Dnase 1

Protocols were adapted for BSL-3 facility regulations. For isolation of cells from whole blood, 250 µL blood were lysed in red blood cell lysis buffer (BioLegend), washed and centrifuged according to the manufacturer’s instructions. Resulting RBC-free pellets were resuspended in low-BSA buffer (1× PBS, 0.04% BSA), filtered with 40 µm FloMi filters (Merck) and counted by hemocytometer in trypan blue.
For isolation of single cells caudal lung lobe was removed and placed in storage medium (1× PBS, 0.5% BSA) until further processing. Storage and isolation media contained 2 µg/mL ActinomycinD. Tissues and cells were centrifuged at 350 × g for 6 min at 4 °C. Lung lobes were mechanically disassociated with tweezers for 2 min in enzymatic digestion medium containing 3.4 mg/mL Collagenase Cls II (Merck) and 1 mg/mL DNase I (PanReac AppliChem) in 2 mL Dispase medium (Corning) per lung lobe followed by 30 min incubation at 37 °C and 5% CO₂. After dissociation of digested lung tissue, cell suspensions were pressed through 70 µm cell strainers with plungers. Red blood cells were lysed (BioLegend), washed with an excess of PBS/BSA and resuspended in low-BSA buffer (1× PBS, 0.04% BSA), and filtered with 40 µm low-volume FloMi filters (Merck). Cells were counted in trypan blue.

3-Mercaptopropionic acid (≥99%), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM·HCl) (96%) and rhodamine B (97%) were purchased from Aldrich. Phosphate-buffered saline (PBS) was purchased from Scharlau. 4-(Dimethylamino)pyridine (DMAP) and tobramycin (97%) were purchased from Acros-Organics. DNase I was purchased from Panreac, Applichem. The water (H₂O) used in the syntheses was deionized water from a Milli-Q A10 Gradient system (Millipore).

siH19-MSC or respective control-MSC were cultured for 10 days under osteogenic-inducing conditions to synthesize ECM. The decellularization process was performed in a flow chamber and 1% (v/v) P/S and 0.5% (v/v) fungizone (Capricorn) were added to each solution. The cell culture media was gently removed, cells were washed with PBS 1 × and treated with 0.2% (v/v) Triton X-100 for 10 min at RT. The solution was carefully removed and a 2 M urea (Sigma-Aldrich)/0.2% (v/v) Triton X-100 solution was slowly added, to avoid disturbance of the matrix, and left to incubate for 10 min with gently agitation. The decellularized matrices were treated with 120 Ku/mL of DNase-I (PanReac AppliChem) for 30 min, at 37 °C, and the insoluble ECM layer was washed three times with de-ionized H₂O to ensure complete removal of the solubilized material.
Decellularization was confirmed by DAPI (1 μg/mL, for 5 min), hematoxylin and eosin staining and by DNA quantification using Pico-Green dsDNA assay (Quant-iT ™ Pico-Green™ dsDNA assay kit, Thermo Fisher Scientific). Non-decellularized matrices were used as control. Further details are provided in the Additional file 1: Methods section.