No funding was received for this study

Animals received humane care in compliance with the Guide for the Principles of Laboratory Animals, prepared by the Institute of Laboratory Animal Resources, and published by the National Institutes of Health. All animal protocols were approved by the responsible local authority (&#8216;&#8216;Amt f&#252;r Gesundheit und Verbraucherschutz, Hansestadt Hamburg&#8217;&#8217;/ Animal protocol # 109/16).
1. Obtain Animals
<ol>
	<li>Commercially obtain female Guinea pigs weighing 500&#8211;600 g.</li>
	<li>House them under conventional conditions in animal cages. Feed standard rat chow and autoclaved water ad libidum.</li>
</ol>
2. Transthoracic Echocardiography
<ol>
	<li>Place the guinea pig in an induction chamber and anesthetize the animal with isoflurane (2&#8211;3%). Check the depth of anesthesia by lack of response to the toe-pinch.&#160;</li>
	<li>Place the anesthetized Guinea pig on a warming platform (40&#8211;42 &#176;C) in a supine position. Continue anesthesia via a nose cone (isoflurane 1.5-2%)</li>
	<li>Shave and depilate the guinea pig&#180;s thorax using an electric animal hair shaver.</li>
	<li>Apply prewarmed (~25 &#176;C) ultrasound transducer gel. Use an echocardiography system that is equipped with a transducer frequency higher than 15 MHz.</li>
	<li>Acquire two-dimensional parasternal long axis views by placing the transducer on the guinea&#180;s pig thorax facing from the right neck towards the left leg and record long axis B-mode images at the plane of the aortic valve with a concurrent visualization of the LV apex. Investigate the pre-operative left-ventricular function.</li>
	<li>Turn the transducer by 90&#176; degrees to obtain a short axis B-mode view at the mid-papillary level. 
	NOTE: The animal is then immediately transferred to the OR table. The animal is continuously anesthetized with 3% isoflurane. 0.05 mg/kg atropine (i.m.) is injected to avoid increased bronchial secretion during mechanical ventilation.</li>
</ol>
3. Surgery
<ol>
	<li>Induction of Myocardial infarction
	<ol>
		<li>Inject 4&#8211;5 mg/kg carprofen and 0.05 mg/kg buprenorphine for analgesia&#160;subcutaneously with a 21 G needle and a 10 mL syringe.&#160;0.5 mg/kg Atropine is injected subcutaneously with a 21 G needle and a 10 mL syringe.</li>
		<li>Place the guinea pig on its back and keep anesthesia with a facemask covering mouth and nose. Check the depth of anesthesia by pinching the hind feet (lack of pedal reflex).</li>
		<li>Spread the guinea pigs&#8217; legs and fix the position using tape.</li>
		<li>Shave the chest and the tracheal region of the anesthetized animal with an electric shaver. Disinfect the area widely using iodine-based scrub, followed by 80% ethanol. Repeat this disinfection steps twice.</li>
		<li>Perform a 1.5 cm vertical incision in the tracheal area and bluntly dissect the muscles covering the trachea until you see the trachea. Puncture the trachea with 18 G i.v. cannula and insert the flexible part of the cannula as a tracheal tube.</li>
		<li>Connect the tracheal tube to an animal respirator to continuously ventilate the guinea pig during the procedure. 
		NOTE: Now the anesthesia is maintained with isoflurane 3% via the tracheal tube (inspiration assisted ventilation with maximum inspiration pressure, respiration rate: 100&#8211;120/min, peak inspiration pressure: 18-22 cm Hg using PEEP-ventilation while the chest is open).</li>
		<li>Identify the 5th intercostal space by counting the rib spaces beginning at the first intercostal space. Perform a 2 cm horizontal incision on the 5th intercostal space on the left side of the guinea pig using scissors and a tweezer. Insert a small animal retractor. Dissect the muscles with an electrocautery until the intercostal muscles are reached, which can be seen after removing the subcutaneous tissue, are reached.</li>
		<li>Gently dissect the intercostal muscles with tweezers until the pleural space is reached and one can see the left lung in front. Insert the retractor between the ribs and open it carefully until a good view of the heart is obtained.</li>
