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 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 &#181;g/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 &#60;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&#38;E) staining9,21.
1. Setup
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Using a pair of tweezers and surgical scissors, remove any pleura that may be lining the exterior of the lung and discard.</li>
</ol>
2. Exposing the airway
<ol>
	<li>Using a spreading technique with the surgical scissors, gently work to expose the additional airway.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Holding the surgical scissors parallel to the airway, insert the closed tips into the tissue directly surrounding the unseen airway.</li>
		<li>Slowly open the surgical scissors to gently pull apart the surrounding membrane. Subsequently, remove the surgical scissors and avoid cutting any tissue whatsoever.</li>
		<li>Repeat this process intermittently throughout the dissection procedure to continue exposing the airway.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Sever regions of the airway once confident that the intact ends will remain identifiable and easily located for further dissection.</li>
	<li>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.</li>
</ol>
3. Exposing and excising regions of the vasculature
<ol>
	<li>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.</li>
	<li>When a branching point in the vascular tree has been exposed, use surgical scissors and tweezers to expose more inferior regions of the vasculature.
	<ol>
		<li>Begin by inserting the closed tips of the surgical scissors just below a branching point and between the two corresponding vasculature regions.</li>
		<li>Slowly open the scissors to spread apart the underlying tissues.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Sever regions of the vasculature once confident that the intact ends will remain identifiable and easily located for further dissection.</li>
</ol>
4. Identifying and excising alveolar tissue
<ol>
	<li>Using a pair of forceps or tweezers, pinch and then gently tear away small regions of alveolar tissue.
	<ol>
		<li>Locate a region of tissue that is not in the direct vicinity of the airway or the vasculature.</li>
		<li>Using the tweezers, pinch a small region of the tissue that appears to be devoid of any vasculature or airway.</li>
		<li>Tear the pinched region of the tissue from the lung.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>

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

<ol>
	<li>L&#243;pez-Campos, J. L., Tan, W., Soriano, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+burden+of+COPD.">Global burden of COPD.</a> Respirology. 21 (1), 14-23 (2016).</li><li>Raherison, C., Girodet, P. -. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+COPD.">Epidemiology of COPD.</a> European Respiratory Review. 18 (114), 213-221 (2009).</li><li>Glass, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Idiopathic+pulmonary+fibrosis:+Current+and+future+treatment.">Idiopathic pulmonary fibrosis: Current and future treatment.</a> The Clinical Respiratory Journal. 16 (2), 84-96 (2022).</li><li>Dickinson, K. M., Collaco, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+Fibrosis.">Cystic Fibrosis.</a> Pediatrics in Review. 42 (2), 55-67 (2021).</li><li>DeFreitas, M. R., McAdams, H. P., Azfar Ali, H., Iranmanesh, A. M., Chalian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complications+of+lung+transplantation:+update+on+imaging+manifestations+and+management.">Complications of lung transplantation: update on imaging manifestations and management.</a> Radiology: Cardiothoracic Imaging. 3 (4), e190252 (2021).</li><li>Young, K. A., Dilling, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+of+lung+transplantation.">The future of lung transplantation.</a> Chest. 155 (3), 465-473 (2019).</li><li>Wagner, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+decellularization+and+recellularization+of+normal+versus+emphysematous+human+lungs.">Comparative decellularization and recellularization of normal versus emphysematous human lungs.</a> Biomaterials. 35 (10), 3281-3297 (2014).</li><li>Booth, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acellular+normal+and+fibrotic+human+lung+matrices+as+a+culture+system+for+in+vitro+investigation.">Acellular normal and fibrotic human lung matrices as a culture system for in vitro investigation.</a> American Journal of Respiratory and Critical Care Medicine. 186 (9), 866-876 (2012).</li><li>Uhl, F. E., Wagner, D. E., Weiss, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+decellularized+lung+matrices+for+cell+culture+and+protein+analysis.">Preparation of decellularized lung matrices for cell culture and protein analysis.</a> Methods in Molecular Biology. 1627, 253-283 (2017).</li><li>Wagner, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+scaffolds+of+acellular+human+and+porcine+lungs+for+high+throughput+studies+of+lung+disease+and+regeneration.">Three-dimensional scaffolds of acellular human and porcine lungs for high throughput studies of lung disease and regeneration.</a> Biomaterials. 35 (9), 2664-2679 (2014).</li><li>Uhl, F. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+role+of+glycosaminoglycans+in+decellularized+lung+extracellular+matrix.">Functional role of glycosaminoglycans in decellularized lung extracellular matrix.</a> Acta Biomaterialia. 102, 231-246 (2020).</li><li>Saldin, L. T., Cramer, M. C., Velankar, S. S., White, L. J., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+hydrogels+from+decellularized+tissues:+structure+and+function.">Extracellular matrix hydrogels from decellularized tissues: structure and function.</a> Acta Biomaterialia. 49, 1-15 (2017).</li><li>Giobbe, G. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+hydrogel+derived+from+decellularized+tissues+enables+endodermal+organoid+culture.">Extracellular matrix hydrogel derived from decellularized tissues enables endodermal organoid culture.</a> Nature Communications. 10 (1), 5658 (2019).</li><li>Petrou, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clickable+decellularized+extracellular+matrix+as+a+new+tool+for+building+hybrid-hydrogels+to+model+chronic+fibrotic+diseases+in+vitro.">Clickable decellularized extracellular matrix as a new tool for building hybrid-hydrogels to model chronic fibrotic diseases in vitro.</a> Journal of Materials Chemistry. B. 8 (31), 6814-6826 (2020).</li><li>Nizamoglu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+model+of+fibrosis+using+crosslinked+native+extracellular+matrix-derived+hydrogels+to+modulate+biomechanics+without+changing+composition.">An in vitro model of fibrosis using crosslinked native extracellular matrix-derived hydrogels to modulate biomechanics without changing composition.</a> Acta Biomaterialia. 147, 50-62 (2022).</li><li>Marhuenda, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+extracellular+matrix+hydrogels+enhance+preservation+of+type+ii+phenotype+in+primary+alveolar+epithelial+cells.">Lung extracellular matrix hydrogels enhance preservation of type ii phenotype in primary alveolar epithelial cells.</a> International Journal of Molecular Sciences. 23 (9), 4888 (2022).</li><li>Zhou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+tissue+extracellular+matrix-derived+hydrogels+protect+against+radiation-induced+lung+injury+by+suppressing+epithelial-mesenchymal+transition.">Lung tissue extracellular matrix-derived hydrogels protect against radiation-induced lung injury by suppressing epithelial-mesenchymal transition.</a> Journal of Cellular Physiology. 235 (3), 2377-2388 (2020).</li><li>Pouliot, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+naturally+derived+lung+extracellular+matrix+hydrogel.">Development and characterization of a naturally derived lung extracellular matrix hydrogel.</a> Journal of Biomedical Materials Research. Part A. 104 (8), 1922-1935 (2016).</li><li>Pouliot, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porcine+lung-derived+extracellular+matrix+hydrogel+properties+are+dependent+on+pepsin+digestion+time.">Porcine lung-derived extracellular matrix hydrogel properties are dependent on pepsin digestion time.</a> Tissue Engineering. Part C, Methods. 26 (6), 332-346 (2020).</li><li>de Hilster, R. H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+lung+extracellular+matrix+hydrogels+resemble+the+stiffness+and+viscoelasticity+of+native+lung+tissue.">Human lung extracellular matrix hydrogels resemble the stiffness and viscoelasticity of native lung tissue.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 318 (4), L698-L704 (2020).</li><li>Hoffman, E. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+and+disease+specific+human+lung+extracellular+matrix+composition.">Regional and disease specific human lung extracellular matrix composition.</a> Biomaterials. 293, 121960 (2023).</li><li>Sicard, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+and+anatomical+variations+in+lung+tissue+stiffness.">Aging and anatomical variations in lung tissue stiffness.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 314 (6), L946-L955 (2018).</li></ol>

