This work was supported by National Institutes of Health Grants R01 HL114940 (Biesiadecki), R01 AG060542 (Ziolo), and T32 HL134616 (Sturgill and Salyer).

All procedures performed in this study were approved by the Institutional Animal Care and Use Committee at the Ohio State University in accordance with NIH guidelines.
1. Solution preparation
NOTE: Please see Table 2 for buffer concentrations.
<ol>
	<li>Pre-isolation (up to 2 weeks in advance of myocyte isolation)
	<ol>
		<li>Perfusion buffer base: Prepare 1 L of perfusion buffer base (NaCl, KCl, NaH2PO4, HEPES, Glucose, and BDM) in 1 L of ultrapure 18.2 M&#937;&#183;cm H2O. Adjust to pH 7.4 prior to 0.22 &#181;m sterile filtration. Store at 4 &#176;C until the day of isolation.</li>
		<li>Clearing buffer: Prepare 1 L of clearing buffer (NaCl, KCl, NaH2PO4, HEPES, Glucose, EDTA, and BDM) in 1 L of ultrapure 18.2 M&#937;&#183;cm H2O. Adjust to pH 7.4 prior to 0.22 &#181;m sterile filtration. Store at 4 &#176;C until the day of isolation.</li>
		<li>100 mM CaCl2 stock solution: Weigh out 1.11 g of CaCl2 and dissolve into 100 mL of ultrapure 18.2 M&#937;&#183;cm H2O. Store at room temperature until the day of isolation.</li>
		<li>Liberase aliquots: Dissolve 5 mg of digestive enzymes in 5 mL of sterile, RNase-free water. Prepare 0.8 mL aliquots and store at -20 &#176;C until the day of isolation.</li>
		<li>Laminate the dishes: Apply 100 &#181;L of 40 &#181;g/mL mouse laminin to sterile, glass-bottom 30 mL Petri dishes. Allow to sit for 30 min and remove excess laminin. Set the laminin on a Petri dish with UV incubation at room temperature for 30 min. Store at 4 &#176;C until the day of isolation (laminated Petri dishes can be used for up to 2 weeks).</li>
		<li>DMEM media: In a biosafety cabinet, add 2.5 mL of FBS, 0.5 mL of Penicillin-Streptomycin (50 U/mL-50 &#181;g), and 1 mL of 500 mM BDM to 46 mL of DMEM. Store at 4 &#176;C until the day of isolation (media can be used for up to 2 weeks).</li>
		<li>M199 media: Prepare 1 L of M199 media (NaHCO3, HEPES, L-glutathione, M199, BDM, and BSA) in 1 L of ultrapure 18.2 M&#937;&#183;cm H2O. Adjust to pH 7.4 prior to sterile filtration (0.22 &#181;m filter). Store at 4 &#176;C until the day of isolation.</li>
		<li>500 mM BDM stock: Add 0.5 g of BDM to 10 mL of ultrapure 18.2 M&#937;&#183;cm H2O. Store at 4 &#176;C until the day of isolation.</li>
		<li>Stabilizing platform creation: Mold playdough around the base of the needle screwed into the Luer stopcock. Mold to the Petri dish that will contain the heart and ensure that the needle is at proper height for ventricle injection (~5 mm above bottom of dish).</li>
	</ol>
	</li>
	<li>The day of isolation
	<ol>
		<li>Perfusion buffer: On the day of the isolation, add 9.5 mg of MgCl2 to 100 mL of perfusion buffer base. Allow to mix completely before removing 30 mL of completed perfusion buffer and place in a clean, wide-mouth, 100 mL glass bottle with a screw cap lid (digestion buffer). 
		&#8203;NOTE: These additions can be made on the day of isolation without additional pH adjustments as their addition negligibly alters the pH.
		<ol>
			<li>Remove 12 mL of perfusion buffer and set to the side (stop buffer). Pour the remaining 58 mL of perfusion buffer into another clean, wide-mouth, 100 mL glass bottle with a screw cap lid. Store the remaining perfusion buffer at 37 &#176;C throughout isolation.</li>
		</ol>
		</li>
		<li>Digestion buffer: Add 7.5 &#181;L of 100 mM CaCl2 to 30 mL of perfusion buffer for a final concentration of 25 &#181;M. Immediately prior to the isolation, add 770 &#181;L of Liberase to the digestion buffer for a final concentration of 26 &#181;g/mL.</li>
		<li>Stop buffer: Dissolve 240 mg of BSA in 12 mL of perfusion buffer. Store at 37 &#176;C throughout isolation.</li>
		<li>Clearing buffer: Prepare a 3 mL syringe of clearing buffer and clear the needle of bubbles. Store on ice until isolation.</li>
	</ol>
	</li>
</ol>
2. Manifold preparation
<ol>
	<li>Clear the temperature-controlled manifold with freshly prepared perfusion buffer, being careful to ensure no bubbles remain. Using a Luer lock connector, screw in a 27 G needle, and clear of bubbles. A cleared manifold is essential for a successful isolation.</li>
</ol>
3. Animal preparation
<ol>
