Name: decellularization of the murine cardiopulmonary complex
Uploaded: 2021-05-30T13:00:00
Description: Watch this Scientific Journal Video about Decellularization of the Murine Cardiopulmonary Complex at JoVE.com

We thank Prof. Ivana Novak and Dr. Nynne Meyn Christensen (Centre for Advanced Bioimaging (CAB), University of Copenhagen) for providing microscope access. This work was supported by the European Research Council (ERC-2015-CoG-682881-MATRICAN; AEM-G, OW, RR and JTE); a PhD fellowship from the Lundbeck Foundation (R286-2018-621; MR); the Swedish Research Council (2017-03389; CDM); the Swedish Cancer Society, Cancerfonden (CAN 2016/783, 19 0632 Pj, and 190007; CDM); German Cancer Aid (Deutsche Krebshilfe; RR); and the Danish Cancer Society (R204-A12454; RR).&#160;

All procedures included here have been reviewed and approved by the ethical committee regulating experimental medicine in the University of Copenhagen and agree with Danish and European legislation. To demonstrate this protocol, we have used female BALB/cJ mice of 8-12 weeks of age and an MMTV-PyMT female mouse of 11 weeks of age.
NOTE: Avoiding bacterial contamination of the decellularized ECM scaffold gives the best imaging outcome and allows long-term sample storage. It is therefore importa...

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	<li>Hynes, R. O. <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:+not+just+pretty+fibrils.">Extracellular matrix: not just pretty fibrils.</a> Science. 326, 1216-1219 (2009).</li><li>Mayorca-Guiliani, A. 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=ISDoT:+in+situ+decellularization+of+tissues+for+high-resolution+imaging+and+proteomic+analysis+of+native+extracellular+matrix.">ISDoT: in situ decellularization of tissues for high-resolution imaging and proteomic analysis of native extracellular matrix.</a> Nature Medicine. 23, 890-898 (2017).</li><li>Mayorca-Guiliani, A. 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=Decellularization+and+antibody+staining+of+mouse+tissues+to+map+native+extracellular+matrix+structures+in+3D.">Decellularization and antibody staining of mouse tissues to map native extracellular matrix structures in 3D.</a> Nature Protocols. 14, 3395-3425 (2019).</li><li>White, L. 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=The+impact+of+detergents+on+the+tissue+decellularization+process:+A+TOF-sims+study.">The impact of detergents on the tissue decellularization process: A TOF-sims study.</a> Acta Biomaterialia. 50, 207-219 (2017).</li><li>Ott, H. C., 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=Perfusion-decellularized+matrix:+using+nature's+platform+to+engineer+a+bioartificial+heart.">Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.</a> Nature Medicine. 14, 213-221 (2008).</li><li>Uygun, B. 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=Organ+re-engineering+through+development+of+a+transplantable+recellularized+liver+graft+using+decellularized+liver+matrix.">Organ re-engineering through development of a transplantable recellularized liver graft using decellularized liver matrix.</a> Nature Medicine. 16, 814-820 (2010).</li><li>Susaki, E. 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=Advanced+CUBIC+protocols+for+whole-brain+and+whole-body+clearing+and+imaging.">Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging.</a> Nature Protocols. 10, 1709-1727 (2015).</li><li>Tomer, R., Ye, L., Hsueh, B., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+CLARITY+for+rapid+and+high-resolution+imaging+of+intact+tissues.">Advanced CLARITY for rapid and high-resolution imaging of intact tissues.</a> Nature Protocols. 9, 1682-1697 (2014).</li><li>Erturk, 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=Three-dimensional+imaging+of+solvent-cleared+organs+using+3DISCO.">Three-dimensional imaging of solvent-cleared organs using 3DISCO.</a> Nature Protocols. 7, 1983-1995 (2012).</li><li>Wershof, 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=A+FIJI+Macro+for+quantifying+pattern+in+extracellular+matrix.">A FIJI Macro for quantifying pattern in extracellular matrix.</a> BioRxiv. , (2019).</li></ol>

Decellularization techniques based on tissue agitation alter ECM structure, making them unsuitable for ECM structure analysis4. Perfusion decellularization, using an anatomical route such as the aorta of the trachea, allows to reach the capillary bed, or terminal alveoli, and facilitates the delivery of decellularizing agents throughout the organ. The use of zwitterionic, anionic and non-ionic detergents to decellularize tissue is reported4,5</s...