		<li>Open the pericardium approximately 1 cm in the region of the anterior left ventricular wall with scissors. Place a compress on the left lung to protect it from damage when inducing the cryoinjury of the left ventricle.</li>
		<li>Place the tip of an eroded metal stamp (aluminum)&#160;with a cross-sectional diameter of 0.5 cm into liquid nitrogen for 3 min.</li>
		<li>Press the nitrogen cooled probe onto the left anterior wall of the heart for 30 s. Then separate it from the heart using an electric soldering iron (250 &#176;C) which is placed inside the stamp to warm it up. Repeat this procedure 3 times to obtain a transmural myocardial injury. Observe the blanching of the myocardium.</li>
		<li>Inflate the lungs with maximum pressure (by clamping the outflow tube of the ventilator for 2 s), to avoid atelectasis of the lung. Remove the retractor from the intercostal space.</li>
		<li>Close the ribs with two 3-0 sutures. Close the muscles over the ribs with a 4-0 running suture. For closure of the skin use 5-0 suture single stitches.</li>
		<li>Reduce the isoflurane to 1%. When the animal is breathing spontaneously, remove the tracheal tube, and continue anesthesia with a facemask (isoflurane 2&#8211;3%).</li>
		<li>Assure the absence of reflexes by pinching the hind limb to monitor sufficient depth of anesthesia. Then use a single 8-0 suture to close the puncture site at the trachea. Close the wound with three single stich 4-0 sutures.</li>
		<li>Use buprenorphine (0.05 mg/kg per 12 h) and carprofen (5 mg/kg per 24 h) for pain medication for the following 5 days.</li>
	</ol>
	</li>
	<li>Implantation of EHT (7 days after cryoinjury)
	<ol>
		<li>Place the guinea pig in an induction chamber and anesthetize the animal with isoflurane Check the depth of anesthesia by the lack of response to the toe-pinch.</li>
		<li>Inject 4&#8211;5 mg/kg carprofen and 0.05 mg/kg buprenorphine subcutaneously with a 21 G needle and a 10 mL syringe after induction of anesthesia. Place the guinea pig on its back and keep anesthesia with a facemask covering mouth and nose. 
		NOTE: Sufficiency of anesthesia should be checked by pinching the hind feet.</li>
		<li>Spread the guinea pig&#8217;s legs and fix the position using tape.</li>
		<li>Perform the pre-surgical preparations as described in step 3.1.4&#8211;3.1.6. Perform a 2 cm horizontal skin incision in the scar area of the left lateral side using scissors and tweezers.</li>
		<li>Gently dissect the extrapleural adhesions using an electrocautery. Carefully open the pleural space with scissors. Insert a rib spreader to expose the heart.</li>
		<li>Visually identify the region of the infarction by its pale color in comparison to the healthy surrounding myocardium. Place the engineered heart tissue patch over the infarction region.</li>
		<li>Secure it with two 8-0 sutures at both sides. Make sure to secure the patch in the non-infarcted area of the heart (Figure 1). Inflate the lungs with pressure, to avoid atelectasis of the lung. Remove the retractor from the intercostal space.</li>
		<li>Close the ribs with two 3-0 sutures. Close the muscles over the ribs with a 4-0 running suture. For closure of the skin use 5-0 suture single stitches.</li>
		<li>Reduce the isoflurane to 1%. When the animal is breathing spontaneously, remove the tracheal tube, and continue anesthesia with a facemask (isoflurane 2&#8211;3%).</li>
		<li>Assure the absence of reflexes by pinching the hind limb to monitor sufficient depth of anesthesia. Then use a single 8-0 suture to close the puncture site at the trachea. Close the wound with three single stitch 4-0 sutures.</li>
		<li>Use buprenorphine (0.05 mg/kg per 12 h) and carprofen (5 mg/kg per 24 h) for pain medication for the following 5 days.</li>
		<li>Four weeks after EHT implantation perform a transthoracic echocardiography (as described in step 2) to monitor LV function over time. 
		NOTE: The LV function is monitored before EHT implantation and 4 weeks after implantation to monitor for improvement of cardiac function by EHT implantation</li>