None of the authors have any conflicts of interest.

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 dynamics and cell-ECM interactions7,8,9,10,11,14,15,16,17,18,19,20. 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 separately21,22. 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.

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><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></table>

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.

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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65276/65276fig01.jpg" /> 
Figure 1: Schematic representation of the whole lung decellularization and dissection process. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65276/65276fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65276/65276fig02.jpg" /> 
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. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65276/65276fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65276/65276fig03.jpg" /> 
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. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65276/65276fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65276/65276fig04.jpg" /> 
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. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65276/65276fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65276/65276fig05.jpg" /> 
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. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65276/65276fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65276/65276fig06.jpg" /> 
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. &#8364; 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. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/65276/65276fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

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.

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 ...

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5.2K Views.  University of Vermont. 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.

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

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Cancer Research

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

Decellularization of the Murine Cardiopulmonary Complex

We present here a decellularization protocol for mouse heart and lungs. It produces structural ECM scaffolds that can be used to analyze ECM topology and composition. It is based on a microsurgical procedure designed to catheterize the trachea and aorta of a euthanized mouse to perfuse the heart and lungs with decellularizing agents. The decellularized cardiopulmonary complex can subsequently be immunostained to reveal the location of structural ECM proteins. The whole procedure can be completed in 4 days.
The ECM scaffolds resulting from this protocol are free of dimensional distortions. The absence of cells enables structural examination of ECM structures down to submicron resolution in 3D. This protocol can be applied to healthy and diseased tissue from mice as young as 4-weeks old, including mouse models of fibrosis and cancer, opening the way to determine ECM remodeling associated with cardiopulmonary disease.

This protocol aims to decellularize the heart and lungs of mice. The resulting extracellular matrix (ECM) scaffolds can be immunostained and imaged to map the location and topology of their components.

