The authors thank Prof Hendrik Tevaearai and the Department of Cardiovascular Surgery for financial support for the initial part of the study.

NOTE: Lewis Male and Female rats, 200-220 g were housed under standard laboratory conditions (12 hr light and dark cycle, ad libitum water and food, IVC cage). All animals were treated in compliance with the recommendations of the FELASA and the Swiss Law on animal protection.
1. Cell Preparation: Mesenchymal Stem Cell Isolation from Bone Marrow
<ol>
	<li>Anesthetize the rats with isoflurane 5% and O2 5 L/min in an induction chamber for 5 min. Place the animal&#8217;s snout into a nose cone connected to the anesthesia system. Perform a toe or tail pinch to confirm sedation.</li>
	<li>Remove the skin from the leg with scissors by cutting the skin from ankle to the hip. Remove the muscles and cut the femoral artery for animal exsanguination. Expose the hip joint and dislocate the femur head. It is important not to damage the femur head. Place the bones in sterile PBS in a 50 ml plastic tube.</li>
	<li>Remove all muscles and ligaments from the bones without breaking them. NOTE: It is important that the bone remains intact to avoid linkage of flushing solution or contamination with ethanol. Rinse the peeled bones in 70% ethanol for 5 min.</li>
	<li>Under the sterile laminar flow hood, cut both tips of the bones with scissors. Flush the bone marrow by injecting sterile PBS from the bone extremity. Collect marrow into a 15 ml tube. Centrifuge 7 min at 300 x g.</li>
	<li>Remove the supernatant. Suspend the pellet in 3 ml of Red Blood Cell Lysis buffer. Incubate the suspension for 1 min at room temperature before spinning again for 7 min at 300 x g. In a 150 ml culture flask, add sterile cell culture medium (395 ml IMDM medium, 5 ml pen/strep, 100 ml FBS) and seed the cells. </li>
	<li>Change medium the second day to remove nonadherent cells.
	<ol>
		<li>Perform medium changes every second day for 2-3 weeks until desired cell quantity is obtained.</li>
		<li>Alternatively, prepare the biograft by seeding the cells onto the solid matrix as needed and described elsewhere3.</li>
	</ol>
	</li>
	<li>The day of cardiac implantation, prepare the cells or harvest the biograft just before epicardial application.
	<ol>
		<li>Collect the cells using Accutase cell detachment solution. Count the cells. Fill a 1.5 ml centrifuge tube with the volume of cell suspension calculated to obtain desired cell quantity. Centrifuge for 7 min at 300 x g and remove the supernatant.</li>
		<li>Alternatively, harvest the biograft from cell culture medium, rinse in sterile PBS, and keep in fresh culture medium without serum.</li>
	</ol>
	</li>
</ol>
2. First Thoracotomy and LAD Ligation
<ol>
	<li>Weigh the rat. Turn on the heating pad at 37 &#176;C. Anesthetize the rat with 5% isoflurane and 5 L/min 100% O2 in an induction chamber for 5-7 min. Perform a toe or tail pinch to confirm sedation. Place the rat on the heating pad.</li>
	<li>Intubate the animal with a 14 G i.v. catheter. Connect the intubation catheter to a rodent ventilator programmed for 2.5 L/min oxygen, 2.5% isoflurane, tidal volume of 2 ml, and a breathing frequency of 90 breaths/min.</li>