		<li>Euthanize the animal which IACUC approved protocol and surgically explant the heart for further analysis.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Kirklin, J. K., 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=Seventh+INTERMACS+annual+report:+15,000+patients+and+counting.">Seventh INTERMACS annual report: 15,000 patients and counting.</a> The Journal of heart and lung transplantation: the official publication of the International Society for Heart Transplantation. 34, 1495-1504 (2015).</li><li>Soonpaa, M. H., Field, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+of+studies+examining+mammalian+cardiomyocyte+DNA+synthesis.">Survey of studies examining mammalian cardiomyocyte DNA synthesis.</a> Circulation research. 83, 15-26 (1998).</li><li>Shiba, Y., Hauch, K. D., Laflamme, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+applications+for+human+pluripotent+stem+cells.">Cardiac applications for human pluripotent stem cells.</a> Current pharmaceutical design. 15, 2791-2806 (2009).</li><li>Shiba, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+ES-cell-derived+cardiomyocytes+electrically+couple+and+suppress+arrhythmias+in+injured+hearts.">Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts.</a> Nature. 489, 322-325 (2012).</li><li>Chong, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic-stem-cell-derived+cardiomyocytes+regenerate+non-human+primate+hearts.">Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts.</a> Nature. 510, 273-277 (2014).</li><li>Kattman, S. 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=Stage-specific+optimization+of+activin/nodal+and+BMP+signaling+promotes+cardiac+differentiation+of+mouse+and+human+pluripotent+stem+cell+lines.">Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines.</a> Cell stem cell. 8, 228-240 (2011).</li><li>Watanabe, T., Rautaharju, P. M., McDonald, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventricular+action+potentials,+ventricular+extracellular+potentials,+and+the+ECG+of+guinea+pig.">Ventricular action potentials, ventricular extracellular potentials, and the ECG of guinea pig.</a> Circulation research. 57, 362-373 (1985).</li><li>Weinberger, F., 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=Cardiac+repair+in+guinea+pigs+with+human+engineered+heart+tissue+from+induced+pluripotent+stem+cells.">Cardiac repair in guinea pigs with human engineered heart tissue from induced pluripotent stem cells.</a> Science translational medicine. 8, 363ra148 (2016).</li><li>Shiba, Y., 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=Electrical+Integration+of+Human+Embryonic+Stem+Cell-Derived+Cardiomyocytes+in+a+Guinea+Pig+Chronic+Infarct+Model.">Electrical Integration of Human Embryonic Stem Cell-Derived Cardiomyocytes in a Guinea Pig Chronic Infarct Model.</a> Journal of Cardiovascular Pharmacology and Therapy. 19, 368-381 (2014).</li><li>van Laake, L. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+cardiomyocytes+survive+and+mature+in+the+mouse+heart+and+transiently+improve+function+after+myocardial+infarction.">Human embryonic stem cell-derived cardiomyocytes survive and mature in the mouse heart and transiently improve function after myocardial infarction.</a> Stem cell research. 1, 9-24 (2007).</li><li>Zimmermann, W. H., 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=Engineered+heart+tissue+grafts+improve+systolic+and+diastolic+function+in+infarcted+rat+hearts.">Engineered heart tissue grafts improve systolic and diastolic function in infarcted rat hearts.</a> Nature medicine. 12, 452-458 (2006).</li><li>Johns, T. N., Olson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+myocardial+infarction.+I.+A+method+of+coronary+occlusion+in+small+animals.">Experimental myocardial infarction. I. A method of coronary occlusion in small animals.</a> Annals of surgery. 140, 675-682 (1954).</li><li>van den Bos, E. J., Mees, B. M., de Waard, M. C., de Crom, R., Duncker, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+model+of+cryoinjury-induced+myocardial+infarction+in+the+mouse:+a+comparison+with+coronary+artery+ligation.">A novel model of cryoinjury-induced myocardial infarction in the mouse: a comparison with coronary artery ligation.</a> Heart and circulatory physiology. , H1291-H1300 (2005).</li></ol>

