Bioorthogonal Labeling and Chemoselective Functionalization of Lung Extracellular Matrix

Abstract

Decellularized extracellular matrix (ECM) biomaterials derived from native tissues and organs are widely used for tissue engineering and wound repair. To boost their regenerative potential, ECM biomaterials can be functionalized via the immobilization of bioactive molecules. To enable ECM functionalization in a chemoselective manner, we have recently reported an effective approach for labeling native organ ECM with the click chemistry-reactive azide ligand via physiologic post-translational glycosylation. Here, using the rat lung as a model, we provide a detailed protocol for in vivo and ex vivo metabolic azide labeling of the native organ ECM using N-Azidoacetylgalactosamine-tetraacylated (Ac₄GalNAz), together with procedures for decellularization and labeling characterization. Our approach enables specific and robust ECM labeling within three days in vivo or within one day during ex vivo organ culture. The resulting ECM labeling remains stable following decellularization. With our approach, ECM biomaterials can be functionalized with desired alkyne-modified biomolecules, such as growth factors and glycosaminoglycans, for tissue engineering and regenerative applications.

Keywords: Bioorthogonal; Chemoselective functionalization; Click chemistry; Decellularization; Extracellular matrix; Lung.

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.

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.

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.

Figure 4.. Surgical procedures during lung harvest.

The PA (blue) is connected to the right ventricle. A small incision is made at the right ventricle immediately next to the PA to allow insertion of a blunt-end needle (for flushing) or cannula into the PA. Prior to the initial flushing, the left atrial appendage should be cut open as the drainage outlet.

Figure 5.. Histological characterization of metabolic azide labeling of the lung ECM.

The representative results show two ECM samples derived from rat lungs following three-day in vivo labeling with or Ac₄GalNAz or DMSO. The potential azide ligand in the ECM were conjugated to biotin and then detected using fluorophore-conjugated streptavidin (Red). The lung ECM was indicated by Laminin antibody staining (green). Scale bars: 200 μm.

Figure 6.. Biochemical characterization of azide labeling in the lung ECM.

The representative results show two ECM samples derived from rat lungs following one-day ex vivo labeling with Ac₄GalNAz or DMSO. A. Detection of the azide ligand by Western blot analysis of the conjugated biotin. B. Protein loading was verified by in-gel staining of total protein with Sypro Ruby.