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Summary

Presented here is a protocol for the isolation of regional decellularized lung tissue. This protocol provides a powerful tool for studying complexities in the extracellular matrix and cell-matrix interactions.

Abstract

Lung transplantation is often the only option for patients in the later stages of severe lung disease, but this is limited both due to the supply of suitable donor lungs and both acute and chronic rejection after transplantation. Ascertaining novel bioengineering approaches for the replacement of diseased lungs is imperative for improving patient survival and avoiding complications associated with current transplantation methodologies. An alternative approach involves the use of decellularized whole lungs lacking cellular constituents that are typically the cause of acute and chronic rejection. Since the lung is such a complex organ, it is of interest to examine the extracellular matrix components of specific regions, including the vasculature, airways, and alveolar tissue. The purpose of this approach is to establish simple and reproducible methods by which researchers may dissect and isolate region-specific tissue from fully decellularized lungs. The current protocol has been devised for pig and human lungs, but may be applied to other species as well. For this protocol, four regions of the tissue were specified: airway, vasculature, alveoli, and bulk lung tissue. This procedure allows for the procurement of samples of tissue that more accurately represent the contents of the decellularized lung tissue as opposed to traditional bulk analysis methods.

Introduction

Lung diseases, including chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and cystic fibrosis (CF), currently remain without a cure^1,2,3,4. Lung transplantation is often the only option for patients in later stages, however this remains a limited option both due to the supply of suitable donor lungs and both acute and chronic rejection after transplantation^3,5,6. As such, there is a critical need for new treatment strategies. One promising approach in respiratory bioengineering is the application of tissue-derived scaffolds prepared from decellularized native lung tissue. As acellular whole lung scaffolds retain much of the complexity of the native extracellular matrix (ECM) composition and bioactivity, they have been intensively studied for whole-organ engineering and as improved models for studying lung disease mechanisms^7,8,9,10. In parallel, there is increasing interest in utilizing decellularized tissues from different organs, including lungs, as hydrogels and other substrates for studying cell-cell and cell-ECM interactions in organoid and other tissue culture models^{11,12,13,14,15,16,17}. These provide more relevant models than commercially available substrates, such as Matrigel, derived from tumor sources. However, information on human lung-derived hydrogels is relatively limited at present. We have previously described hydrogels derived from decellularized pig lungs and have characterized both their mechanical and material properties, as well as demonstrated their utility as cell culture models^18,19. A recent report detailed the initial mechanical and viscoelastic characterization of hydrogels derived from decellularized normal and diseased (COPD, IPF) human lungs²⁰. We have also presented initial data characterizing the glycosaminoglycan content of decellularized normal and COPD human lungs, as well as their applicability for studying cell-cell and cell-ECM interactions¹¹.

These examples illustrate the power of utilizing decellularized human lung ECMs for investigative purposes. However, the lung is a complex organ, and both the structure and function vary in different regions of the lung, including ECM composition and other properties such as stiffness^21,22. As such, it is of interest to study the ECM in individual regions of the lung, including the trachea and large airways, medium-sized and small airways, and alveoli, as well as large, medium-sized, and small blood vessels. To this end, we have developed a reliable and reproducible method for dissecting decellularized human and pig lungs and subsequently isolating each of those anatomic regions. This has allowed detailed differential analysis of regional protein content in both normal and diseased lungs²¹.

Protocol

All animal studies have been performed in accordance with the IACUC of University of Vermont (UVM). All human lungs were acquired from UVM Autopsy Services and related studies were performed as per the guidelines of IRB of UVM.

NOTE: Decellularization of pig and human lungs has been previously described by our group^{7,8,9,10, 21}. In brief, whole lung lobes are decellularized through sequential perfusion of the airways and vasculature with a series of 2 L detergent and enzyme solutions using a peristaltic pump: 0.1% Triton-X 100, 2% sodium deoxycholate, 1 M sodium chloride, 30 µg/mL DNase/1.3 mM MgSO₄/2 mM CaCl₂, 0.1% peracetic acid/4% ethanol, and a deionized water wash. Standard methods for confirming efficient decellularization include the determination of <50 ng/mg residual double-stranded DNA within decellularized lungs and the absence of DNA fragments by gel electrophoresis, and nuclear staining by hematoxylin and eosin (H&E) staining^9,21.