None of the authors has competing financial interests or other conflicts of interest to declare.

A variety of small animal models are available to study the effect that cell transplantation exerts on injured hearts9,10,11. We chose a guinea pig model because of all small animal models its (electro)physiology resembles most closely that of humans. The advantages of small animal models are simple housing, manageable costs, and few workforces. In comparison with mice and rats, guinea pigs&#180; cardiac (electro)physiology is m...

The number of patients with heart failure is increasing in our aging population. For end-stage heart failure, orthotopic heart transplantation is the only curative treatment option. However, especially in European countries, there is an increasing organ donor shortage. Therefore, alternative treatment options are necessary. Recent achievements in mechanical circulatory support are promising, but especially in the long-term run, limited by its side effects like bleeding, pump thrombosis and infectious complications1.
The endogenous regeneration capacity of the adult human heart is extremely limited. Therefore, cardiac regeneration therapies might become an alternative treatment option for end-stage heart failure patients2,3. Different techniques including stem cell-based cell injection or tissue engineering approaches have been described3,4,5.
Human induced pluripotent stem cells (hiPSC), as well as human embryonic stem cells (hESC) can be effectively differentiated to spontaneously beating human cardiomyocytes6, which has been a major achievement in the field of cardiac regenerative therapies.
To replace myocardium after a myocardial infarction and to improve the function of a failing heart, survival of a suitable number of cardiomyocytes and their mechanical and electrical coupling with the native heart is essential. To investigate the potential of cardiac regenerative therapies with human iPS cell derived cardiomyocytes, a suitable research model is needed. The ideal model should be cost-effective and have a human-like physiology and electrophysiology. Large animal models like pigs would be ideal from that point of view, however, those experiments are very expensive and large amounts of cardiomyocytes would be necessary to replace a relevant number of cardiomyocytes in order to see effects on the left ventricular function in a pig infarction model.
To answer elementary biological questions towards human cell-based cardiac regeneration, e.g., cell survival, vascularization, and electrical coupling, small animal models are more suitable. From the available small animal models, the guinea pig is the most useful species, as compared to rats and mice, as their electrophysiology more closely resembles the situation in humans7. In this guinea pig model, we induced a transmural cryoinjury of the left ventricle. One week after induction of myocardial infarction implantation of a three-dimensional, spontaneously beating hiPS-cell derived cardiomyocyte patch was performed. Cardiomyocyte cell survival was evaluated 28 days after implantation by histological examination.

<table><tbody><tr><td>Ventilator (VetFlo Dual Mode)</td><td>Kent Scientific</td><td></td><td></td></tr><tr><td>Forene</td><td>abbvie</td><td>1000009819</td><td></td></tr><tr><td>Carprofen</td><td>Zoetis</td><td>256692</td><td></td></tr><tr><td>Atropin</td><td>Braun</td><td>PZN 00648037</td><td></td></tr><tr><td>Buprenorphin</td><td>Sigma</td><td></td><td></td></tr><tr><td>Metal stamp</td><td></td><td></td><td></td></tr><tr><td>Electric soldering iron</td><td>Claytools</td><td></td><td></td></tr><tr><td>3-0 prolene suture</td><td>Ethicon</td><td></td><td></td></tr><tr><td>4-0 prolene suture</td><td>Ethicon</td><td>662SLH</td><td></td></tr><tr><td>5-0 prolene suture</td><td>Ethicon</td><td>8710H</td><td></td></tr><tr><td>8-0 prolene suture</td><td>Ethicon</td><td>8841H</td><td></td></tr><tr><td>Tungsten Carbide Scissor&amp;nbsp;</td><td>FST</td><td>No. 14568-12</td><td></td></tr><tr><td>Stainless sterilization Container</td><td>FST</td><td>No. 20890-51</td><td></td></tr><tr><td>Graefe Forceps</td><td>FST</td><td>No.11652-10</td><td></td></tr><tr><td>Extra fine Graefe Forceps</td><td>FST</td><td>No.11150-10</td><td></td></tr><tr><td>Forceps</td><td>FST</td><td>No. 11022-15</td><td></td></tr><tr><td>Halsted- Mosquito</td><td>FST</td><td>No. 13009-12</td><td></td></tr><tr><td>Forceps</td><td>FST</td><td>No.13003-10</td><td></td></tr><tr><td>Baby Mixter</td><td>FST</td><td>No. 13013-14</td><td></td></tr><tr><td>Needle holder (Castroviejo with Tungsten Casbide Jaws)&amp;nbsp;</td><td>FST</td><td>No. 12565-14</td><td></td></tr><tr><td>Needle Holder (Halsey)</td><td>FST</td><td>No. 12501-13</td><td></td></tr><tr><td>Alm Retractor with Blumt Teeth</td><td>FST</td><td>No. 17008-07</td><td></td></tr><tr><td>Spring Scissor</td><td>FST</td><td>No. 15000-00</td><td></td></tr><tr><td>Compress 5x5</td><td>Fink + Walter</td><td>PZN 08821417</td><td></td></tr><tr><td>Venflon Pro Safety</td><td>Becton Dickinson</td><td>PZN11123964</td><td></td></tr><tr><td>Cautery High Temp 2&quot;</td><td>Bovie Medical Corporation</td><td>0100607151011055</td><td></td></tr></tbody></table>