	<li>Prepare a buprenorphine solution at 0.1 mg/kg. Inject subcutaneously one third of the solution. Shave the left part of the thorax. Disinfect with a 1% Betadine solution.</li>
	<li>Incise the skin perpendicular to the sternum at the fourth intercostal space. Separate the 3 layers of thoracic muscle (pectoralis major, ascending pectoralis, and external oblique pectoralis). Open the fourth intercostal space (between ribs 4 and 5).</li>
	<li>Use a small retractor to spread the ribs and to expose the heart. Open the pericardium carefully. Locate and ligate the left anterior descending coronary artery (LAD) with a 7.0 suture 4 mm below the atrium.</li>
	<li>Close the intercostal space with two stitches using 3.0 sutures. Position the two sutures proximally and distally from the sternum. First, tighten the distal suture. Clamp the ventilator exhaust tubing for 2 sec in order to inflate the chest and avoid any pneumothorax. Tighten the second suture. Position the muscles layers back in place (no sutures are needed). Close the skin with a 5.0 suture.</li>
	<li>Disinfect the suture with 1% Betadine solution. Inject the rest of the buprenorphine solution. Turn off the anesthesia system. Remove intubation catheter.</li>
	<li>Keep the rat in the cage under a warm Lamp for 1-2 hr. Keep a thermometer in the cage and control the distance between the warm lamp and the cage in order to avoid over heating. Return the rat in the IVC unit. Inject subcutaneously Buprenorphine 0.05-0.1 mg/kg post &#8211;operatively every 6-12 hr for 48 hr.</li>
</ol>
3. Epicardial Administration of the Treatment Via a Second Thoracotomy
<ol>
	<li>Repeat steps 2.1-2.3.</li>
	<li>Incise the skin perpendicular to the sternum at the fifth intercostal space. Separate the 3 layers of thoracic muscle (pectoralis major, ascending pectoralis and external oblique pectoralis). Open the fifth intercostal space (between ribs 5 and 6).</li>
	<li>Use a small retractor to spread the ribs and to expose the heart. If there is some adherence, carefully remove them with fine forceps.</li>
	<li>Locate the infarct area that appears as a pale area below the ligature. NOTE: When necessary, use a 10 cm piece of suture (7.0) inserted at the apex to better visualize the left ventricle. Pull gently to the suture maintained loosely by a clamp to expose the heart.</li>
	<li>Apply one of the following treatments:
	<ol>
		<li>Position the biograft at the surface of the heart. Use the prefilled fibrin sealant syringe prewarmed at room temperature for 10 min. Apply a drop (50-100 &#956;l) of fibrin sealant under the biograft using a 25 G Luer needle. Verify that the biograft is perfectly sealed.</li>
		<li>Alternatively, apply the cell pellet collected with a pipette tip at the surface of the heart onto the pale area below the ligature. Apply a drop (50-100 &#956;l) of fibrin sealant onto the cell pellet.</li>
	</ol>
	</li>
	<li>Remove the piece of suture from the apex.</li>
	<li>Repeat steps 2.6-2.8.</li>
</ol>