1. Setup

1. Gather all necessary equipment required for the dissection procedure, including a glass casserole dish, two pairs of surgical tweezers, one pair of forceps, and one pair of surgical scissors, and autoclave before use.
2. Obtain a section of the lung, place it in the glass casserole dish, and orient it so that the superior end of the airway can be seen clearly.
3. Identify the proximal end of the vasculature and keep it intact until later steps. The end of the vasculature should be clearly visible and entirely opaque white in color.
4. Using a pair of tweezers and surgical scissors, remove any pleura that may be lining the exterior of the lung and discard.

2. Exposing the airway

1. Using a spreading technique with the surgical scissors, gently work to expose the additional airway.
   1. Locate the largest airway, which will typically have a diameter of approximately 2-4 cm. Another way to identify an airway is through the observation of cartilage rings, which can be detected visually or via palpation of the tissue.
   2. Using a pair of forceps, palpate down the length of the airway in order to determine the location of the unseen airway to a depth of approximately 1 in.
      NOTE: Being lined with cartilage rings, the airway is characteristically harder than the other lung tissues. As such, finding and palpating the unseen airway should be relatively simple.
   3. Holding the surgical scissors parallel to the airway, insert the closed tips into the tissue directly surrounding the unseen airway.
   4. Slowly open the surgical scissors to gently pull apart the surrounding membrane. Subsequently, remove the surgical scissors and avoid cutting any tissue whatsoever.
   5. Repeat this process intermittently throughout the dissection procedure to continue exposing the airway.
2. Using the surgical scissors, cut the airway at the branching points and dissect along either branch independently.
   NOTE: A branching point is a location at which one airway splits into two separate airways.
3. Sever regions of the airway once confident that the intact ends will remain identifiable and easily located for further dissection.
4. Place severed regions of the airway into the corresponding tube. The size of the severed regions will vary depending on the sample but, in general, will range between 1-5 cm in length. The width varies based on the relative location along the airway tree, with the distal regions maintaining smaller widths than the more proximal regions.

3. Exposing and excising regions of the vasculature

1. Apply gentle pressure to the vasculature and slowly pull away from the airway. Allow the vasculature to stretch slightly and use surgical scissors to further separate the vasculature from the airway.
   NOTE: Too much pressure will rip the vasculature. If the vasculature rips, simply place that section of vasculature in the corresponding labeled tube and identify its intact end.
2. When a branching point in the vascular tree has been exposed, use surgical scissors and tweezers to expose more inferior regions of the vasculature.
   1. Begin by inserting the closed tips of the surgical scissors just below a branching point and between the two corresponding vasculature regions.
   2. Slowly open the scissors to spread apart the underlying tissues.
   3. Intermittently, use a pair of tweezers to remove the tissue that was spread apart using the surgical scissors, as well as any other tissue directly surrounding the vasculature.
3. When the vasculature is covering regions of the airway or becoming cumbersome to any step in the dissection procedure, cut the vasculature at a branching point and further dissect along either branch independently.
4. Sever regions of the vasculature once confident that the intact ends will remain identifiable and easily located for further dissection.

4. Identifying and excising alveolar tissue

1. Using a pair of forceps or tweezers, pinch and then gently tear away small regions of alveolar tissue.
   1. Locate a region of tissue that is not in the direct vicinity of the airway or the vasculature.
   2. Using the tweezers, pinch a small region of the tissue that appears to be devoid of any vasculature or airway.
   3. Tear the pinched region of the tissue from the lung.
2. Observe the region of tissue removed and confirm whether or not it is alveolar tissue.
   NOTE: Alveolar tissue is present throughout the lung, so it can and should be removed throughout the dissection procedure. Any tissue that cannot easily be identified as primarily alveoli, vasculature, or airway should be categorized as bulk tissue and placed in the corresponding labeled tube.

