Steps | Culture protocol | Culture media composition | Culture duration | CM labels |
EB formation | SHEF-2 cells were pooled from 3 T25 flasks and seeded in 10 cm non-adherent petridish in 20 ml of MEF-CM. | MEF-CM composed of: KO-DMEM + 20% SR + 8 ng/ml bFGF + 0.1 mM β-mercaptoethanol + 1% L-glutamine + 1% NEAA. | Day 0-1 | - |
FBS treatment of EBs | EBs were collected and cultured in 20 ml of 10%FBS supplemented KO-DMEM. | KO-DMEM + 10% FBS + 0.1 mM β-mercaptoethanol + 1% L-glutamine + 1% NEAA. | Day 1-3.5 | - |
Activin A treatment of EBs | EBs were collected and treated with 100 ng/ml human Activin A. | KO-DMEM + 10% SR + 100 ng/ml human Activin A + 0.1 mM β-mercaptoethanol + 1% L-glutamine + 1% NEAA. | Day 3.5-8 | Day-8 CM |
EB suspension culture | EBs were collected and cultured in KO-DMEM | KO-DMEM + 10% SR + 0.1 mM β-mercaptoethanol + 1% L-glutamine + 1% NEAA. | Day 8-11 | Day-11 CM |
EB suspension culture | EBs were collected and cultured in KO-DMEM. | KO-DMEM + 10% SR + 0.1 mM β-mercaptoethanol + 1% L-glutamine + 1% NEAA. | Day 11-16 | Day-16 CM |
EB suspension culture | EBs were collected and cultured in KO-DMEM. | KO-DMEM + 10% SR + 0.1 mM β-mercaptoethanol + 1% L-glutamine + 1% NEAA. | Day 16-22 | Day-22 CM |