<ol>
	<li>Gjesdal, O., Bluemke, D. A., Lima, J. 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+remodeling+at+the+population+level--risk+factors,+screening,+and+outcomes.">Cardiac remodeling at the population level--risk factors, screening, and outcomes.</a> Nat. Rev. Cardiol. 8, 673-685 (2011).</li><li>Alcon, A., Cagavi Bozkulak, E., Qyang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerating+functional+heart+tissue+for+myocardial+repair.">Regenerating functional heart tissue for myocardial repair.</a> Cell Mol. Life Sci. 69, 2635-2656 (2012).</li><li>Guex, A. G., Fortunato, G., Hegemann, D., Tevaearai, H. T., Giraud, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+Protocol+for+the+Culture+of+Cells+on+Plasma-Coated+Electrospun+Scaffolds.">General Protocol for the Culture of Cells on Plasma-Coated Electrospun Scaffolds.</a> Methods Mol. Biol. 1058, 119-131 (2013).</li><li>Selye, H., Bajusz, E., Grasso, S., Mendell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+techniques+for+the+surgical+occlusion+of+coronary+vessels+in+the+rat.">Simple techniques for the surgical occlusion of coronary vessels in the rat.</a> Angiology. 11, 398-407 (1960).</li><li>Gomes, A. C., Falcao-Pires, I., Pires, A. L., Bras-Silva, C., Leite-Moreira, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+heart+failure:+an+updated+review.">Rodent models of heart failure: an updated review.</a> Heart Fail. Rev. 18, 219-249 (2013).</li><li>Horstick, G., 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=Surgical+procedure+affects+physiological+parameters+in+rat+myocardial+ischemia:+need+for+mechanical+ventilation.">Surgical procedure affects physiological parameters in rat myocardial ischemia: need for mechanical ventilation.</a> Am. J. Physiol. 276, 472-479 (1999).</li><li>Duchatelle, J. P., Vivet, P., Cortes, M., Groussard, O., Pocidalo, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+and+Hemodynamic-Effects+of+Lateral+Thoracotomy+or+Sternotomy+in+Mechanically+Ventilated+Rats.">Respiratory and Hemodynamic-Effects of Lateral Thoracotomy or Sternotomy in Mechanically Ventilated Rats.</a> Eur. Surg. Res. 17, 10-16 (1985).</li><li>Giraud, M. N., 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=Hydrogel-based+engineered+skeletal+muscle+grafts+normalize+heart+function+early+after+myocardial+infarction.">Hydrogel-based engineered skeletal muscle grafts normalize heart function early after myocardial infarction.</a> Artif. Organs. 32, 692-700 (2008).</li><li>Siepe, 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=Myoblast-seeded+biodegradable+scaffolds+to+prevent+post-myocardial+infarction+evolution+toward+heart+failure.">Myoblast-seeded biodegradable scaffolds to prevent post-myocardial infarction evolution toward heart failure.</a> J. Thorac. Cardiov. Sur. 132, 124-131 (2006).</li><li>Guex, A. G., 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=Plasma-functionalized+electrospun+matrix+for+biograft+development+and+cardiac+function+stabilization.">Plasma-functionalized electrospun matrix for biograft development and cardiac function stabilization.</a> Acta Biomater. 10, (2014).</li><li>Terrovitis, 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=Noninvasive+Quantification+and+Optimization+of+Acute+Cell+Retention+by+In+Vivo+Positron+Emission+Tomography+After+Intramyocardial+Cardiac-Derived+Stem+Cell+Delivery.">Noninvasive Quantification and Optimization of Acute Cell Retention by In Vivo Positron Emission Tomography After Intramyocardial Cardiac-Derived Stem Cell Delivery.</a> J. Am. Coll. Cardiol. 54, 1619-1626 (2009).</li><li>Guo, H. D., Wang, H. J., Tan, Y. Z., Wu, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+marrow-derived+cardiac+stem+cells+carried+in+fibrin+improves+cardiac+function+after+myocardial+infarction.">Transplantation of marrow-derived cardiac stem cells carried in fibrin improves cardiac function after myocardial infarction.</a> Tissue Eng. Part A. 17, 45-58 (2011).</li><li>Qiao, 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=Death+and+Proliferation+Time+Course+of+Stem+Cells+Transplanted+in+the+Myocardium.">Death and Proliferation Time Course of Stem Cells Transplanted in the Myocardium.</a> Mol. Imaging Biol. 11, 408-414 (2009).</li><li>Conradi, L., Pahrmann, C., Schmidt, S., Deuse, T., Hansen, A., Eder, 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=Bioluminescence+Imaging+for+Assessment+of+Immune+Responses+Following+Implantation+of+Engineered+Heart+Tissue+(EHT).">Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT).</a> J. Vis. Exp. 52 (e2605), (2011).</li></ol>

