Penicillin streptomycin glutamine (psg)

As previously described, peripheral blood was collected in S-Monovette K3 EDTA blood collection tubes (Sarstedt) (Anft et al., 2020 (link); Paniskaki et al., 2022 (link)). Collected blood was prediluted in PBS/BSA (Gibco) at a 1:1 ratio and underlaid with 15 mL of Ficoll-Paque Plus (GE Healthcare). Tubes were centrifuged at 800 g for 20 min at room temperature. Isolated PBMCs were washed twice with PBS/BSA and stored at −80°C until use. The cryopreserved PBMCs were thawed by incubating cryovials 2–3 min at 37°C in bead bath, washed twice in 37°C RPMI 1640 media (Life Technologies) supplemented with 1% penicillin–streptomycin-glutamine (Sigma-Aldrich), and 10% fetal calf serum (PAN-Biotech) medium, and incubated overnight at 37°C.

As a control for our AMOs, we generated cortical hiPSC organoids from the same hiPSC line used to derive smNPCs for AMO line 2 (Reinhardt et al., 2013b (link)). After manual 2D culture of hiPCs, all steps were fully automated using our liquid handling system (Beckman Coulter) with attached incubator (Thermo Fisher). Generally, we followed the protocol previously published by Paşca et al., 2015 (link). (and also described in more detail by Sloan et al., 2018 (link)), with adaptations for our automation pipeline (see Figure 7—figure supplement 1). Starting with 90–100% confluent cultures, we detached hiPSCs with accutase and seeded 10,000 cells per well in ultra-low attachment U-bottom plates (Corning). Cortical organoid medium consisted of DMEM F-12, 20% Knock-out Serum replacement (GIBCO), 1% penicillin/streptomycin/glutamine, 1% Non-essential amino acids (Sigma-Aldrich), and 0.2% 2-Mercaptoethanol (Thermo Fisher). For the first 6 days, we supplemented the cortical organoid medium with 5 μM dorsomorphin (Enzo Life Sciences) and 10 μM SB-431542 (Biomol). During seeding only, we also added 10 μM ROCK inhibitor Y-27632. Aggregates were fed every 3 days using an automated liquid handling system. From day 6 to 24, culture medium supplements were exchanged to EGF and FGF2 (both 20 ng/ml, PeproTech) and afterwards BDNF and NT3 (metabion) (both 20 ng/ml).

Embryonic mouse oligodendrocyte precursor cells (Oli-neu) were grown in DMEM (Gibco-Life Technologies 41965062) containing N2 supplement (Life Technologies), penicillin–streptomycin–glutamine (Life Technologies 10378016), T3 (Sigma-Aldrich; 340 ng ml⁻¹), T4 (Sigma-Aldrich 89430; 400 ng ml⁻¹), bFGF (PeproTech; 10 ng ml⁻¹) and PDGF-ββ (R&D Systems; 1 ng ml⁻¹) on plates coated with poly-l-lysine (Sigma-Aldrich; 0.01%). Cells were differentiated with differentiation medium (DMEM containing N2 supplement; penicillin–streptomycin–glutamine; T3 (Sigma-Aldrich; 340 ng ml⁻¹); T4 (Sigma-Aldrich; 400 ng ml⁻¹); ErbB inhibitor (Santa Cruz sc-204170; 1 µM)) for 1 day.

Ten million E6.1 Jurkat Cells (Fred Hutchinson Cancer Research Center derived clone, Cell Services, CRUK) were cultured in RPMI media (Cat no 31870-082, Life Technologies, Inc., USA) containing 10% FBS, Penicillin/Streptomycin/Glutamine (Sigma-Aldrich G6784) at 1% and 2-Mercaptoethanol (50 μM) at 37 °C/5% CO2. For cells requiring a phase block the cells were incubated with 50 μM Nocodazole for 20 h at 37 °C per 5% CO₂, counted and checked for viability using a Vi-Cell counter (Beckman Coulter, Inc., USA). Cells were washed once in PBS containing 2% FBS (wash buffer) and the cellular suspensions were divided in two.

Both differentiated and undifferentiated HepaRG cells were purchased from BIOPREDIC INTERNATIONAL. After thawing, the undifferentiated HepaRG cells were cultured in complete William's E medium (William's E medium (Gibco) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin/glutamine solution, 5 mg/mL insulin (Sigma-Aldrich), and 0.5 μM hydrocortisone hemisuccinate (Sigma-Aldrich)), as previously described 15 (link). The differentiated HepaRG were maintained in HepaRG™ Maintenance/Metabolism Medium (BIOPREDIC INTERNATIONAL, France) for further hepatocyte function analysis. HepG2 cell lines were obtained from ATCC and were cultured in DMEM (Gibco) containing 10% FBS and 1% penicillin-streptomycin (Gibco).

The PDAC-derived cell line BxPC3 (classical subtype) and Panc-1 (basal-like subtype) were cultured in DMEM high glucose with 10% fetal bovine serum and 1% Penicillin-Streptomycin-Glutamine (Sigma-Aldrich, St. Louis, USA) at 37°C, 5% CO₂ in a humidified atmosphere. Mycoplasma contamination was excluded in both cell lines by PCR (MycoScope PCR Detection Kit, Genlantis, San Diego, CA). Cell line authentication was performed by short tandem repeat (STR) profiling using the PowerPlex^® 21 System (Promega, Madison, USA) according to the manufacturer’s instructions.
To generate single cell-derived cell lines (SCDCLs), limited dilution of both parental cell lines was performed in 96-well plates. For Panc-1 two and for BxPC3 four 96-well plates were seeded initially. Each well was examined by phase-contrast microscopy three hours after plating to ensure that only wells harboring a single cell were used for further cultivation. Once the single-cell clones reached approximately 80% confluency in a 96-well plate, they were transferred to a 6-well plate. Upon reaching 80% confulency in the 6-well-plate, the SCDCLs were further transferred to a T-25 flask. After reaching 80% confluency in the T-25 flask, both RNA and protein samples were collected.