	<li>Inject the mouse intraperitoneally with 100 mg/kg ketamine and 20 mg/kg xylazine by body weight immediately prior to the isolation. This procedure used a male, 4-month-old C57Bl/6 mouse.</li>
	<li>If needed, place the mouse on a heated surgical pad to encourage sedation. Tent the arms of the mouse, and tape the limbs and base of the tail to a blue lab diaper. Ensure that the animal is fully anesthetized&#160;by the withdrawal of&#160;toe-pinch reflex. Apply a sterile drape around the surgical area.</li>
</ol>
4. Cardiomyocyte isolation procedure
<ol>
	<li>Expose the sternum of the mouse and, lateral to the midline, cut proximally through the ribs and to the axilla. Gently cut fully through the diaphragm, ensuring shallow cuts to avoid the heart. Clamp the sternum with a hemostat and fold the ribs backwards to expose the thoracic cavity.</li>
	<li>Gently remove the pericardium from the heart and cut fully through the inferior vena cava, immediately distal to the heart. Use a 3 mL syringe with a 27 G needle to quickly inject 3 mL of ice-cold clearing buffer into the right ventricle of the heart over 1 min.</li>
	<li>Gently hold the heart using tweezers and pull away from the body, exposing as much of the aorta as possible. Using a hemostat, clamp the ascending aorta, being careful not to clamp the atria, and excise the heart from the chest.</li>
	<li>Quickly transfer the clamped heart to the lid of a polypropylene Petri dish with approximately 10 mL of warm perfusion buffer. Place the clamped heart into the supporting platform and use a stabilized 27 G needle attached to the manifold to inject 10 mL of temperature-controlled 37 &#176;C perfusion buffer into the left ventricle over 5 min.</li>
	<li>Transfer the clamped heart and supporting platform to a Petri dish with approximately 5 mL of digestion buffer. Exchange input syringes for a 50 mL syringe with 25 mL of digestion buffer.</li>
	<li>Clear the manifold of any bubbles and remaining perfusion buffer before injection. Carefully replace the needle into the same needle position in the left ventricular apex.</li>
	<li>Use a perfusion pump to inject temperature-controlled 37 &#176;C digestion buffer into the left ventricle for 15 min using a 50 mL syringe. Control the temperature of the solution with a water jacket previously calibrated to eject 37 &#176;C solution at the end of the needle.</li>
	<li>Remove the ventricles from the atria and transfer to a 10 mL beaker. Add 3 mL of digestion buffer to the beaker and, using sharp scissors, cut the ventricles into large chunks. Cover the beaker with aluminum foil and place in a shaking water bath preheated to 37 &#176;C for 5 min.</li>
	<li>Discard the supernatant, being careful to avoid removing any chunks of tissue. Resuspend the tissue chunks in 3 mL of digestion buffer and triturate for approximately 4 min or until a homogenous mixture is achieved.</li>
	<li>Filter the cells through a 70 &#181;m nylon cell strainer into a 50 mL polypropylene tube.</li>
	<li>Return the filtrate to a 14 mL round-bottom polypropylene tube and centrifuge for 1 min at 100 rpm. Discard the supernatant and resuspend the pellet in 3 mL of stop buffer.</li>
	<li>Add 54 &#181;L of 100 mM CaCl2 stock to the resuspended cells for a final CaCl2 concentration of 1.8 mM. This step can also be performed stepwise to increase cell yield.</li>
	<li>Allow live cells to settle by gravity for 10 min before removing the supernatant containing dead cells. Resuspend the pellet in storage solution (perfusion solution with 200 &#181;M CaCl2). These cells can now be used for functional experiments (i.e., calcium imaging/contraction, patch clamp, etc.), culture, etc. 
	&#8203;NOTE: The cells may also be resuspended in lab- or experiment-dependent solutions.</li>
</ol>
5. Cell culture
<ol>
	<li>Count the cells using a hemocytometer or by a field view count using a gridded cover slip. As myocytes are very large, counting using a hemocytometer is not always accurate. This is used to get an idea of approximately how many cells were obtained from the isolation.</li>
	<li>Dilute the cells to ~25,000 cells/mL with DMEM media. Add 1 mL of cell solution to each well.</li>
	<li>Incubate at 37 &#176;C, 95% O2, 5% CO2 for 2 h.</li>
	<li>Gently aspirate DMEM media from each well and add 2 mL of M199 media.</li>
	<li>Incubate at 37 &#176;C, 95% O2, 5% CO2 for up to 24 h.</li>
</ol>