implantation of hipsc-derived cardiac-muscle patches after myocardial injury in a guinea pig model

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss of relevant amounts of heart muscle mass can lead to the heart failure. Besides heart transplantation, there is no curative treatment option for the end-stage heart failure. In times of organ donor shortage, organ independent treatment modalities are needed. Left-ventricular assist devices are a promising therapy option, however, especially as destination therapy, limited by its side-effects like stroke, infections and bleedings. In recent years, several cardiac repair strategies including stem cell injection, cardiac progenitors or myocardial tissue engineering have been investigated. Recent improvements in cell biology allow for the differentiation of large amounts of cardiomyocytes derived from human induced pluripotent stem cells (iPSC). One of the cardiac repair strategies currently under evaluation is to transplant artificial heart tissue. Engineered heart tissue (EHT) is a three-dimensional in vitro created cardiomyocyte network, with functional properties of native heart tissue. We have created EHT-patches from hiPSC derived cardiomyocytes. Here we present a protocol for the induction of left ventricular myocardial cryoinjury in a guinea pig, followed by implantation of hiPSC derived EHT on the left ventricular wall.

This guinea pig model is a suitable model to investigate cardiac regeneration after implantation of hiPSC derived EHT-patches. It reproducibly leads to large transmural myocardial injuries. Scar size is evaluated by histology four weeks after cryoinjury. Mason trichrome staining reveals large transmural scars (Figure 2). Scar size was similar over a large number of injured animals reflecting a high degree of reproducibility8. On average 25% of the left ventricular myo...

Watch this Scientific Journal Video about Implantation of hiPSC-derived Cardiac-muscle Patches after Myocardial Injury in a Guinea Pig Model at JoVE.com

Implantation of hiPSC-derived Cardiac-muscle Patches after Myocardial Injury in a Guinea Pig Model

Here we present a protocol for the induction of left ventricular cryoinjury followed by the implantation of a cardiac muscle patch, derived from human iPS-cell cardiomyocytes in a guinea pig model.

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss ...

implantation-hipsc-derived-cardiac-muscle-patches-after-myocardial

Research

JoVE Journal

Medicine

7.2K Views.  University Heart Center Hamburg. Het doel van de methode is om de functionele gevolgen van gemanipuleerde hartweefselimplantatie in een klein cryoinfarctmodel te evalueren. Het voordeel van deze methode is dat het zorgt voor de inductie van relatief reproduceerbare hartinfarcten met stabiele infarct maten. Na bevestiging van een gebrek aan reactie op teen knijpen, plaats de verdoofde cavia op een 40 tot 42 graden Celsius opwarming platform in de supine positie.Breng oogzalf aan en scheer en ontpil de thorax. Breng 25 ...

Video: Implantation of hiPSC-derived Cardiac-muscle Patches after Myocardial Injury in a Guinea Pig Model

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), and smooth muscle cells (SMCs) are often limited by low yield, purity, and/or poor phenotypic stability. Here, we present novel protocols for generating hiPSC-CMs, -ECs, and -SMCs that are substantially more efficient than conventional methods, as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells, by sealing the needle track to prevent the cells from being squeezed out of the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury, the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch, and treatment with both the cells and the patch, but not with the cells alone, was associated with significant improvements in cardiac function and infarct size.

We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine delivery.

