Name: Decellularization of the Murine Cardiopulmonary Complex (Video) | JoVE Journal
Uploaded: 2021-05-30T13:00:00.000+00:00
Duration: 8 min 34 s
Description: Learn decellularization of mouse heart and lungs to create ECM scaffolds for tissue engineering research. This method preserves structural integrity for biochemical analysis and regenerative medicine applications.

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5 minutes

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 important to keep all the steps sterile. As such, all instruments and surgical material, including suture, micro-suture, solutions, tubing, Luer connectors and catheters, must be sterile. Surfaces, including a polystyrene tray, must be disinfected with 70% ethanol, and the perfusion should preferably be carried out under a laminar flow hood. All procedures take place at room temperature unless otherwise indicated.
1. Post-mortem microsurgery
<ol>
	<li>Euthanize the mouse using a CO2 chamber.
	<ol>
		<li>Place mouse into a 4 L chamber, and begin to&#160;fill with 100% CO2, starting at 0.2 L/min for 2 min,&#160;increasing until reaching a flow of 0.8 L/min after 3 min.&#160;The mouse should fall unconscious during the first 2 min, and then respiration&#160;should cease (usually around 5 min, but flow can be maintained as necessary). Confirm death prior to proceeding to the next step.</li>
	</ol>
	</li>
	<li>Shave the thorax, abdomen and back of the mouse with the hair clipper and disinfect with 70% ethanol. Shaving greatly reduces the number of artifacts due to the presence of hair either on samples for imaging or biochemical analysis.</li>
	<li>Pin the mouse to a polystyrene tray, extending its fore- and hindlimbs, as well as its head and tail. Place it under the microsurgery microscope.</li>
	<li>Using a Mayo straight pattern scissors establish surgical access with a cutaneous incision running from the submandibular region to the lower abdomen and dissect subcutaneously to expose the thoracic wall and peritoneum.</li>
	<li>Using microsurgical scissors, cut the pectoralis major and pectoralis minor muscles along the sixth intercostal space on both sides of the thoracic wall.</li>
	<li>Using straight-pattern scissors, cut the sternum along the previous incisions, and then complete a sternotomy by cutting the sternum along its long axis, then elevate and pin both sides of the thoracic wall to expose the cardiopulmonary complex.</li>
	<li>Using round-tipped micro-forceps (or Dumont micro-forceps), excise the thymus and surrounding adipose tissue by delicately pulling them off their attachments. This will reveal the major vessels.</li>
	<li>Using the cautery, cauterize the descending cava vein and, using straight pattern scissors, cut the esophagus.</li>
	<li>Using sharp micro-forceps, separate the brachiocephalic veins and the brachiocephalic, left common carotid and left subclavian arteries from the underlying tissue to facilitate ligation and cauterization.</li>
	<li>Using micro-needle holder, sharp micro-forceps and 9-0 suture place stitches above the emergence of the brachiocephalic, left common carotid and left subclavian arteries.</li>
	<li>Cauterize the brachiocephalic veins.</li>
	<li>Separate the submandibular salivary glands along the midline to expose the neck muscles and the trachea. Separate the muscles to expose the cricothyroid ligament. Using micro-scissors, open an entrance by sectioning the ligament.</li>
	<li>Introduce a 27 G catheter in the trachea and delicately push until the trachea branches into the bronchi (i.e., until resistance to the catheter is met, then retreat 3 mm). Be careful not to disrupt the bronchi. Using a 6-0 suture, place 3 stitches around the trachea to secure the catheter.</li>
	<li>Section the mouse at the height of the 12th thoracic vertebra. The descending aorta runs anteriorly to the spine and should be sectioned here along with the spine. Set the lower half apart.</li>
	<li>Retrogradely catheterize the aorta and push the catheter until it reaches the aortic arc. Using 9-0 suture, place 4 stitches around the aorta, beginning 5 mm below the catheter tip.</li>
</ol>
2. Decellularization
<ol>
	<li>Connect the mouse to a pump system using silicone tubing and Luer connectors. Perfuse with deionized water at 200 &#181;L/min for 15 min. Maintain this flow rate during decellularization.</li>
	<li>Change the perfusion agent to 0.5% sodium deoxycholate (DOC) diluted in deionized water and perfuse overnight.</li>
	<li>Change the perfusion agent to 0.1% sodium dodecyl sulphate (SDS) diluted in deionized water and perfuse for 8 hours.</li>
	<li>Perfuse with deionized water overnight to wash away SDS and DOC for 24 h.</li>
	<li>Resect the decellularized heart and lungs by sectioning its attachments to the thorax using a curved scissors and store in a sterile cryo-tube with deionized water with 1% (v/v) penicillin-streptomycin and 0.3 &#181;M sodium azide at 4 &#176;C. ECM scaffolds can be stored for at least for 12 weeks1. If the scaffold will be used for biochemical analysis (e.g., mass spectrometry), snap freeze in liquid nitrogen.</li>
</ol>
3. Immunostaining
<ol>
	<li>Plan the imaging: determine primary antibody (or antibodies) and the combination of fluorescently conjugated secondary antibodies to match each other and to fit the laser lines of the fluorescence microscope.</li>
	<li>Block the sample by immersing it in a cryotube containing 6% (v/v) donkey serum - 3% (w/v) bovine serum albumin (BSA) overnight.</li>
	<li>Incubate with primary antibody (or antibodies) in 3% donkey serum in PBS for 24 h.</li>
	<li>Wash 5 times for 1 h each time in 0.05% tween 20 in PBS (PBST).</li>
	<li>Incubate the sample with fluorescently conjugated secondary antibody (or antibodies) in 3% donkey serum in PBS for 24 h.</li>
	<li>Wash 5 times for 1 hour in 0.05% (PBST). Wait 1 h between each wash.</li>
	<li>Add deionized water and store at 4 &#176;C away from direct light. At this point, the scaffold is ready to image.</li>
</ol>
4. Imaging
<ol>
	<li>Place the sample in a glass-bottomed dish and humidify it with two droplets of storing solution (PBS or deionized water).</li>
	<li>Prepare the objective. We recommend using a water immersion objective.</li>
	<li>Inspect the sample using fluorescence light.</li>
	<li>Switch to computer control. Turn on lasers and adjust laser intensity, pinhole aperture, detectors wavelengths, gain, resolution and zoom. Set the number and step size for z-stack and begin acquisition. We recommend using multiphoton laser excitation to increase tissue penetration and to minimize scattering of light, bleaching and tissue damage.</li>
</ol>
5. Hematoxylin-eosin staining
<ol>
	<li>Excise 1 lung lobe from a euthanized mouse.</li>
	<li>Place in a 10 mm x 10 mm x 5 mm cryomold and cover it with approximately 500 &#181;L of OCT compound.</li>
	<li>Freeze on dry ice (-70 &#176;C) and maintain the sample at that temperature.</li>
	<li>Excise one decellularized lung lobe from a processed mouse according to step 2.5.</li>
	<li>Place in a cryomold with the largest surface area down and cover it with OCT compound as specified in step 5.2.</li>
	<li>Freeze on dry ice (-70&#176;C) and maintain the sample at that temperature until otherwise required. The sample can be stored for at least 12 weeks.</li>
	<li>Section frozen tissue blocks at -20 &#176;C in a cryostat with 5 &#181;m thickness and place sections on adhesive glass slides and store at -80 &#176;C.</li>
	<li>Take slides to room temperature until air dried (approximately 20 min).</li>
	<li>Shortly immerse in PBS and fix by immersing the slides in 4% paraformaldehyde in PBS for 15 min. Wash once in PBS for 5 min, then twice in distilled water for 5 min.</li>
	<li>Immerse in Mayer&#39;s hematoxylin solution for 10 min. This time can be optimized according to tissue source and stain preparation.</li>
	<li>Wash in a Coplin jar under running distilled water for 10 min.</li>
	<li>Immerse in eosin solution for 7 min. This time can be optimized according to tissue source and stain preparation.</li>
	<li>Dip in 50% ethanol to remove excess Eosin and dehydrate by shortly dipping in 70% ethanol, and in 96% and 100% ethanol for 30 seconds. Dip in xylene several times.</li>
	<li>Apply few drops of DPX mounting medium and place a glass coverslip.</li>
	<li>Leave slides to dry overnight under a chemical hood.</li>
	<li>Scan slides in a slide scanner.</li>
</ol>