Representative Results

An overall schematic of the protocol is depicted in Figure 1. Once mastered, the regional dissection of decellularized lung tissue is easily reproducible. Determining the categorization of each severed tissue sample is imperative to the success of the dissection procedure. Vascular tissue is substantially more elastic than airway, so using forceps to stretch the tissue is often a strong indicator of whether a particular sample is vasculature or airway. Typically, vascular tissue runs parallel to the airway (Figure 2A). Vascular tissue also tends to appear whiter and opaquer in color (Figure 2B) than airway tissue (Figure 2C). The surgical scissor spreading technique described in the protocol is depicted in Figure 3. Larger samples of the airway are encompassed by rings of cartilage which appear slightly whiter than the airway itself. Thus, observing cartilage rings is an immediate indicator that the sample in question is the airway (Figure 4). Determining whether a sample is primarily alveoli is somewhat more complicated due to alveoli being present throughout the lung and too small to observe with bare eyes. Once removed, alveolar tissue tends to retreat into a bulb-like shape and appears relatively homogenous (Figure 5). Sometimes, alveolar tissue can appear speckled, but it should never contain visible streaks of white as this may suggest the presence of medium-to-large sized airway or vasculature. In cases where white streaks or other unidentifiable structures are observed, the tissue sample should be categorized as bulk lung and placed in the corresponding labeled tube. This dissection process is inexact and, as such, we classify the alveolar tissue category as alveolar-enriched. Using this protocol, it is impossible to obtain a 100% pure alveolar tissue sample. However, we have previously shown using mass spectrometry that ECM composition varies between individual regions of decellularized lungs, including whole lung ECM (wECM), alveolar-enriched ECM (aECM), airway ECM (airECM), and vasculature ECM (vECM) (Figure 6A–F)²¹. In particular, in decellularized lungs obtained from patients with no history of lung disease, we have characterized an enrichment of basement membrane associated proteins (i.e., laminins) in aECM, while airECM is enriched in cartilage-associated ECM proteins, such as aggrecan (ACAN), and vECM is enriched with fibronectin (FN1) and other soluble ECM proteins associated with blood vessels (Figure 6G,H)²¹. Moreover, we have previously demonstrated that ECM composition is altered in patients with IPF or COPD in a region-specific manner, highlighting the necessity of methods to interrogate individual lung regions as described here²¹.

Figure 1: Schematic representation of the whole lung decellularization and dissection process. Please click here to view a larger version of this figure.

Figure 2: Example showing the difference between decellularized airway and vascular tissue during dissection. (A) Initial anatomy with the airways and vasculature juxtaposed. (B) Airway and (C) vasculature each being held with forceps. Please click here to view a larger version of this figure.

Figure 3: Procedure for identifying and harvesting vasculature. (A) Large region of pulmonary vasculature being held upright with forceps. There are no cartilage rings and the tissue has some degree of elasticity, confirming the sample is vasculature. (B) Surgical scissors are used to carefully sever the upper portion of the vasculature. (C) More than enough vasculature is retained below the cut so that it can easily be relocated and further dissected. (D) A section of more distal vasculature that can be sectioned. Please click here to view a larger version of this figure.

Figure 4: Procedure for identifying and harvesting airway. (A) Large region of the airway being held upright with forceps. The image shows clear cartilage rings, confirming the sample is airway. (B) Surgical scissors are used to carefully sever the upper portion of the airway. (C) More than enough airway is retained below the cut so that it can easily be relocated and further dissected. (D) The severed airway is placed in the corresponding labeled tube. Please click here to view a larger version of this figure.

Figure 5: Example showing a representative sample of alveolar tissue. Isolated alveolar tissue being held up for examination with a pair of forceps. The alveolar tissue sample is spherical after extraction from the lung and has uniform coloration. Please click here to view a larger version of this figure.

