Cells in 75 cm2 culture flasks were digested with trypsin, counted and mixed with alginate solution. Alginate cell suspension was added dropwise to calcium chloride solution using a syringe to make microbeads, which were then suspended and cultured.

Operation method

Alginate coating

Principle

Cells in 75 cm2 culture flasks were digested with trypsin, counted and mixed with alginate solution. Alginate cell suspension was added dropwise to calcium chloride solution using a syringe to make microbeads, which were then suspended and cultured.

Materials and Instruments

D-PBSA Trypsin
Culture medium suitable for cell growth Sodium alginate solution
Sterile filters

Move

Salt solution containing:

(a) NaCl, 8.0 g

(b) D-glucose, 1.0 g

(c) Prepare 1 L of UPW

(d) Adjust pH to 7.2~7.4 with HCl or NaOH

(e) Autoclave

0.1 mol/L _CaCl2 containing:

(a) Salt solution, 500 ml

(b) _CaCl2-2H2O, 7.35 g

(c) Adjust pH to 7.2~7.4 with HCl or NaOH (d) Autoclave 1. Dissolve alginate at 1.5 % concentration in salt solution. NaOH to adjust pH to 7.2-7.4

(d) Autoclaving

1. Dissolve the alginate in the salt solution at a concentration of 1.5 % and stir the solution at room temperature for at least 4 h until the alginate is completely dissolved. The alginate dissolved in the salt solution can be stored at 4 °C for 3 d.

2. Filter the alginate 3 times through a sterilized, non-thermally active 0.45 um filter. The last filtration should be done before the alginate is used.

3. Cells are cultured to confluence.

4. Cells are digested with 3 ml of trypsin (at a concentration suitable for cell passaging culture), and then cells are counted.

5. After centrifugation of the cells (900 r/min for 4 min ), the trypsin-containing supernatant is completely discarded.

6. The cell density is adjusted by gently suspending the cells in the alginate, so that the cells are mixed with the alginate. Adjust the cell density to ^2x106 cells/ml by gently suspending the cells in alginate. At this point, it is critical that no air bubbles are generated in the alginate, as air bubbles will result in the formation of voids within the beads.

7. Transfer the alginate-suspended cells into a sterile syringe fitted with a 27G needle.

8. Using a tortuous modeling motion, add a droplet of alginate cell suspension to a beaker containing 0.1 mol/L _CaCl2. _CaCl2 induces the formation of alginate beads.

9. The beads were gelatinized for 10 min and then washed three times with D-PBSA.

10. Finally, the beads were washed with growth medium.

11. The beads were placed into 175 ^cm2 culture flasks containing growth medium and incubated at 37°C, 100 % relative humidity and 5 % _CO2.

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