We present here a decellularization protocol for mouse heart and lungs. It produces structural ECM scaffolds that can be used to analyze ECM topology and ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Engineered Lung Tissues Prepared from Decellularized Lung Slices

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex vivo. Recently developed organoid models have facilitated the expansion and study of lung epithelial progenitors in vitro, but these platforms typically rely on mouse tumor-derived matrix and/or serum, and incorporate just one or two cellular lineages. Here, we describe a protocol for generating engineered lung tissues (ELTs) based on the multi-lineage recellularization of decellularized precision-cut lung slices (PCLS). ELTs contain alveolar-like structures comprising alveolar epithelium, mesenchyme, and endothelium, within an extracellular matrix (ECM) substrate closely resembling that of native lung. To generate the tissues, rat lungs are inflated with agarose, sliced into 450 &#181;m-thick slices, cut into strips, and decellularized. The resulting acellular ECM scaffolds are then reseeded with primary endothelial cells, fibroblasts, and alveolar epithelial type 2 cells (AEC2s). AEC2s can be maintained in ELT culture for at least 7 days with a serum-free, chemically-defined growth medium. Throughout the tissue preparation and culture process, the slices are clipped into a cassette system that facilitates handling and standardized cell seeding of multiple ELTs in parallel. These ELTs represent an organotypic culture platform that should facilitate investigations of cell-cell and cell-matrix interactions within the alveolus as well as biochemical signals regulating AEC2s and their niche.

This protocol describes a method to generate reproducible, small-scale engineered lung tissues, by repopulating decellularized precision-cut lung slices with alveolar epithelial type 2 cells, fibroblasts, and endothelial cells.

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex ...

Comparative Study of Decellularization Techniques for Bovine Lung ECM Hydrogels

Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells&#39; natural environment in vitro. However, maintaining the native biochemical content of the ECM, achieving mechanical stability, and comprehending the impact of the decellularization process on the mechanical properties of the ECM hydrogels are challenging. Here, a pipeline for decellularization of bovine lung tissue using two different protocols, downstream characterization of the effectiveness of decellularization, fabrication of reconstituted decellularized lung ECM hydrogels and assessment of their mechanical and cytocompatibility properties were described. Decellularization of the bovine lung was pursued using a physical (freeze-thaw cycles) or chemical (detergent-based) method. Hematoxylin and Eosin staining was performed to validate the decellularization and retention of major ECM components. For the evaluation of residual collagen and sulfated glycosaminoglycan (sGAG) content within the decellularized samples, Sirius red and Alcian blue staining techniques were employed, respectively. Mechanical properties of the decellularized lung ECM hydrogels were characterized by oscillatory rheology. The results suggest that decellularized bovine lung hydrogels can provide a reliable organotypic<span style="font-size: 11pt; font-family: Calibri, sans-serif; color: black; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&#160;alternative to commercial ECM products by retaining most native ECM components. Furthermore, these findings reveal that the decellularization method of choice significantly affects gelation kinetics as well as the stiffness and viscoelastic properties of resulting hydrogels.

Author Spotlight: Optimizing Bovine Lung Decellularization for Organotypic Hydrogels

The protocol elucidates two distinct decellularization methodologies applied to native bovine pulmonary tissues, providing a comprehensive account of their respective characterizations.

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells&#39; natural ...

Transplantation of Bioengineered Lung Using Decellularized Mouse Lungs and Primary Human Endothelial Cells

Lung transplantation is a critical treatment for patients with end-stage lung diseases like idiopathic pulmonary fibrosis, but challenges such as donor shortages and posttransplant complications persist. Bioengineered lungs, integrating patient-specific cells into decellularized animal scaffolds, present a promising alternative. Despite progress in using bioengineered lungs in animal models, functionality and structure remain immature. This protocol addresses a critical barrier in organ bioengineering: the need for a cost-effective experimental platform. By using mouse models instead of larger animals like rats or swine, researchers can significantly reduce the resources required for each experiment, accelerating research progress. 
The protocol outlines a detailed procedure for lung bioengineering using mouse heart-lung blocks and human primary cells, focusing on isolation strategy for the mouse heart-lung block, decellularization, bioreactor setup, perfusion-based organ culture, and orthotopic transplantation of bioengineered lungs. This mouse-scale platform not only reduces experimental costs but also provides a viable framework for optimizing cell types and numbers for recellularization, testing different cell types using histological and molecular methods, and ensuring blood flow post-transplantation. The method holds potential for broad applications, including studying cell interactions in three-dimensional culture conditions, cell-matrix interactions, and ex vivo cancer modeling, thereby advancing the field of organ bioengineering.

This paper describes how to create bioengineered mouse lungs using decellularization and recellularization methods. It also details subsequent orthotopic lung transplantation.

Lung transplantation is a critical treatment for patients with end-stage lung diseases like idiopathic pulmonary fibrosis, but challenges such as donor ...

Cardiopulmonary Complex Decellularization: A Technique for Decellularizing Heart and Lungs from Murine Model

Source: Mayorca-Guiliani, A. E. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/61854" target="_blank">Decellularization of the Murine Cardiopulmonary Complex</a>. J. Vis. Exp. (2021)
This video demonstrates a method of producing decellularized extracellular matrix or ECM scaffolds by applying antegrade perfusion of detergents through the vasculature. The structural ECM scaffolds can then be used to analyze ECM remodeling associated with cardiopulmonary disease.

Source: Mayorca-Guiliani, A. E. et al. Decellularization of the Murine Cardiopulmonary Complex. J. Vis. Exp. (2021)
This video demonstrates a method of ...