Reagents and equipment

• 5 + 9d, adherent cultures of EBs: these consist of EBs plated at day 5 into 60 mm tissue-culture dishes, and incubated for 9 d further (Protocol 3)

• Trypsin/EDTA solution (Protocol 1)

• Hepatocyte culture medium (HCM): Hepa-tocyte basal, modified Williams 'E' medium (HBM medium; Cat. No. CC-3199, Cambrex) supplemented per 500 ml with the following quantities of sterile additives (from Cambrex, unless otherwise indicated): 0.5 ml ascorbic acid (Cat. No. CC-4316); 10 ml bovine serum albumin (BSA), fatty-acid-free (Cat. No. CC-4362); 0.5 ml hydrocortisone (Cat. No. CC-4335); 0.5 ml transferrin (Cat. No. CC-4313); 0.5 ml insulin (Cat. no. CC-4321); 0.5 ml human epidermal growth factor (Cat. No. CC-4317); 0.5 ml gentamycin-amphotericin (Cat. No. CC-4381); 0.05 mg/ml streptomycin and 0.03 mg/ml penicillin (Protocol 1)a

• 0.02 N acetic acid in distilled water, filter sterilized

• Collagen I solution: 0.05 mg/ml collagen Type I (Cat. No. 354236, BD Biosciences) in

• 60 mm tissue-culture dishes

• Sterile, 5 ml glass transfer pipette

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