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