The ECM is a three-dimensional network made of proteins and glycans that accommodates all cells in a multicellular organism, giving organs their shape and regulating cell behavior throughout life1. From egg fertilization onwards, cells build and remodel the ECM, and are in turn strictly controlled by it. The purpose of this protocol is to open a way to analyze and map mouse ECM, as mice are the most used model organism in mammalian pathophysiology.
The development of this method was driven by the need to characterize and isolate metastasis-associated native ECM2. As tumors lack proper anatomical vascularization and mice are relatively small organisms, microsurgical procedures were designed to retrogradely catheterize the aorta, while isolating the circulation of the major vessel leading to a tumor (e.g., the pulmonary veins), thus focusing reagent flow and allowing tumor decellularization. This method produces ECM scaffolds with a conserved structure2 that can be immunostained and imaged, allowing ECM structure mapping in submicron detail. To carry out this protocol, it is necessary to acquire surgical and microsurgical skills (dissection, microsuturing and catheterization) that may represent a potential limitation to its use. To our knowledge, this method represents the state-of-the-art for native ECM structure imaging analysis2,3.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>MICROSURGERY</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 suture, triangular section needle (Vicryl)</td>
			<td>Ethicon</td>
			<td>6301124</td>
			<td></td>
		</tr>
		<tr>
			<td>9-0 micro-suture (Safil)</td>
			<td>B Braun</td>
			<td>G1048611</td>
			<td></td>
		</tr>
		<tr>
			<td>Adson forceps</td>
			<td>Fine Science Tools</td>
			<td>11006-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Adson forceps with teeth</td>
			<td>Fine Science Tools</td>
			<td>11027-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo microneedle holder</td>
			<td>Fine Science Tools</td>
			<td>no. 12061-01</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 ventilation chamber for mouse euthanasia</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water (Milli-Q IQ 7000, Ultrapure lab water system)&#160;</td>
			<td>Merck</td>
			<td>ZIQ7000T0</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable polystyrene tray (~30 &#215; 50 cm)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope (Greenough, with two-armed gooseneck)</td>
			<td>Leica</td>
			<td>S6 D</td>
			<td></td>
		</tr>
		<tr>
			<td>Double-ended microspatula</td>
			<td>Fine Science Tools</td>
			<td>10091-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont microforceps (two)</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont microforceps with 45&#176; tips (two)</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair clippers</td>
			<td>Oster</td>
			<td>76998-320-051</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsey needle holder (with tungsten carbide jaws)</td>
			<td>Fine Science Tools</td>
			<td>12500-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Intravenous 24-gauge catheter (Insyte)</td>
			<td>BD</td>
			<td>381512</td>
			<td></td>
		</tr>
		<tr>
			<td>Intravenous 26-gauge catheter (Terumo)</td>
			<td>Surflo-W</td>
			<td>SR+DM2619WX</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayo scissors (tough cut, straight)</td>
			<td>Fine Science Tools</td>
			<td>14110-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Microforceps with ringed tips (two)</td>
			<td>Aesculap</td>
			<td>FM571R</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-spring scissors (Vannas, curved)</td>
			<td>Fine Science Tools</td>
			<td>15001-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Minicutter</td>
			<td>KLS Martin</td>
			<td>80-008-03-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Molt Periostotome</td>
			<td>Aesculap</td>
			<td>D0543R</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles (27 gauge; Microlance)</td>
			<td>BD</td>
			<td>21018</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper towel (sterile) or surgical napkin&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Serrated scissors (CeramaCut, straight)</td>
			<td>Fine Science Tools</td>
			<td>14958-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Spatula (Freer-Yasargil)</td>
			<td>Aesculap</td>
			<td>OL166R</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes (1 mL; Plastipak)</td>
			<td>BD</td>
			<td>3021001</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes (10 mL; Plastipak)</td>
			<td>BD</td>
			<td>3021110</td>
			<td></td>
		</tr>
		<tr>
			<td>Tendon scissors (Walton)</td>
			<td>Fine Science Tools</td>
			<td>14077-09</td>
			<td></td>
		</tr>
		<tr>
			<td>IMMUNOSTAINING</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey anti-guinea pig IgG</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11055</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 donkey anti-rabbit IgG</td>
			<td>Life Technologies</td>
			<td>A11037</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA(albumin bovine fraction V, standard grade, lyophilized)&#160;</td>
			<td>Serva</td>
