Gibco™Advanced DMEM/F-12

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost in advance the cultrex growth factor reduced BME type 2 gel on ice or in fridge	Use of pre‐warmed 24‐well suspension culture plates

Upstream tips
Defrost in advance the cultrex growth factor reduced BME type 2 gel on ice or in fridge
Protocol tips
Use of pre‐warmed 24‐well suspension culture plates

Publication protocol

Processing of solid lung tissue:
1. Solid lung tissue was minced, washed with 10 ml AdDF+++ (Advanced DMEM/F12 containing 1× Glutamax, 10 mM HEPES, and antibiotics), and digested in 10 ml AO medium (Table EV1) containing 1–2 mg ml−1 collagenase (Sigma‐C9407) on an orbital shaker at 37°C for 1–2 h.
2. The digested tissue suspension was sequentially sheared using 10‐ and 5‐ml plastic and flamed glass Pasteur pipettes. After every shearing step, the suspension was strained over a 100‐μm filter with retained tissue pieces entering a subsequent shearing step with ˜10 ml AdDF+++.
3. 2% FCS was added to the strained suspension before centrifugation at 400 rcf. The pellet was resuspended in 10 ml AdDF+++ and centrifuged again at 400 rcf.
4. In case of a visible red pellet, erythrocytes were lysed in 2 ml red blood cell lysis buffer (Roche‐11814389001) for 5 min at room temperature before the addition of 10 ml AdDF+++ and centrifugation at 400 rcf.
Organoid culture
1. Lung cell pellets were resuspended in 10 mg ml−1 cold Cultrex growth factor reduced BME type 2 (Trevigen‐3533‐010‐02), and 40 μl drops of BME‐cell suspension were allowed to solidify on pre‐warmed 24‐well suspension culture plates (Greiner‐M9312) at 37°C for 10–20 min.
2. Upon completed gelation, 400 μl of AO medium was added to each well and plates transferred to humidified 37°C/5% CO2 incubators at ambient O2.
3. Medium was changed every 4 days and organoids were passaged every 2 weeks: Cystic organoids were resuspended in 2 ml cold AdDF+++ and mechanically sheared through flamed glass Pasteur pipettes. Dense (organoids were dissociated by resuspension in 2 ml TrypLE Express (Invitrogen‐12605036), incubation for 1–5 min at room temperature, and mechanical shearing through flamed glass Pasteur pipettes.
4. Following the addition of 10 ml AdDF+++ and centrifugation at 300 or 400 rcf respectively, organoid fragments were resuspended in cold BME and reseeded as above at ratios (1:1–1:6) allowing the formation of new organoids. Single‐cell suspensions were initially seeded at high density and reseeded at a lower density after ˜1 week. Success rate was determined by dividing the number of successfully established, expanded, and cryopreserved AO lines by the number of attempts.
5. NSCLC organoids could be distinguished from normal regular cystic organoids by morphology (size, irregular shape, thick organoid walls, dense) as well as histology.
6. Separation from normal AOs was achieved by manual separation and in case of TP53 mutations by the addition of 5 μM Nutlin‐3a (Cayman Chemicals‐10004372) to the culture medium. For the R‐spondin withdrawal assay, established organoid lines were trypsinized to single cells and grown in AO medium ± R‐spondin until organoids were depleted. Intestinal organoids were cultured as previously described (Sato et al, 2011).
7. Unless specified, airway organoids were analyzed after at least 7 days post‐splitting at the indicated passage



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