Name: decellularization of the murine cardiopulmonary complex
Uploaded: 2021-05-30T13:00:00.000+00:00
Duration: 8 min 34 s
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 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/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>

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

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 ...

decellularization-of-the-murine-cardiopulmonary-complex

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2.7K Views.  University of Copenhagen (UCPH). 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.

Video: Decellularization of the Murine Cardiopulmonary Complex

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

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.

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

Bioengineering

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

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

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.

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

Developmental Biology

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 ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

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

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

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

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.

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.

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components ...

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

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart disease. The development of the primitive cardiac valves involves the epithelial-to-mesenchymal transition (EMT) of endocardial cells from the atrioventricular canal (AVC) and outflow tract (OFT) regions of the heart in response to local inductive myocardial and endocardial signals. Once the cells delaminate and invade the extracellular matrix (cardiac jelly) located between the endocardium and the myocardium, the primitive endocardial cushions (EC) are formed. This process implies that the endocardium has to fill the gaps left by the delaminated cells and has to reorganize itself to converge (narrow) or extend (lengthen) along an axis. Current research has implicated the planar cell polarity (PCP) pathway in regulating the subcellular localization of the factors involved in this process. Classically, the initial phases of cardiac valve development have been studied in cross-sections of embryonic hearts or in ex vivo AVC or OFT explants cultured on collagen gels. These approaches allow the analysis of apico-basal polarity but do not allow the analysis of cell behavior within the plane of the epithelium or of the morphological changes of migrating cells. Here, we show an experimental approach that allows the visualization of the endocardium at valvulogenic regions as a planar field of cells. This experimental approach provides the opportunity to study PCP, planar topology, and intercellular communication within the endocardium of the OFT and AVC during valve development. Deciphering new cellular mechanisms involved in cardiac valve morphogenesis may contribute to understanding congenital heart disease associated with endocardial cushion defects.

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

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.

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart ...

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

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ultimately rejection can occur. Creating a functional, autologous bio-artificial heart could solve these challenges. Biofabrication of a heart comprised of scaffold and cells is one option. A natural scaffold with tissue-specific composition as well as micro- and macro-architecture can be obtained by decellularizing hearts from humans or large animals such as pigs. Decellularization involves washing out cellular debris while preserving 3D extracellular matrix and vasculature and allowing &#34;cellularization&#34; at a later timepoint. Capitalizing on our novel finding that perfusion decellularization of complex organs is possible, we developed a more &#34;physiological&#34; method to decellularize non-transplantable human hearts by placing them inside a pressurized pouch, in an inverted orientation, under controlled pressure. The purpose of using a pressurized pouch is to create pressure gradients across the aortic valve to keep it closed and improve myocardial perfusion. Simultaneous assessment of flow dynamics and cellular debris removal during decellularization allowed us to monitor both fluid inflow and debris outflow, thereby generating a scaffold that can be used either for simple cardiac repair (e.g. as a patch or valve scaffold) or as a whole-organ scaffold.

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.

The ultimate solution for patients with end-stage heart failure is organ transplant. But donor hearts are limited, immunosuppression is required, and ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

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.

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

Biology

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

We developed a simple method for isolating high quality individual mouse heart cells by the antegrade perfusion technique. This method is Langendorff-free and useful for isolating ventricular and atrial myocytes or interstitial cells, such as cardiac fibroblasts or progenitors.

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating ...

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/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 ...

Decellularization of the Murine Cardiopulmonary Complex

Summary

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Chapters in this video

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

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

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

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

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

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

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

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

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