Alginate Culture of Chondrocytes
Dominik Haudenschild, September 18, 1996

Materials Needed
• Salt Solution
0.15M NaCl
25mM Hepes pH 7.4
• Alginate
1.2% Alginate in Salt Solution, autoclaved then 0.4mm filtered
• 102mM Calcium Chloride in Water
• 55mM Sodium Citrate in Salt Solution
** All reagents can be at room temperature **

Putting cells into Alginate Beads
Trypsinize cells pellet gently in centrifuge, and aspirate media
Resuspend cells in Alginate at 1 million cells per ml
Aspirate cells into 10ml syringe, then attach 22 gauge needle
Add cells in alginate to 2 volumes of Calcium Chloride solution (in either a 50 ml tube or 125ml
bottle) dropwise with constant mixing
Allow beads to cure for 5 to 10 minutes at room temperature
Pour off liquid
Wash beads 2-3 times with 1 volume Salt Solution
Wash beads 1-2 times with 1 volume media (with serum)
Put 10 million cells into T-175 flask, and feed with 100ml media

Release of cells from Alginate Beads
Wash beads once or twice with salt solution
Resuspend beads in 2-5 volumes of Sodium Citrate in Salt solution
Agitate gently at room temperature or 27°C until beads dissolve
Pellet cells in clinical centrifuge
Remove supernatant