Name: Author Spotlight: Dissection and Isolation of Region-Specific Decellularized Lung Tissue
Uploaded: 2023-09-29T14:50:00
Description: Watch this Scientific Journal Video about Dissection and Isolation of Region-Specific Decellularized Lung Tissue at JoVE.com

The authors thank the UVM autopsy services for human lung procurement and Robert Pouliot PhD for contributions to the overall dissection techniques. These studies were supported by R01 HL127144-01 (DJW).

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 group7,8,9,10, 21. In ...

Regional Decellularized Lung Tissue Isolation to Study Cell-Cell and Cell-ECM Interactions

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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 hydrogels12,13. 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...

Lung diseases, including chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and cystic fibrosis (CF), currently remain without a cure1,2,3,4. 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 transplantation3,5,6. 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 mechanisms7,8,9,10. 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 models11,12,13,14,15,16,17. 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 models18,19.&#160;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 stiffness21,22. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bonn Scissors</td>
			<td>Fine Science Tools</td>
			<td>14184-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 - Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps, Curved, S/S, Blunt, Serrated - 130mm</td>
			<td>CellPath</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hardened Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>14090-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Moria Iris Forceps</td>
			<td>Fine Science Tools</td>
			<td>11373-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrex Glass Casserole Dish</td>
			<td>Cole-Parmer</td>
			<td>3175-10</td>
			<td></td>
		</tr>
	</tbody>
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dissection and isolation of region-specific decellularized lung tissue

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.

Author Spotlight: Dissection and Isolation of Region-Specific Decellularized Lung Tissue  

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. For the lung, 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 extracellular matrix interactions on corresponding cell behavior.

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...

Watch this Scientific Journal Video about Dissection and Isolation of Region-Specific Decellularized Lung Tissue at JoVE.com

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.

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 ...

dissection-and-isolation-of-region-specific-decellularized-lung-tissue

Research

JoVE Journal

Bioengineering

Exposing the Airway and Excising the Vasculature and Alveolar Tissue from a Human and Pig Lung

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 two to four 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 one to five 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. A mass spectrometry analysis of the excised 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.