<ol><li>Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+matrix%3A+not+just+pretty+fibrils%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((ISDoT%3A+in+situ+decellularization+of+tissues+for+high-resolution+imaging+and+proteomic+analysis+of+native+extracellular+matrix%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">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/pubmed?term=((Decellularization+and+antibody+staining+of+mouse+tissues+to+map+native+extracellular+matrix+structures+in+3D%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">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/pubmed?term=((The+impact+of+detergents+on+the+tissue+decellularization+process%3A+A+TOF-sims+study%5BTitle%5D)+AND+%22Acta+Biomaterialia%22%5BJournal%5D)">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/pubmed?term=((Perfusion-decellularized+matrix%3A+using+nature%27s+platform+to+engineer+a+bioartificial+heart%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">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/pubmed?term=((Organ+re-engineering+through+development+of+a+transplantable+recellularized+liver+graft+using+decellularized+liver+matrix%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">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/pubmed?term=((Advanced+CUBIC+protocols+for+whole-brain+and+whole-body+clearing+and+imaging%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">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/pubmed?term=((Advanced+CLARITY+for+rapid+and+high-resolution+imaging+of+intact+tissues%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">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/pubmed?term=((Three-dimensional+imaging+of+solvent-cleared+organs+using+3DISCO%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">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/pubmed?term=((A+FIJI+Macro+for+quantifying+pattern+in+extracellular+matrix%5BTitle%5D)+AND+%22BioRxiv%22%5BJournal%5D)">A FIJI Macro for quantifying pattern in extracellular matrix.</a> BioRxiv. , (2019).</li>
</ol>

The authors have nothing to disclose.