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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=Myocytes+isolated+from+rejecting+transplanted+rat+hearts+exhibit+a+nitric+oxide-mediated+reduction+in+the+calcium+current.">Myocytes isolated from rejecting transplanted rat hearts exhibit a nitric oxide-mediated reduction in the calcium current.</a> Journal of Molecular and Cellular Cardiology. 33 (9), 1691-1699 (2001).</li><li>Traynham, C. 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=Diesterified+nitrone+rescues+nitroso-redox+levels+and+increases+myocyte+contraction+via+increased+SR+Ca(2+)+handling.">Diesterified nitrone rescues nitroso-redox levels and increases myocyte contraction via increased SR Ca(2+) handling.</a> PLoS One. 7 (12), 52005 (2012).</li><li>Nixon, B. R., 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=Combined+troponin+I+Ser-150+and+Ser-23/24+phosphorylation+sustains+thin+filament+Ca(2+)+sensitivity+and+accelerates+deactivation+in+an+acidic+environment.">Combined troponin I Ser-150 and Ser-23/24 phosphorylation sustains thin filament Ca(2+) sensitivity and accelerates deactivation in an acidic environment.</a> Journal of Molecular and Cellular Cardiology. 72, 177-185 (2014).</li><li>Swager, S. 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=Claudin-5+levels+are+reduced+from+multiple+cell+types+in+human+failing+hearts+and+are+associated+with+mislocalization+of+ephrin-B1.">Claudin-5 levels are reduced from multiple cell types in human failing hearts and are associated with mislocalization of ephrin-B1.</a> Cardiovascular Pathology. 24 (3), 160-167 (2015).</li><li>Pinckard, K. 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=A+novel+endocrine+role+for+the+BAT-released+lipokine+12,13-diHOME+to+mediate+cardiac+function.">A novel endocrine role for the BAT-released lipokine 12,13-diHOME to mediate cardiac function.</a> Circulation. 143 (2), 145-159 (2021).</li><li>Roof, S. R., Shannon, T. R., Janssen, P. M., Ziolo, M. 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No conflicts of interest to disclose.

The principal advantage of our Langendorff-free cardiomyocyte isolation technique is that it limits hypoxia and ischemic time by not requiring cannulation to a Langendorff apparatus. Alternatively to classical Langendorff techniques that take several minutes to remove, clean, and hang the heart, often resulting in ischemic damage to the myocyte, our method includes an in vivo clearing of blood via an ice-cold clearing solution. The ice-cold clearing buffer contains ethylenediaminetetraacetic acid (EDTA)...

