| 生长方案: |
Human lung organoids (HLO) were derived from hPSCs as previously described (Carvalho et al., 2019; Jacob et al., 2017; Miller et al., 2019), with minor modifications. Briefly, H9 hPSCs were maintained in mTeSR Plus media and were seeded on Day 0 onto matrigel (Corning) coated surface. On Days 1-4 cells were treated with 100 ng/mL Activin A and increasing concentration of defined FBS (dFBS, HyClone) in RPMI 1640 medium (Thermo), to induce definitive endoderm formation (Day 1: 3 µM CHIR99021 and no dFBS; Day 2: 0.2% dFBS; Days 3-4: 2% dFBS). On Day 4, cells were >95% double positive for CXCR4/CD184 and c-Kit/CD117 as determined by flow cytometry. On Days 5-9 cells were treated with anterior foregut induction medium, containing 10 µM SB431542, 100 nM LDN193189, 2 µM CHIR99021, 1 µM SAG, 500 ng/mL FGF4 in Foregut Basal Medium (Advanced DMEM, 1x B27, 1x N2, 10 mM HEPES, 1x Glutagro (all Thermo), 50 µg/mL ascorbic acid (Sigma), 0.4 µM monothioglycerol (Sigma)). On Day 9, anterior foregut spheroids were harvested by gentle pipetting and transferred into an ultra-low attachment plate, in Lung Organoid Medium I (3 µM CHIR99021, 10 ng/mL BMP4, 10 ng/mL FGF7, 10 ng/mL FGF10, 50 nM all-trans retinoic acid, in Foregut Basal Medium). The medium was changed every two days until Day 15. On Day 15, the medium was changed to Lung Organoid Medium II (3 µM CHIR99021, 10 ng/mL FGF7, 10 ng/mL FGF10, in Foregut Basal Medium). On Day 25 the organoids were embedded in matrigel, in transwell inserts, and grown in Lung Organoid Medium III (3 µM CHIR99021, 10 ng/mL FGF7, 10 ng/mL FGF10, 50 nM dexamethasone (Sigma), 100 µM 8-bromo-cAMP (Sigma), 100 µM IBMX (Sigma), in Foregut Basal Medium). The medium was changed every 3 days. The CHIR99021 was withdrawn on Days 35-42. All media components were from Stem Cell Technologies unless noted otherwise. |
| 提取方案: |
All tubes and pipet tips used for cell harvesting were pre-treated with 1% BSA in 1X PBS. The HLOs were transferred with a wide end pipet tip from matrigel to 1 mL Organoid Harvesting solution (Cultrex) and incubated 1h at +4C, with end-to-end rotation. Then the cells were dissociated in Accutase (Stem Cell) with DNaseI (200 U/mL, Worthington) and Dispase (100 U/mL, Corning) at 37C, in 10 min increments, with end-to-end rotation, until single cell suspension was obtained. The cells were washed in Cell Staining Buffer (Biolegend) and stained with TotalSeq HTO antibodies for 30 min on ice. The cells were washed twice in Cell Staining Buffer and filtered through a 40 µm pipet tip strainer (BelArt). The cells were counted using Trypan Blue dye and hemocytometer and pooled for sequencing. |
