation of the new cell membrane during cell
division. For dividing cells, capsules are better suited. A procedure for the production of alginate-PLL
capsules is described in section 6-14.
1. Prepare all needed materials as described in section 6-10; reaction vessel, pressure bottle, 60 mL
syringe, beakers, graduated cylinders, etc. Autoclave the reaction vessel.
2. Prepare all needed encapsulation reagents.
For this run: 50 mL 1.5 % alginate solution, low viscosity grade, sterile filtered
500 mL 100 mM CaCl
2
polymerization solution (non-sterile)
600 mL 0.9 % NaCl + 10 mM CaCl
2
washing solution (non-sterile)
3. Switch on the control unit. Set the vibration frequency, the electrostatic tension and the pumping
rate to the appropriate values as previously determined.
4. Place 500 mL of polymerization solution in the pressure bottle and close it. Attach the silicone
tubing to the liquid membrane filter. Pump the polymerization solution into the reaction vessel.
5. Detach the silicone tubing from the membrane filter and place the reaction vessel in a sterile
biological hood.
6. Prepare 10 mL of concentrated microorganism suspension. The suspension should be free of
bi- or trivalent cations (e.g. Ca, Mg, Al, Fe) or contain them in a very low concentration, to avoid
preliminary polymerization reactions with the alginate. Chose the desired microorganism concen-
tration the way that it is in the final polymer mixture < 1010 cells/mL (for animal cells <107 cells/
mL). Carefully mix the 10 mL microorganism suspension with 50 mL 1.5 % sterile alginate solution
gently to minimize the formation of air bubbles.
7. Fill a sterile 60 mL syringe with the polymer-product mixture aseptically. Attach the syringe to the
bead producing unit. Attach the reaction vessel (with the attached syringe) to the control unit.
Advance the syringe pump arm, so that it touches the plunger. Start the magnetic stirrer so that a
slight vortex is visible. Activate the vibration and the syringe pump. Press the “turbo” button until
a continuous liquid jet is formed. Activate the electrostatic dispersion unit. If needed, modify the
pumping speed or/and the frequency to obtain a clear bead chain down to the collection cup.