For decades, an essential idea in cardiac biology literature is the molecular mechanism of action. The mechanism of action must be established in order to publish reliable studies. A well-established strategy to determine molecular mechanism is isolated cardiomyocyte studies, which require high-quality cardiomyocytes for attaining trustable data. Cellular and molecular experiments performed on cardiomyocytes to determine mechanism of action are the gold standard for investigating contraction1, electrophysiology2, calcium (Ca2+) cycling3, myofilament Ca2+ sensitivity4, cytoskeleton5, metabolism6, effects of hormones7, signaling molecules8, drug studies9, etc. The mouse has become the species of choice for most cardiac biology experiments due to the ease of genetic manipulation, its small size, its relatively short lifespan, low cost, etc10. However, the reliable isolation of high-quality mouse cardiomyocytes is not trivial with current techniques.
Labs have been isolating cardiomyocytes for almost 70 years11. Virtually all techniques to isolate cardiomyocytes rely on digestion of the heart via various enzymes (collagenase, protease, trypsin, etc.). In the early periods (1950s-1960s), the chunk method was employed, which involved removing the heart, cutting into much smaller pieces and incubating in solution with collagenase/protease/trypsin12. In the 1970s labs implemented the ameliorated &#8220;Langendorff&#8221; method13, which isolated cardiomyocytes using a coronary artery perfusion-based isolation technique (retrograde perfusion with enzyme via the Langendorff apparatus); this technique remains the dominant method of myocyte isolation in the field today, ~50 years later14,15,16. Recent work has shifted to cannulating the heart in vivo to limit hypoxia time and ischemic damage resulting in superior cardiomyocyte isolations (better yields and higher quality)17. Recently, this has evolved into performing in vivo, Langendorff-free heart perfusions18,19,20,21,22. We have evolved the Langendorff-free cardiomyocyte isolation technique based on the Ackers-Johnson et al.18 technique and adapted various components from the many previous isolation techniques. These key adaptations include the injection of an ice-cold clearing buffer and the incorporation of a supporting platform to stabilize the needle, allowing for decreased manipulation of the heart. Also detailed in this technique is&#160; temperature control of injected buffers (37 &#176;C), which decreased the time between in vivo injection and digestion due to less EDTA perfusion as previously published18. By decreasing manipulation of the heart and therefore minimizing puncture site size, thorough and constant perfusion of the coronary arteries is obtained. We also refined the technique with a secondary chunk method digestion,&#160;the amount of EDTA in the injected clearing buffer, and changed the pH. Our described technique is more reliable, more efficient, and does not require the extensive training/practice compared to the using the Langendorff apparatus (Table 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 cc Bd Luer-Lok Syringe</td>
			<td>Fisher Sci</td>
			<td>14-827-52</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Pyrex Low-Form Beaker</td>
			<td>Cole-Palmer</td>
			<td>UX-34502-01</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL polypropylene cap glass media storage bottle</td>
			<td>DWK Life Sciences</td>
			<td>UX-34523-00</td>
			<td></td>
		</tr>
		<tr>
			<td>14 mL Round-Bottom Polypropylene Test Tubes With Cap</td>
			<td>Fisher Sci</td>
			<td>14-959-11B</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3-Butanedione Monoxime</td>
			<td>Sigma</td>
			<td>B0753</td>
			<td>&#62;98%</td>
		</tr>
		<tr>
			<td>3 cc BD Luer-Lok Syringe</td>
			<td>Fisher Sci</td>
			<td>14-823-435</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm glass bottom dishes</td>
			<td>MatTek Corporation</td>
			<td>P35G-1.0-20-C</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL BD Syringe without Needle</td>
			<td>Fisher Sci</td>
			<td>13-689-8</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Conical Centrifuge Tubes</td>
			<td>Cole-Palmer</td>
			<td>EW-22999-84</td>
			<td></td>
		</tr>
		<tr>
			<td>95% O2 5% CO2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AIMS Space Gel Heating Pad</td>
			<td>Fisher Sci</td>
			<td>14-370-223</td>
			<td></td>
		</tr>
		<tr>
			<td>BD PrecisionGlide 27 G X 1/2&#34; Hypodermic Needles</td>
			<td>Becton Dickinson</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A3803</td>
			<td>Heat shock fraction, lyophilized powder, essentially fatty acid free, &#62;98%</td>
		</tr>
		<tr>
			<td>Calcium Chloride dihydrate</td>
			<td>Sigma</td>
			<td>C7902</td>
			<td>&#62;99%</td>
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		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td>Suitable for cell culture, &#62;99.5%</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Fisher Sci</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Sci</td>
			<td>AAA1071336</td>
			<td></td>
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		<tr>
			<td>Falcon 100 mm TC-treated Cell Culture Dish</td>
			<td>Corning</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>R&#38;D Systems (Bio-techne)</td>
			<td>S11195</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Isotemp Heated Immersion Circulators</td>
			<td>Fisher Sci</td>
			<td>13-874-432</td>
			<td></td>
		</tr>
		<tr>
			<td>Hartman Mosquito Hemostatic Forceps</td>
			<td>World Precision Instruments</td>
			<td>15921</td>
			<td></td>
		</tr>
		<tr>
			<td>Hausser Scientific Hy-Lite Counting Chamber Set</td>
			<td>Fisher Sci</td>
			<td>02-671-11</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td>&#62;99.5%</td>
		</tr>
		<tr>
			<td>Labeling Tape</td>
			<td>Fisher Sci</td>
			<td>15-901-10R</td>
			<td></td>
		</tr>
		<tr>
			<td>Legato 100 Syringe Pump</td>
			<td>kdScientific</td>
			<td>788100</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutathione</td>
			<td>Fisher Sci</td>
			<td>ICN19467980</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase TH Research Grade</td>
			<td>Sigma</td>
			<td>5401135001</td>
			<td>High thermolysin concentration</td>
		</tr>
		<tr>
			<td>M199</td>
			<td>Fisher Sci</td>
			<td>MT10060CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Invitrogen</td>
			<td>AM9530G</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Laminin</td>
			<td>Corning</td>
			<td>354232</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Fisher Sci</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td>&#62;99%</td>
		</tr>
		<tr>
			<td>Precision Digital Reciprocating Water Bath</td>
			<td>ThermoFisher Scientific</td>
			<td>TSCIR19</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td>Suitable for cell culture</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma</td>
			<td>S5886</td>
			<td>&#62;99%</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>Sigma</td>
			<td>S5011</td>
			<td>&#62;99%</td>
		</tr>
		<tr>
			<td>Sterile Cell Strainer 70 &#181;m</td>
			<td>Fisher Sci</td>
			<td>22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>Student Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>91460-11</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Absorbent Underpads</td>
			<td>Fisher Sci</td>
			<td>NC9481815</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple and effective method to consistently isolate mouse cardiomyocytes

