Amaxa p4 primary cell 4d nucleofector kit

To generate e-iHeps, 1.0 × 10⁶ MEFs were transfected with 5.0 μg of the episomal vector using the Amaxa P4 Primary Cell 4D-Nucleofector kit (Lonza) according to the manufacturer's instructions. A total of 1.5 × 10⁵ transfected cells were plated onto collagen-coated 35 mm cell culture dishes and cultured in MEFM for 48 hrs [13 (link)]. After 48 hrs of transfection, the cells were cultured in HCM with the small molecules A83-01, BMP4, and CHIR99201 (ABR). 1a e-iHeps can be expanded stably over 20 passages in the presence of ABR. For r-iHep generation, the MEFs were transduced with retroviral particles and cultured as previously described [31 (link)]. Briefly, 5 × 10⁴ MEFs were seeded onto 0.1% gelatin-coated 35 mm cell culture dishes and incubated with retrovirus-containing MEFM together with 8 μg/ml of protamine sulfate (Sigma) for 48 hrs. After 48 hrs of viral infection, the medium was replaced with HCM containing ABR. To generate d-iHeps, 5 × 10⁴ cells of MEFs were seeded onto 0.1% gelatin-coated 35 mm cell culture dishes and incubated with dox-inducible lentivirus-containing MEFM together with 8 μg/ml of protamine sulfate (Sigma) for 48 hrs. After 48 hrs, the medium was replaced with HCM containing 1 μg/ml of doxycycline (Tocris). The culture medium was changed every other day.

The coding regions of Hnf4a, Foxa1, Foxa3, Gata4, Hnf1a, and mCherry were amplified by PCR and cloned into pCR8/GW/TOPO (Invitrogen) to create donor plasmids. The inserts in the donor plasmids were transferred into the destination plasmids pCXLE-gw (37626) (Addgene) and pMX-gw (18656) (Addgene) by the LR reaction.
To generate e-iHeps, 1.5 × 10⁵ fibroblasts were transfected with 1.5 μg of each episomal vector using the Amaxa P4 Primary Cell 4D-Nucleofector kit (Lonza) according to the manufacturer’s instructions. The transfected cells using the program setting CZ-167 were plated onto collagen-coated dishes and cultured in MEF medium for 2 days. On day 2 post-transfection, HCM was supplied to the cells, and the medium was changed every other day. To generate r-iHeps, fibroblasts were seeded at the density of 2.5 × 10⁴ cells per well of 6-well plates and incubated with retrovirus-containing supernatants together with 8 μg/ml of protamine sulfate (Sigma) for 2 days. On day 2 post-transduction, the medium was replaced with HCM, and the medium was changed every other day.