Cell lifter

Establishment of the airway and lung organoids was based on the protocol described by Sachs et al., (Sachs et al., 2019 (link)) with minor modifications (Hoareau et al., 2021 (link)). Briefly, the human derived lung tissue was minced using a pair of surgical scalpels (Swann-Morton, 0507, UK), lifted with a cell lifter (VWR,76036-004, USA) into a 50 ml falcon tube and washed with 10 ml AdDF+++ (Gibco, 12634028, USA) supplemented with 10 mM HEPES (Gibco, 15630106, USA), penicillin-streptomycin and 1x GlutaMax (Gibco, 35050061, China) followed by centrifugation at 400 x g for 5 min. The minced lung tissue was resuspended in AdDF+++ containing 2 mg/ml collagenase (Sigma-Aldrich, C9407, USA) and placed on an orbital shaker at 200 rpm, at 37°C for 2.5 h. After digestion, the tissue was sheared with a 10 ml glass Pasteur pipette and strained using a pre-wet 100 µm filter (Corning, 431752, USA). The strained suspension was collected and 2% FBS was added, followed by 400 x g centrifugation for 5 min. 10 ml of AdDF+++ was then added to the pellet before centrifugation. The pellet was resuspended in 2 ml of red blood cell lysis buffer (Roche, 11814389001, Germany) for 5 min at room temperature (RT) to lyse residual erythrocytes, followed by addition of 10 ml AdDF+++ and finally centrifugation at 400 x g for 5 min.