Figure 6: Decellularized lung matrisome varies depending on anatomical region. Representative image of a decellularized human lung on the ventral (A) and dorsal (B) side and subsequent dissection to isolated airway (C) and vascular (D) trees. € Liquid nitrogen milled ECM powders of whole decellularized lung ECM (wECM), as well as ECM from alveolar-enriched (aECM), airway-enriched (airECM), and vasculature-enriched (vECM) regions. (F) Principal component analysis (PCA) plot demonstrating the similarity of the total matrisome composition amongst region-specific samples. (G) Ratio of mean basement membrane composition from decellularized lung-specific regions. (H) Heatmap of top 25 matrisome proteins across all decellularized lung regions. This figure is reprinted from Hoffman et al.²¹. Please click here to view a larger version of this figure.

Discussion

Decellularized tissues from humans and other species are frequently utilized as biomaterials for studying ECM composition as well as cell-ECM interactions in ex vivo culture models, including 3D hydrogels^12,13. Similar to other organs, decellularized lungs have previously been utilized to determine ECM compositional differences in healthy versus diseased (i.e., emphysematous and IPF) lungs and are increasingly being utilized as hydrogels for studying ECM dynamics and cell-ECM interactions^{7,8,9,10,11,14,15,16,17,18,19,20}. However, proteomic and hydrogel studies in decellularized lungs have only considered the lung as a whole, and thus are unable to determine the role of individual anatomical regions on overall study results. As the lung is a complex organ that drastically varies in physiological role, composition, and structure between anatomical regions, it is critical to develop methods to study these individual regions separately^21,22. Herein, we describe an innovative method of deriving individual anatomical regions (alveolar-enriched, airway, and vasculature regions) from decellularized lungs for a variety of downstream applications, including both proteomic characterization and ex vivo hydrogel studies²¹

Following whole lung decellularization, our method describes a stepwise dissection protocol to enrich airway and vasculature trees. Proceeding with care is the most important aspect of the dissection procedure so as not to misidentify a particular sample nor to haphazardly sever a region of tissue. A potential consequence of the latter is to lose track of where the airway or vasculature is routed within the lung. Currently, it is best practice to hold the airway or vasculature with forceps until the tissue is exposed to the point where it can be collected. In the future, modifications to this protocol could include applying a clamp to the exposed end of the airway or vasculature, which would allow for constant identification of the tissue being actively dissected. Past modifications included the introduction of a spreading technique with surgical scissors, which is described in the protocol. Prior to the use of a spreading technique, a manual method of exposing the airway or vasculature was performed which involved ripping away surrounding tissue. This modification is more effective in exposing the airway or vasculature and limits the amount of damage to surrounding tissues, which can then be further dissected into region-specific samples.

One limitation of this procedure is that avoiding the accidental severance of small vasculature and airway can be difficult, as once these reach smaller diameters, they become increasingly delicate. As such, it becomes more beneficial to forego the procurement of extremely small airway and vasculature and to categorize the region as bulk instead. This is completely acceptable, as bulk lung tissue is meant to contain a mixture of all lung tissue types. Another limitation is the difficulty in isolating pure alveolar tissue without the sample containing trace amounts of vasculature or airway. An easy way to avoid this is to collect small samples of alveolar tissue (approximately 5 mm³), such that the center of the tissue sample is less likely to contain unwanted tissue types.

The methods described are novel and we are not currently aware of any other region-specific lung dissection methods. This protocol provides the ability to distinguish between different regions of the lung, which establishes a more robust scientific understanding of the composition.

This dissection protocol may have applications in the generation of hydrogels for 2D and 3D cell culture applications, as well as the development of bioinks for 3D printing applications.

Materials

Bonn Scissors	Fine Science Tools	14184-09
Dumont #5 – Fine Forceps	Fine Science Tools	11254-02
Forceps, Curved, S/S, Blunt, Serrated – 130mm	CellPath	N/A
Hardened Fine Scissors	Fine Science Tools	14090-11
Moria Iris Forceps	Fine Science Tools	11373-22
Pyrex Glass Casserole Dish	Cole-Parmer	3175-10