-
27G × 1/2 tuberculin syringe (BD, catalog number: 305620)
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Reusable Stainless Steel Dispensing Needle with Luer Lock Connection, Blunt, 18 Gauge (McMaster Carr, catalog number: 6710A44)
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Dremel rotary tool kit (Dremel, catalog number: 8220)
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Luer-Lok Syringe 1 ml (BD, catalog number: 309628)
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Luer-Lok Syringe 10 ml (BD, catalog number: 302995)
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Luer-Lok Syringe 30 ml (BD, catalog number: 302832)
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Petri Dish, PS, 145/20 mm (Greiner Bio-One, catalog number: 639102)
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2-0 PERMA-HAND Silk Suture (Ethicon, catalog number: LA55G)
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Sterile Gauze Sponges (Covidien, catalog number: 2187)
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PVDF Transfer Membrane (Thermo Fisher Scientific, catalog number: 88518), storage: RT
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Sprague Dawley rats (100-250 gram) (Charles River Laboratories, Strain Code: 400)
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N-azidoacetylgalactosamine-tetraacylated (Ac4GalNAz) (Click Chemistry Tools, catalog number: 1086), storage: -20 °C
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Dimethyl Sulfoxide (DMSO) (Fisher Scientific, catalog number: BP231-100), storage: room temperature (RT)
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Dulbecco′s Phosphate Buffered Saline (DPBS), 1× without calcium and magnesium (Corning, catalog number: 21-031-CV), storage: RT
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Dulbecco′s Modification of Eagle′s Medium (DMEM) (Corning, catalog number: 10-013-CV), storage: 2-8 °C
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FetalClone I Serum (Cytiva, catalog number: SH30080.03), storage: -20 °C
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Antibiotic-Antimycotic (100×) (Thermo Fisher Scientific, catalog number: 15240062), storage: -5 °C to -20 °C
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Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: 74255), storage: 2-8 °C
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Triton X-100 (Sigma-Aldrich, catalog number: T8787), storage: RT
-
Phosphate Buffered Saline (PBS), 20× (Boston BioProducts, catalog number: BM-220), storage: RT
-
Paraformaldehyde (Sigma-Aldrich, catalog number: P6148), storage: 2-8 °C
-
Click-iT Cell Reaction Buffer Kit (Invitrogen, catalog number: C10269), storage: 2-6 °C
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Biotin-PEG4-Alkyne (Click Chemistry Tools, catalog number: TA105), storage: -20 °C
-
Bovine Serum Albumin (BSA) (Fisher Scientific, catalog number: BP9706100), storage: 2-8 °C
-
Rabbit Polyclonal Laminin Antibody (Novus, catalog number: NB300-144), storage: -20 °C
-
Streptavidin, Alexa Fluor 647 conjugate (Thermo Fisher Scientific, catalog number: S21374), storage: -5 °C to -30 °C and protect from light
-
Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor Plus 488 (Thermo Fisher Scientific, catalog number: A32790), storage: 4 °C and protect from light
-
DAPI Fluoromount-G (SouthernBiotech, catalog number: 0100-20), storage: RT
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Urea, ultrapure, 99% (Alfa Aesar, catalog number: J65769), storage: RT
-
HEPES (1 M) (Thermo Fisher Scientific, catalog number: 15630-080), storage: 2-8 °C
-
Protease Inhibitor Cocktail (100×) (Thermo Fisher Scientific, catalog number: 87786), storage: 4 °C
-
BCA Protein Assay Kit (Thermo Fisher Scientific, catalog number: 23225), storage: RT
-
4-15% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad Laboratories, catalog number: 4561085), storage: 4 °C
-
Tris Buffered Saline-Tween (TBST, 10×, with 0.5% Tween-20, pH 7.4) (Boston BioProducts, catalog number: IBB-181), storage: RT
-
High Sensitivity Streptavidin-HRP (Thermo Fisher Scientific, catalog number: 21130), storage: 4 °C
-
SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, catalog number: 34580), storage: RT
-
SYPRO Ruby Protein Gel Stain (Thermo Fisher Scientific, catalog number: S12000), storage: RT
-
Ac4GalNAz Injection Solution (see Recipes)
-
Control Injection Solution (see Recipes)
-
Ex vivo Culture Medium (see Recipes)
-
Decell Solution (see Recipes)
-
2× Decell Solution (see Recipes)
-
1× PBS (pH 7.4) (see Recipes)
-
Fixation Solution (see Recipes)
-
Biotin-PEG4-Alkyne Click Reaction Mixture (see Recipes)
-
Urea Extraction Buffer (see Recipes)
-
1× TBST (pH 7.4) (see Recipes)
-
Blocking Buffer (see Recipes)
-
HRP-Imaging Mixture (see Recipes)
-
Iris Scissors, 11.5 cm, straight (World Precision Instruments, catalog number: 503708-12)
-
Pulmonary artery (PA) cannula (Figure 1)