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

Developmental Biology

Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

Cardiac cell therapy has gained increasing interest and implantation of biomaterials associated with cells has become a major issue to optimize myocardial cell delivery. Rodent model of myocardial infarction (MI) consisting of Left Anterior Descending Artery (LAD) ligation has commonly been performed via a thoracotomy; a second open-heart surgery via a sternotomy has traditionally been performed for epicardial application of the treatment. Since the description of LAD ligation model, post-surgery mortality rate has dropped from 35-13%, however the second surgery has remained critical. In order to improve post-surgery recovery and reduce pain and infection, minimally invasive surgical procedures are presented.&#160;Two thoracotomies were performed, the initial one for LAD ligation and the second one for treatment epicardial administration. Biografts consisting of cells associated with solid or gel type matrices were applied onto the infarcted area.&#160;LAD ligation resulted in loss of heart function as confirmed by echocardiography performed after 2 and 6 weeks. Goldner trichrome staining performed on heart sections confirmed transmural scar formation. First and second surgeries resulted in less that 10%&#160;post-operative mortality.&#160;

Implantation of a biograft to treat myocardial infarction induced by LAD ligation in a rodent model has conventionally required two open-heart surgeries. In order to reduce mortality and provide optimal conditions for fixation of solid and gelatinous biomatrices associated with cells, minimally invasive procedures have been developed.

Cardiac cell therapy has gained increasing interest and implantation of biomaterials associated with cells has become a major issue to optimize myocardial ...

Bioengineering

Intramyocardial Cell Delivery: Observations in Murine Hearts

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.
Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe.
Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load.

Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents.

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue ...

LAD-Ligation: A Murine Model of Myocardial Infarction

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.
Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.
The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

Biology

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

Sterile Pericarditis in Aachener Minipigs As a Model for Atrial Myopathy and Atrial Fibrillation

Atrial fibrillation (AF) is the most common arrhythmia caused by structural remodeling of the atria, also called atrial myopathy. Current therapies only target the electrical abnormalities and not the underlying atrial myopathy. For the development of novel therapies, a reproducible large animal model of atrial myopathy is necessary. This paper presents a model of sterile pericarditis-induced atrial myopathy in Aachener minipigs. Sterile pericarditis was induced by spraying sterile talcum and leaving a layer of sterile gauze over the atrial epicardial surface. This led to inflammation and fibrosis, two crucial components of the pathophysiology of atrial myopathy, making the atria susceptible to the induction of AF. Two pacemaker electrodes were positioned epicardially on each atrium and connected to two pacemakers from different manufacturers. This strategy allowed for repeated non-invasive atrial programmed stimulation to determine the inducibility of AF at specified time points after surgery. Different protocols to test AF inducibility were used. The advantages of this model are its clinical relevance, with AF inducibility and the rapid induction of inflammation and fibrosis-both present in atrial myopathy-and its reproducibility. The model will be useful in the development of novel therapies targeting atrial myopathy and AF.

We describe a sterile pericarditis model in minipigs to study atrial myopathy and atrial fibrillation (AF). We present surgical and anesthetic techniques, strategies for vascular access, and a protocol to study the inducibility of AF.

Atrial fibrillation (AF) is the most common arrhythmia caused by structural remodeling of the atria, also called atrial myopathy. Current therapies only ...

Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising cell source for cardiac repair after ischemic damage. However, a low grafted rate is a significant obstacle for effective cardiac tissue regeneration after transplantation. This protocol shows that multiple hiPSC-CM ultrasound-guided percutaneous injections into an MI area effectively increase cell transplantation rates. The study also describes the entire hiPSC-CM culture process, pretreatment, and ultrasound-guided percutaneous delivery methods. In addition, the use of human mitochondrial DNA help detect the absence of hiPSC-CMs in other mouse organs. Lastly, this paper describes the changes in cardiac function, angiogenesis, cell size, and apoptosis at the infarcted border zone in mice 4 weeks after cell delivery. It can be concluded that echocardiography-guided percutaneous injection of the left ventricular myocardium is a feasible, relatively invasive, satisfactory, repeatable, and effective cellular therapy.

Ultrasound-guided cell delivery around the site of myocardial infarction in mice is a safe, effective, and convenient way of cell transplantation.

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem ...

Implantation of hiPSC-derived Cardiac-muscle Patches after Myocardial Injury in a Guinea Pig Model

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