TABLE 1.
Three‐dimensional differentiation protocol of hiPSC in Biomimesys®.
| Days 45 | Incubation conditions | Milieu | Cytokines/molecules |
|---|---|---|---|
| D‐3 | Hypoxia (4% O2, 5% CO2) | StemMACS™ iPS‐Brew XF medium (Miltenyi) | ROCK inhibitor (Y27632, 10 μM) (Cell Guidance Systems) |
| D‐2 | StemMACS™ iPS‐Brew XF medium (Miltenyi) | ‐ | |
| D‐1 | ‐ | ‐ | |
| D0 |
Normoxia (20% O2, 5% CO2) |
RPMI 1640 medium (Life Technologies) + B27 (with insulin) (Life Technologies) |
Activin A (100 ng/ml) (Miltenyi) FGF2 (40 ng/ml) (Miltenyi) BMP4 (20 ng/ml) (Miltenyi) |
| D1 | ‐ | ‐ | |
| D2 | RPMI 1640 medium (Life Technologies) + B27 (with insulin) (Life Technologies) | Activin A (10,000 ng/ml) (Miltenyi) | |
| D3 | ‐ | ‐ | |
| D4 | ‐ | ‐ | |
| D5 |
Hypoxia (4% O2, 5% CO2) |
RPMI 1640 medium (Life Technologies) + B27 (with insulin) (Life Technologies) |
VEGF (200 ng/ml) (Miltenyi) FGF2 (50 ng/ml) (Miltenyi) BMP4 (100 ng/ml) (Miltenyi) |
| D6 | ‐ | ‐ | |
| D7 | RPMI 1640 medium (Life Technologies) + B27 (with insulin) (Life Technologies) |
VEGF (200 ng/ml) (Miltenyi) FGF2 (50 ng/ml) (Miltenyi) BMP4 (100 ng/ml) (Miltenyi) |
|
| D8 | ‐ | ‐ | |
| D9 | ‐ | ‐ | |
| D10 | RPMI 1640 medium (Life Technologies) + B27 (with insulin) (Life Technologies) |
HGF (100 ng/ml) (Miltenyi) SB431542 (10 μg/ml) (Cell Guidance Systems) |
|
| D11 | ‐ | ‐ | |
| D12 | ‐ | ‐ | |
| D13 | RPMI 1640 medium (Life Technologies) + B27 (with insulin) (Life Technologies) | HGF (100 ng/ml) (Miltenyi) | |
| D14 | ‐ | ‐ | |
| D15 | ‐ | ‐ | |
| D16 | RPMI 1640 medium (Life Technologies) + B27 (with insulin) (Life Technologies) |
HGF (100 ng/ml) (Miltenyi) OSM (20 ng/ml) (Miltenyi) |
|
| D17 | ‐ | ‐ | |
| D18 | ‐ | ‐ | |
| D19 |
Normoxia (20% O2, 5% CO2) |
Hepatocyte culture medium (HCM) (Lonza) without EGF |
HGF (100 ng/ml) (Miltenyi) OSM (200 ng/ml) (Miltenyi) DEX (0,5 μM) (SIGMA) |
| D20 | ‐ | ‐ | |
| D21 | ‐ | ‐ | |
| D22 | Hepatocyte culture medium (HCM) (Lonza) without EGF |
HGF (100 ng/ml) (Miltenyi) OSM (200 ng/ml) (Miltenyi) DEX (1 μM) (SIGMA) |
|
| D23 | ‐ | ‐ | |
| D24 | ‐ | ‐ | |
| D25 | Hepatocyte culture medium (HCM) (Lonza) without EGF |
OSM (200 ng/ml) (Miltenyi) DEX (1 μM) (SIGMA) |
|
| D26 | ‐ | ‐ | |
| D27 | ‐ | ‐ | |
| D28 | Hepatocyte culture medium (HCM) (Lonza) without EGF | ‐ |