Rat tail collagen type 1

Rat tail collagen type-1 (Corning, Inc. NY) was purchased from the manufacturer as a concentrated stock solution. In order to study collagen in macromolecular form, the stock solution was diluted to a final collagen concentration of 3 mg/mL in 20 mM acetic acid. The resulting molecular solution of collagen remained unpolymerized when kept at room temperature for the duration of experiments. In addition, hydrogels exhibiting fibril and matrix-level structures of collagen were prepared according to manufacturer’s protocol. Appropriate amounts of 10X Dulbecco's Phosphate-Buffered Saline (10X DPBS, Life Technologies), 1.0 N NaOH, cell culture grade distilled water, and collagen stock solution were mixed together in a centrifuge tube on ice in the given order, resulting in a solution with neutral pH and isotonic ionic strength. This neutralized collagen solution had a final collagen concentration of either 3 or 6 mg/mL. Prepared solution was then dispensed into a chamber slide (Nunc Lab-Tek II, Thermo Scientific) and allowed to polymerize for 1 hour at 37°C in a CO₂ incubator. Afterwards, isotonic saline solution (1X DPBS) was added on top of the hydrogel to prevent dehydration and incubation was continued until the experiments next day.

ECFC were isolated as described previously.^{41 (link)} In brief, 8–16 mL of venous cord blood was collected in lithium heparin-coated tubes (Greiner Bio-One, Kremsmünster, Austria) immediately after delivery of the placenta. Mononuclear cells (MNC) were separated via density gradient centrifugation using Lymphoprep™ Density Gradient Medium (Axis Shild, Alere Technologies AS, Oslo, Norway). The emerged buffy coat was washed and resuspended in culture medium (Endothelial Cell Growth Medium MV Kit (PromoCell, Heidelberg, Germany) supplemented with 0.1% Gentamycin (ThermoFisher Scientific, Waltham, MA)). 2 × 10⁷ cells were seeded per well of six-well culture plates (ThermoFisher Scientific) pre-coated with rat tail collagen type 1 (Corning, Corning, NY) and cultured at 37 °C, 21% O₂, 5% CO₂ in a humidified incubator. After overnight incubation, the culture medium was changed and then twice a week.

Thoracic and lumbar DRGs were extracted from P1-P2 rat pups into Leibovitz medium on ice. Excess axonal tissue was trimmed off. For the explants, DRGs were digested for 12 min in 0.25% trypsin at 37°C and then washed three times in Leibovitz and kept at 4°C until use. For the dissociated cells, DRGs were digested for 45 min in 0.25% trypsin at 37°C and then triturated through a fire-polished pipette. Cells were washed three times in opti-MEM and concentrated by centrifugation to a smaller volume (≈ 200 μL).
Collagen was prepared, on ice, with the following concentrations: 0.2% rat tail collagen type 1 (Corning), 0.1% sodium bicarbonate, 1×opti-MEM and 1×penicillin/streptomycin. After addition of NGF to the collagen, the dissociated cells were added to the collagen and mixed thoroughly. 750 μL of the collagen was spread on a 35 mm petri dish and allowed to set. A second layer of 750 μL of collagen was added and 5 − 12 DRG explants were added within this second layer. After the collagen set, the dishes were transferred to an incubator for 2 days (37°C and 5% CO₂).

Normal and AML patient BMMCs were purchased from Folio Conversant and ProteoGenex. Recombinant human fibronectin was purchased from Millipore. Rat tail collagen type 1 and matrigel were purchased from Corning Inc. CellTiter‐Glo^® 3D cell viability assay reagent was purchased from Promega. Human thrombopoietin, human interleukin‐3 (IL3), human stem cell factor, human granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and human macrophage colony‐stimulating factor (M‐CSF) were purchased from Life Technologies. Rosiglitazone, human erythropoietin (EPO), granulocyte colony‐stimulating factor (G‐CSF), interleukin‐7 (IL‐7) and receptor activator of nuclear factor kappa‐Β ligand (RANKL) were purchased from R&D systems. Human FLT3 ligand was purchased from TONBO Biosciences. Qubit™ dsDNA HS Assay Kit and MirVana miRNA isolation kit were purchased from Thermo Fisher. Organoid harvest media was purchased from Trevigen. RNeasy Plus Micro Kit, QIAshredder mini spin column and AllPrep DNA/RNA Mini were purchased from Qiagen. Agilent RNA 6000 Pico Kit was ordered from Agilent Technologies. FITC‐anti‐human MPO flow kit was purchased from BioLegend. FITC‐labelled anti‐human CD71, FITC‐labelled anti‐terminal deoxynucleotidyl transferase (TDT), and anti‐human CD110 were purchased from BD Biosciences. TRAP staining kit was purchased from B‐Bridge International, Inc.

Transwell inserts (0.4 um pores, PET, Corning) were coated with 0.2 mg/mL rat tail collagen type 1 (Corning) prior to seeding of intestinal stem cells. Collagen was added to plasma treated transwells for 15 minutes, and excess solution was removed. Transwells were dried in a cell culture hood for 2 hours. Cryopreserved myofibroblasts were seeded at 50,000 cells per cm² in tissue culture 24 well plates. Myofibroblasts were allowed to attach to the plates for 2 to 3 hours before the addition of transwells and seeding of intestinal stem cells. Intestinal stem cells were removed from Matrigel^® and dissociated with EDTA in HBSS followed by 5 minutes of 0.05% Trypsin in HBSS. Cells were triturated into a single cell solution. Approximately 250,000 cells were seeded per transwell (SA: 0.33 cm²) in intestinal stem cell culture media supplemented with 10 μM rho kinase inhibitor (Tocris Y-27632) to prevent apoptosis. Media was replaced after 24 hours with experimental media to promote differentiation, which contained 10% FBS and 10% WRN conditioned media in Advanced DMEM with 1% penicillin – streptomycin. Cells were grown for 3 days before analysis.

PAA gels with a storage modulus of 500 Pa or 10 kPa were made as described (67 (link)). Cells were also cultured on glass (~GPa) as a nonphysiologically stiff control. Glass coverslips and PAA gels activated with sulfo-SANPAH (Thermo Fisher Scientific, Waltham, MA) were incubated with 0.1 mg/mL rat tail collagen type 1 (Corning, Edison, NJ) for 2 h at room temperature to coat the surface with extracellular matrix ligands and enable cell adhesion. Gels were sterilized by UV exposure for 30 min.

Human small airway epithelial cells (#CC-2547 and #CC-2934, Lonza, Basel, Switzerland) of three healthy donors and three COPD patients (Table S3) were cultured as described previously [34 (link)]. As stated by Lonza, the cells were isolated from donated human tissue after obtaining permission for their use in research applications by informed consent or legal authorization. SAECs were grown in PneumaCult™-Ex Plus Medium (Stemcell Technologies, Vancouver, Canada) on rat tail collagen type 1 (Corning Life Sciences B.V., Amsterdam, the Netherlands) coated transwells (#3460, Corning Life Sciences B.V., Amsterdam, the Netherlands) until confluent. Cells were further cultivated in air–liquid interface using Pneumacult-ALI-S Medium (Stemcell Technologies, Vancouver, Canada) for four weeks until fully differentiated.