			<td>11930.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen IV polyclonal antibody (RRID: AB_2276457)&#160;</td>
			<td>Millipore</td>
			<td>AB756P</td>
			<td>Host: rabbit</td>
		</tr>
		<tr>
			<td>PBS (pH 7.4, 10&#215;, Gibco)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>70011044</td>
			<td>Host: goat</td>
		</tr>
		<tr>
			<td>Periostin polyclonal antibody (a kind gift from Manuel Koch. RRID:AB2801621)</td>
			<td></td>
			<td></td>
			<td>Host: guinea pig</td>
		</tr>
		<tr>
			<td>Scalpel disposable with blade no.11 (pcs. 10)</td>
			<td>VWR</td>
			<td>233-5364)</td>
			<td></td>
		</tr>
		<tr>
			<td>Serum (normal donkey serum)&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>IMAGING</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Detectors (hybrid detector (Leica, HyD S model) and photomultiplier tubes (PMTs; )&#160;</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Fluorescence light source&#160;</td>
			<td>Leica</td>
			<td>EL6000</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Microscope (inverted multiphoton microscope)&#160;</td>
			<td>Leica</td>
			<td>SP5-X MP</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Objective (lambda blue, 20&#215;, 0.70 numerical aperture (NA) IMM UV)&#160;</td>
			<td>Leica</td>
			<td>HCX PL APO</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Two-photon Ti&#8211;sapphire laser (Spectra-physics, Mai Tai DeepSee model)&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;White-light laser (WLL)&#160;</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DECELLULARIZATION</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol (absolute alcohol 99.9%); absolute alcohol must be adjusted to 70% (vol/vol) using deionized water&#160;</td>
			<td>Plum</td>
			<td>1680766</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water (Milli-Q IQ 7000, Ultrapure lab water system)&#160;</td>
			<td>Merck</td>
			<td>ZIQ7000T0</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer-to-tubing male fittings (1/8 inch)</td>
			<td>World Precision Instruments</td>
			<td>13158-100</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (pH 7.4, 10&#215;, Gibco)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>70011044</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump (with 12 channels)</td>
			<td>Ole Dich</td>
			<td>110AC(R)20G75</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone tubing (with 2-mm i.d. and 4 mm o.d.)</td>
			<td>Ole Dich</td>
			<td>31399</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma-Aldrich</td>
			<td>08591-1ML-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate (DOC)</td>
			<td>Sigma-Aldrich</td>
			<td>D6750-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulphate</td>
			<td>Sigma-Aldrich</td>
			<td>L3771-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>H&#38;E STAINING</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4% PFA</td>
			<td>Fisher Scientific</td>
			<td>15434389</td>
			<td></td>
		</tr>
		<tr>
			<td>96% Ethanol</td>
			<td>Plum</td>
			<td>201446-5L</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute ethanol</td>
			<td>Plum</td>
			<td>201152-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips (24x50mm; 1000 pcs)</td>
			<td>Hounisen</td>
			<td>422.245</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryomolds Intermediate (15 x 15 x 5 mm; 100 pcs)</td>
			<td>Tissue-Tek</td>
			<td>4566</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM3050S</td>
			<td></td>
		</tr>
		<tr>
			<td>DPX mounting medium</td>
			<td>Hounisen</td>
			<td>1001.0025</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y solution alcoholic 0.5%</td>
			<td>Sigma</td>
			<td>1024390500</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather microtome blade stainless steel,C35 (50 pcs)</td>
			<td>Pfm medical</td>
			<td>207500003</td>
			<td></td>
		</tr>
		<tr>
			<td> 
			Fisherbrand Superfrost Plus slides (25 x 75 mm; 144 pcs)</td>
			<td>Thermofisher</td>
			<td>6319483</td>
			<td></td>
		</tr>
		<tr>
			<td>Mayers hematoxylin</td>
			<td>Sigma</td>
			<td>MHS32-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT compound</td>
			<td>VWR</td>
			<td>361603E</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide scanner (Nanozoomer)</td>
			<td>Hamamatsu Photonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sigma</td>
			<td>534056-4L</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Cardiopulmonary decellularization 
After successfully completing the protocol, the heart and lungs, as well as annex tissue such as the aortic arc, will be free of cells. Decellularization can be validated by hematoxylin-eosin staining (Figure 1) of the ECM scaffolds showing removal of the nuclei comparing to the native tissue. These scaffolds retain the dimensions of fresh organs and its insoluble ECM structure is intact<sup class="xref...

Watch this Scientific Journal Video about Decellularization of the Murine Cardiopulmonary Complex at JoVE.com

Decellularization of the Murine Cardiopulmonary Complex

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 ...

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