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.

There are a few elements to examine when determining the success of an isolation. First, the cardiomyocytes must be rod-shaped with no membrane blebs, such as the cells isolated in Figure 1. A typical isolation will yield ~80% of the myocytes being rod-shaped. If the isolation yields anything less than 50% rod-shaped cells, then it is considered an unsuccessful isolation and cardiomyocytes are not used. Lastly, the cardiomyocytes should be quiescent. Spontaneously contraction myocytes demons...

Watch this Scientific Journal Video about A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes at JoVE.com

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and ...

a-simple-effective-method-to-consistently-isolate-mouse

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

2.7K Views.  The Ohio State University. The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

Video: A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma (glioma) and medulloblastoma. The widely used stereotactic approach only allows for the injection of a single animal at a time, is labor intensive and requires highly specialized equipment. The guide screw method, initially developed by Lal et al.,1 was developed to eliminate cumbersome stereotactic procedures. We now describe a modified guide screw approach that is rapid and exceptionally safe; both of which are critical ethical considerations. Notably, our procedure now incorporates an infusion pump that allows up to 10 animals to be simultaneously injected with tumor cells.
To demonstrate the utility of this procedure, we established human U87MG glioma cells as intracranial xenografts in mice, which were then treated with AMG102; a fully human antibody directed to HGF/scatter factor currently undergoing clinical evaluation2-5. Systemic injection of AMG102 significantly prolonged the survival of all mice with intracranial U87MG xenografts and resulted in a number of complete cures. 
This study demonstrates that the guide screw method is an inexpensive, highly reproducible approach for establishing intracranial xenografts. Furthermore, it provides a relevant physiological model for validating novel therapeutic strategies for the treatment of brain cancers.

In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches.

