Dominik Haudenschild, January 11, 1996 at Genzyme

• I need about 250ml of cells/microcarriers for complete cell line characterization.

• Remove cells from the reactor in five 50ml tubes, using sterile procedure.

• Within about 15 minutes of being removed from the reactor, gently spin the cells and microcarriers at about 250g in a centrifuge

• Removed the cell culture supernatant until the total volume was about 12ml

• Add 1.5 ml bovine serum and gently break up the cell pellet

• Add 1.5 ml DMSO and gently mix

• Aliquot suspension into 1.5ml tubes with O-ring seal, filling each one to about 1.25ml so they don't burst when frozen

• Freeze in vapor phase of liquid nitrogen cryocontainer

• Using sterile technique, transfer about 20ml of cell/microcarrier suspension into a roller bottle

• Add growth media (925+10% serum)