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><tbody><tr><td>&lt;strong&gt;MICROSURGERY&lt;/strong&gt;</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>CO&lt;font class=&quot;font6&quot;&gt;&lt;sub&gt;2&lt;/sub&gt;&lt;/font&gt;&lt;font class=&quot;font5&quot;&gt; ventilation chamber for mouse euthanasia&lt;/font&gt;</td><td></td><td></td><td></td></tr><tr><td>Deionized water (Milli-Q IQ 7000, Ultrapure lab water system)&amp;nbsp;</td><td>Merck</td><td>ZIQ7000T0</td><td></td></tr><tr><td>Disposable polystyrene tray (~30 &amp;times; 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&amp;deg; 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&amp;nbsp;</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>&lt;strong&gt;IMMUNOSTAINING&lt;/strong&gt;</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)&amp;nbsp;</td><td>Serva</td><td>11930.03</td><td></td></tr><tr><td>Collagen IV polyclonal antibody (RRID: AB_2276457)&amp;nbsp;</td><td>Millipore</td><td>AB756P</td><td>Host: rabbit</td></tr><tr><td>PBS (pH 7.4, 10&amp;times;, Gibco)&amp;nbsp;</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)&amp;nbsp;</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>&lt;strong&gt;IMAGING&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;Detectors (hybrid detector (Leica, HyD S model) and photomultiplier tubes (PMTs; )&amp;nbsp;</td><td>Leica</td><td></td><td></td></tr><tr><td>&amp;nbsp;Fluorescence light source&amp;nbsp;</td><td>Leica</td><td>EL6000</td><td></td></tr><tr><td>&amp;nbsp;Microscope (inverted multiphoton microscope)&amp;nbsp;</td><td>Leica</td><td>SP5-X MP</td><td></td></tr><tr><td>&amp;nbsp;Objective (lambda blue, 20&amp;times;, 0.70 numerical aperture (NA) IMM UV)&amp;nbsp;</td><td>Leica</td><td>HCX PL APO</td><td></td></tr><tr><td>&amp;nbsp;Two-photon Ti&amp;ndash;sapphire laser (Spectra-physics, Mai Tai DeepSee model)&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;White-light laser (WLL)&amp;nbsp;</td><td>Leica</td><td></td><td></td></tr><tr><td>&lt;strong&gt;DECELLULARIZATION&lt;/strong&gt;</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&amp;nbsp;</td><td>Plum</td><td>1680766</td><td></td></tr><tr><td>Deionized water (Milli-Q IQ 7000, Ultrapure lab water system)&amp;nbsp;</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&amp;times;, Gibco)&amp;nbsp;</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>&lt;strong&gt;H&amp;amp;E STAINING&lt;/strong&gt;</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>&lt;br/&gt; 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

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...

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 ...

decellularization-of-the-murine-cardiopulmonary-complex

Research

JoVE Journal

Cancer Research

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

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

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

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

This method enables decellularization of a complex solid organ using a simple protocol based on osmotic shock and perfusion of ionic detergent with minimal organ matrix disruption. It comprises a novel decellularization technique for human hearts inside a pressurized pouch with real-time monitoring of flow dynamics and cellular debris outflow.

Decellularization of Whole Human Heart Inside a Pressurized Pouch in an Inverted Orientation

Classically, the endocardium of the mouse embryonic valve primordium has been analyzed using transversal, coronal, or sagittal sections. Our novel approach for en face, two-dimensional imaging of the endocardium at valvulogenic regions allows planar polarity and cell rearrangement analysis of the endocardium during valve development.

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

This protocol demonstrates microscopy-guided isolation and immunofluorescence staining of murine pulmonary veins. We prepare tissue samples containing the left atrium, pulmonary veins, and the corresponding lungs and stain them for cardiac Troponin T and Connexin 43.

Microdissection and Immunofluorescence Staining of Myocardial Sleeves in Murine Pulmonary Veins

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

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

Learn decellularization of mouse heart and lungs to create ECM scaffolds for tissue engineering research. This method preserves structural integrity for biochemical analysis and regenerative medicine applications.

Decellularization of the Murine Cardiopulmonary Complex

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