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

A Modified Precipitation Method to Isolate Urinary Exosomes

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. Urinary exosome vesicles, 40-100 nm in size, are released into the urine under normal conditions by cells from all nephron segments and may contain protein, mRNA and microRNA representative of their cell type of origin. Under conditions of renal dysfunction or injury, exosomes may contain altered proportions of these components, which may serve as biomarkers for disease. There are currently several methods available for isolation of urinary exosomes, and we have previously conducted an experimental comparison of each of these approaches, including three based on ultracentrifugation, one using a nanomembrane ultrafiltration concentrator, one using a commercial precipitation reagent and one using a modification of the precipitation technique using ExoQuick reagent that we developed in our laboratory. We found the modified precipitation method produced the highest yield of exosome particles, miRNA, and mRNA, making this approach suitable for the isolation of exosomes for subsequent RNA profiling. We conclude that the modified exosome precipitation method offers a quick, scalable, and effective alternative for the isolation of exosomes from urine. In this report, we describe our modified precipitation technique using ExoQuick reagent for isolating exosomes from human urine.

This manuscript describes a protocol for isolating exosomes from human urine using a modified precipitation technique.

Identification of biomarkers that allow early detection of kidney diseases in urine and plasma has been an area of active interest for several years. ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card,&#160;tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with conjunctiva. Patients suffer from recurrent corneal defects, pain, inflammation, and loss of vision.
Previously, a murine model of LSCD was described and compared to two other models. The goal was to produce a consistent mouse model of LSCD that both mimics the phenotype in humans and lasts long enough to make it possible to study the disease pathophysiology and to evaluate new treatments. Here, the technique is described in more detail.
A motorized tool with a rotating burr has been designed to remove the rust rings from the corneal surface or to smooth the pterygium bed in patients. It is a suitable device to create the desired LSCD model. It is a readily available, easy-to-use tool with a fine tip that makes it appropriate for working on small eyes, as in mice. Its application prevents unnecessary trauma to the eye and it does not result in unwanted injuries, as often is the case with chemical injury models. As opposed to a blunt scraper, it removes the epithelium with the basement membrane. In this protocol, the limbal area was abraded two times, and then the whole corneal epithelium was shaved from limbus to limbus. To avoid stroma injury, care was taken not to brush the corneal surface once the epithelium was already removed.

The following article provides an easy, reproducible technique to effectively create a sustainable mouse model of limbal stem cell deficiency (LSCD). This animal model is useful in testing and comparing the efficacy of treatments for limbal stem cell diseases.

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with ...

Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, pancreatitis, and pancreatic cancer. All three of these diseases mark active areas of study, not only to develop immediate therapy, but also to better understand their pathophysiology. There are few tools to further these areas of study. Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. The technique requires the intricate dissection of the second portion of the duodenum and ampulla, followed by the occlusion of the bile duct and the cannulation of the pancreatic duct. Although the technique is technically challenging at first, the applications are myriad. Ambiguity in the specifics of the procedure between groups highlighted the need for a standard protocol. This work describes the expression of a green fluorescent protein (GFP) within the pancreas after the pancreatic duct infusion of a viral vector expressing GFP versus a sham surgery. The infusion and therefore expression is specific to the pancreas, without expression present in any other tissue type.

Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. A pancreatic duct infusion technique for drug and viral delivery to pancreatic cells is presented here.

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS images are constructed from calibrated freehand 2D B-mode ultrasound images, which are positioned into a voxel array. Ultrasound (US) imaging allows quantification of muscle size, fascicle length, and angle of pennation. These morphological variables are important determinants of muscle force and length range of force exertion. The presented protocol describes an approach to determine volume and fascicle length of m.&#160;vastus lateralis and m.&#160;gastrocnemius medialis. 3DUS facilitates standardization using 3D anatomical references. This approach provides a fast and cost-effective approach for quantifying 3D morphology in skeletal muscles. In healthcare and sports, information on the morphometry of muscles is very valuable in diagnostics and/or follow-up evaluations after treatment or training.

3D ultrasound imaging (3DUS) allows fast and cost-effective morphometry of musculoskeletal tissues. We present a protocol to measure muscle volume and fascicle length using 3DUS.

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS ...

Isolation and Cultivation of Adult Rat Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).
The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.
Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are &#946;-myosin heavy chain (&#946;-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

Here, we present a protocol for the isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). Isolated ARVC can be used for short and long-term cultivation. The isolation and cultivation of ARVC can play a key role in developing new treatment regimens for cardiac diseases.