Human hepatocyte spheroids treated by Fc(aMD4)B3 or HGF

The changes in gene expression induced by Fc(aMD4)B3 or HGF (Hepatocyte growth factor) were examined in primary human hepatocyte spheroid culture by RNA-Seq. HGF is a natural ligand of c-Met receptor. Fc(aMD4)B3 is a surrogate c-Met receptor agonist made by lasso-grafting c-Met-binding macrocyclic peptide aMD4 into B3 position of Fc. Cryopreserved human hepatocytes were obtained from Gibco. Cells were seeded into ultra-low attachment 96-well plates at 1,500 viable cells per well and subsequently centrifuged at 200 × g for 2 min. Cells were seeded in 100 ul Williams E medium supplemented with 2 mM GlutaMAX, 100 units/ml penicillin, 100 ug/ml streptomycin, 4 ug/ml insulin, 1 uM dexamethasone, 15 mM HEPES (pH7.4), and 10% FBS. From five days after seeding, when the spheroids were sufficiently compact, 50% of the medium was exchanged every two days for a serum-free Williams E medium supplemented with 2 mM GlutaMAX, 100 units/ml penicillin, 100 ug/ml streptomycin, 6.25 ug/ml insulin, 6.25 ug/ml transferrin, 6.25 ug/ml selenous acid, 1.25 mg/ml BSA, 5.35 ug/ml linoleic acid, 100 nM dexamethasone, and 15 mM HEPES (pH7.4). Spheroids were treated in a serum-free medium with 0.3 nM HGF or 30 nM Fc(aMD4)B3 on day 7 and day 9. Forty-eight spheroids were collected for each group (non-stimulated control, Fc(aMD4)B3, HGF) and subjected to RNA isolation at day 8 (24h) or day 10 (72h).