Figure 1. Custom-made PA cannula. A. Diagram. The cannula is composed of a top luer connector (1/8’, for connection with the bioreactor), a middle transparent tubing segment (1/8’, for detecting potential air bubbles), and a bottom curved metal needle (18 gauge, for connection with the PA). A small side hole at the end of the metal needle is generated using Dremel rotary tool with a sanding disc, to avoid potential perfusion blockage upon insertion into the PA. A series of grooves are created using a wire cutter around the tip of the metal needle to help stabilize the suture. B. An actual image of the cannula.
-
Adson Forceps, straight, serrated (World Precision Instruments, catalog number: 503719-12)
-
SILASTIC Thin-wall Silicone Laboratory Tubing (Dow Corning, catalog number: 508-009)
-
Easy-Load Pump Head with 2-Channels for Precision Pump Tubing (Cole Parmer, catalog number: EW-77202-60)
-
Masterflex L/S Digital Precision Modular Drive with Remote I/O and Benchtop Controller (Cole Parmer, catalog number: UX-07557-00)
-
Autoclavable three-way valve (Cole Parmer, catalog number: EW-31200-80)
-
Organ culture bioreactor (Figure 2)

Figure 2. Organ culture bioreactor. A. Driven by a peristaltic pump, the culture medium is aspirated from the organ chamber, and perfuses through a series of thin-wall silicone tubing (for oxygenation) into the cannula leading to the PA of the lungs in culture. The filter on the organ chamber is for pressure equilibration. B. An actual image of the perfusion bioreactor.
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Decellularization chambers (Figure 3)

Figure 3. Decellularization chambers. A. There are two chambers in the system: the reservoir chamber containing fresh solutions for decellularization; and the collection chamber containing the lungs to be decellularized. The reservoir chamber should be placed approximately 50 cm higher than the collection chamber, which generates the gravity pressure to drive the fluid flow from the reservoir chamber into the lungs through the PA cannula. The filters on both chambers are for pressure equilibration. B. An actual image of the decellularization chambers.
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Semi-Automated Rotary Microtome (Leica Biosystems, catalog number: RM2245)
-
EVOS FL Auto 2 Imaging System (Thermo Fisher Scientific, catalog number: AMAFD2000)
-
Microcentrifuge (Eppendorf, catalog number: 5415R)
-
BeadBug Microtube Homogenizer (Benchmark Scientific, catalog number: D1030)
-
Prefilled 2.0 ml Tubes, with Acid Washed Silica (Glass) Beads, 1.0 mm (Benchmark Scientific, catalog number: D1031-10)
-
Mini-PROTEAN Tetra Vertical Electrophoresis Cell for Mini Precast Gels, with Mini Trans-Blot Module (Bio-Rad Laboratories, catalog number: 1658036)
-
ChemiDoc Gel and Western blot imaging system (Bio-Rad Laboratories, catalog number: 12003153)
-
Reach-in IR CO2 Incubator (Caron, catalog number: 7400-25-1)
This works is supported by the Department of Biomedical Engineering at Carnegie Mellon University. Z.L. and Y.X. are supported by scholarships from the China Scholarship Council. This protocol is originally adapted from Ren et al. (2018). We thank Pitt Biospecimen Core (University of Pittsburgh) for histological processing. We also thank the Vivarium at Carnegie Mellon University for animal husbandry. Finally, we thank Piyumi Wijesekara and Wai Hoe Ng for their assistance in gathering information regarding materials and reagents.
X.R. has a patent application (US20170362266A1) related to this work.
All animal procedures were performed according to the protocol approved by the Institutional Animal Care and Use Committee at Carnegie Mellon University (Approval ID#TR202000006, valid 2018-2022).