Dissection and Isolation of Region-Specific Decellularized Lung Tissue

Decellularized organs are continually being used in a variety of tissue engineering applications. However, most of these studies consider the organ as a whole, and not the individual anatomical regions. We developed this method to look at individual anatomical regions of decellularized human lungs for more precise model systems for downstream applications.

When developing model systems, it's important to consider different design aspects so that your system best recapitulates normal biology. Further along, this includes factors such as environmental stress, cyclic mechanical stress, and elasticity, which may differ between independent regions of the lung. Utilizing this method, we've been able to show that the extracellular matrix derived from individual anatomical lung regions, from both healthy and diseased lungs, have distinct proteomic signatures.

This allows us to further understand lung disease and potentially come up with novel therapeutic avenues. To continue with this research, our lab is currently developing three-dimensional hydrogels from healthy and diseased lung extracellular matrix. These hydrogels allow us to perform in vitro organoid modeling in order to elucidate the role of cell-extracellular matrix interactions on corresponding cell behavior Begin by using a spreading technique with surgical scissors to carefully expose the airway of the procured pig lung.

First, locate the largest airway, which typically has a diameter of 2 to 4 centimeters. Then use a pair of forceps to palpate down the length of the airway to a depth of one inch. Finally, hold the surgical scissors parallel to the airway and insert the closed tips into the tissue surrounding the unseen airway.

Gently open the surgical scissors to pull apart the surrounding membrane. Take out the scissors and refrain from cutting any tissue. Repeat this process intermittently to continue exposing the airway.

Using the surgical scissors, cut the airway at the branching points, and dissect along either branch independently. Sever regions of the airway, once confident that the intact ends will remain identifiable and easily located. Place severed regions of the airway into the corresponding tube, with the size of the severed regions, ranging between 1 to 5 centimeters in length, and the width varying based on the relative location along the airway tree.

Gently apply pressure to the vasculature to expose it and slowly pull it away from the airway. Allow the vasculature to stretch slightly, and use surgical scissors to further separate it from the airway. Avoid applying too much pressure as it may rip the vasculature.

Once the branching point in the vascular tree has been exposed, insert the closed tips of the surgical scissors just below the branching point and between the two corresponding vasculature regions. Then, slowly open the scissors to spread apart the underlying tissues. Intermittently, use a pair of tweezers to remove the tissue spread apart and any other tissue directly surrounding the vasculature.

Cut the vasculature at a branching point when it covers regions of the airway or becomes cumbersome to any dissection step. Subsequently, dissect along either branch independently. Confirm that the two ends of the vasculature remain identifiable and easily located for further dissection, then, sever the regions of the vasculature.

To excise the alveolar tissue, locate a region of the tissue that is not in the vicinity of the airway or vasculature. Pinch a small region of the tissue that appears to be devoid of vasculature or airway, and tear the pinched tissue from the lung. Alveolar tissue is present widely in the lung and should be removed throughout the dissection.

Observe the region of tissue removed, and confirm whether or not it is alveolar tissue. This procedure was applied to a human lung model. The mass spectrometry analysis of the excise tissues indicated that ECM composition varies between individual regions of decellularized lungs, including whole lung ECM, alveolar-enriched ECM, airway ECM, and vasculature ECM In decellularized lungs obtained from patients with no history of lung disease, basement membrane-associated proteins increased in alveolar-enriched ECM.

At the same time, airway ECM was enriched in cartilage-associated ECM protein, such as aggrecan. The vasculature ECM was enhanced with fibronectin and other soluble ECM proteins associated with blood vessels.