Dissection and Isolation of Region-Specific Decellularized Lung Tissue

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.

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 paralle...

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...

Materials

Name	Company	Catalog Number	Comments
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