Tryple express

PBMCs were incubated with immunogenic peptide pools for nine days with three cycles of stimulations. After stimulation, PBMCs were washed and stained with anti CD3 AF700, anti CD8 APC, and Propidium iodide (PI, BD). Peptide specific live T cells (CD3⁺CD8⁺PI⁻) were FACS isolated for organoid killing assay. Organoids were dissociated to single cells with Tryple Express, labeled with CFSE (BD), and cocultured in at least triplicate with sorted T cells at a 10:1 effector:target ratio in T cell culture medium. To analyze the activity of ICIs, 5 µg mL⁻¹ PD‐1 inhibitor and/or 10 µg mL⁻¹ CTLA‐4 inhibitor was added during the progress of stimulation and killing assay. Each organoid killing assay was repeated twice. After 3 days of coculture, organoids and T cells were collected. Cells were washed in FACS buffer and stained with anti CD45 APC‐Cy7, Annexin V PE, 7‐AAD (BD) in Annexin V buffer (BD) for 15 min at RT. The apoptosis of organoids labeled with CFSE were analyzed with flow cytometer Canto II (BD) or CytoFLEX (Beckman). FITC⁺CD45 APC‐Cy7⁻ was used to detect organoids and Annexin V⁻7‐AAD⁻ was used to define live cells.

Endothelial differentiation was performed as described previously with some modifications^{24 (link)}. The iPSCs were cultured in mTeSR1 medium (Stemcell Technologies) on dishes coated with iMatrix511 (Nippi; (0.5 µg/cm²). After the colony size was grown up to 750–1000 μm in diameter, the medium was changed to Essential8 medium (Thermo Fisher Scientific) containing 4 μM of CHIR99021 (Wako), 80 ng/mL of BMP4 (HumanZyme), and 80 ng/mL of VEGF (Thermo Fisher Scientific). Then 48 to 51 h later, cells were detached using TrypLE Express (Thermo Fisher Scientific) and seeded onto dishes coated with LM411-E8 (Nippi; 0.4 μg/cm²) with a density of 2500 ~ 10,000/cm² in Stempro-34 SFM (Thermo Fisher Scientific) containing 80 ng/ml of VEGF. Four to six days later, cells were detached using TrypLE Express and sorted using Alexa Fluor 647 mouse anti-human CD31 antibody (1 μg/1 × 10⁶ cells; BD Biosciences) and PE mouse anti-human CD144 antibody (0.5 μg/1 × 10⁶ cells; BD Biosciences) with a FACSAria II flow cytometer (BD Biosciences).

hESC H1 line was cultured feeder-free on hESC-qualified Matrigel (BD Biosciences) in E8 media (Stemcell Technologies) with 30% of irradiated mouse embryonic fibroblasts (iMEFs) conditional media. Cells were passaged every 3–5 days at 80% confluent with TrypLE Express (Invitrogen). After dissociation, the cells were plated in E8 media with 10 μM Y-27632, (StemGent) for 24 h. After 24 h media without Y-27632 was replenished daily. iMEF conditional media was prepared by incubating iMEFs with hESC media without bFGF for 24 h for 7 days. Collected media were filtered, flash frozen and stored at −80 °C. To initiate differentiation, the cells were dissociated using TrypLE Express to single cells and seeded at 150,000 cell/cm2 onto 1:30 dilution of growth factor reduced Matrigel (BD Biosciences) in DMEM/F12 in E8-MEF conditional media with 10 µM Y-27632. Two days following seeding the differentiation was started. Day 1 cells were exposed to RPMI + 3 µM CHIR-99021 (Stemgent) + 100 ng/ml rhActivinA (R&D Systems). Days 2–3: + 100 ng/ml rhActivinA + 0.2% FBS. Day 4–5: + 2% FBS + 50 ng/ml KGF (Peprotech). Days 6–9: DMEM/B27 + 50 ng/ml KGF + 2 μM RA (Sigma) + 0.25 μM SANT-1 (Sigma) + 100 ng/ml rhNoggin (R&D Systems). Days 10–14: DMEM/B27 + PdBU (1 µM) + Alk5i (1 µM) + 100 ng/ml rhNoggin (R&D Systems). PPs are defined as Day 9 and EPs as Day 14 of differentiation, respectively.