The authors have nothing to disclose.

The permanent ligation of LAD causes irreversible myocardial injury. The first animal model was described in 19604. Since then, it has been considered as a standard and suitable model for chronic MI. Its stability and reproducibility allowed experimental evaluation of therapies for MI5. Improved procedures following the initial description reported an operative mortality rate of 35-13%6.
As expected, LAD ligation induced impaired heart function observed within ...

Since the end of the 19th century, cardiovascular disease has remained the number one serial killer in industrialized countries. Among them, coronary artery disease represents the main etiology. The acute phase results in myocardial infarction (MI) and is followed by maladaptive remodeling that progressively develops toward a chronic phase and severe heart failure. Despite recent significant technological and therapeutic advances, the morbidity and mortality due to the progression of heart failure is still growing1. In this context, cell therapy has gained increasing interest as a new therapeutic option to stop the progression of the disease toward heart failure and to stimulate the recently identified regenerative capacity of the myocardium. Experimental and clinical investigations have provided compelling evidence of the beneficial effects obtained after cardiac transplantation of various cell types. Major outcomes included improved cardiac contractile function, decreased left ventricular remodeling, reduced infarct size, and increased vascular density in the infarcted area. However, the low cell number retention after cell injection remained an important drawback. Association of cells with a biomatrix to improve of cell delivery2 have recently fostered researcher and clinical interests.
Ligation of the left anterior descending coronary artery (LAD) is a reference method for MI in small animal model that results in transmural infarction and a mature scar. Cell therapy applied in the chronic phase of MI requires a second surgical intervention. A median sternotomy is usually performed to allow intramyocardial injection of the cells or epicardial implantation of biografts. Such invasive surgical procedures increase the mortality rate, post-surgery recovery time, pain, and risk of infection. The minimally invasive approach presented here not only prevents such bias but also provides optimal accessibility of the heart for treatment application. MI and epicardial implantation of cells associated with a gel type biomatrix are performed on a beating heart via left intercostal thoracotomies.

<table><tbody><tr><td>Polysorb 3-0 suture</td><td>Covidien</td><td>UL 204</td><td></td></tr><tr><td>Polysorb 5-0 suture</td><td>Covidien</td><td>UL 202</td><td></td></tr><tr><td>Surgipro II 7-0 Suture</td><td>Covidien</td><td>VP904X</td><td></td></tr><tr><td>Catheter Insyte 14 G</td><td>BD</td><td>381267</td><td></td></tr><tr><td>Inspira Advanced Volume Controlled Ventilator</td><td>Harvard Apparatus</td><td>557058</td><td></td></tr><tr><td>Dumont #7 Forceps</td><td>FST Germany</td><td>11274-20</td><td></td></tr><tr><td>Fibrin Sealant</td><td>Baxter</td><td>1501441</td><td>TISSEEL Glue, Frozen -20 &amp;deg;C</td></tr><tr><td>Castroviejo Eye Specula</td><td>Harvard Apparatus</td><td>72-8925</td><td>use as retractor for the ribs</td></tr><tr><td>IMDM GlutaMAX</td><td>Gibco</td><td>31980</td><td></td></tr><tr><td>Pen/Strep</td><td>Gibco</td><td>15140</td><td></td></tr><tr><td>FBS</td><td>PAA Clone</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; A15-101</td><td></td></tr><tr><td>Bone Scissors</td><td>Fine Science Tools</td><td>16044-10</td><td></td></tr><tr><td>Red Blood Cell Lysis Solution</td><td>Gentra Systems</td><td>D-50k1</td><td></td></tr><tr><td>Accutase Cell Detachment Solution</td><td>Stem Cell Technology</td><td>7920</td><td></td></tr></tbody></table>

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;

All animals recovered within 1 hr&#160;after thoracotomies. The wound healing was rapid. No infection or edema was observed.
The double left thoracotomy allowed optimal access to the heart (Figure 1). Pain and post-surgery mortality were low. The animal recovered quickly from the surgery and gained weight (Figure 2). Kaplan Meir survival percent was 96% for the first thoracotomy. Four rats over 104 died within the 24 hr following treatment application via<...

Watch this Scientific Journal Video about Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix at JoVE.com

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

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 ...

cell-based-therapy-for-heart-failure-rat-double-thoracotomy-for

Research

JoVE Journal

Bioengineering

11.9K Views.  University of Fribourg. 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.

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

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

Medicine

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

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Biology

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 ...

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) ...

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection fraction (HFrEF). Before they can be considered for use, and implemented in humans, extensive preclinical studies are required in large animal models to evaluate the safety, efficacy, and fate of the injectate (e.g., stem cells) once delivered into the myocardium. Small rodent models offer advantages (e.g., cost effectiveness, amenability for genetic manipulation); however, given inherent limitations of these models, the findings in these rarely translate into the clinic. Conversely, large animal models such as rabbits, have advantages (e.g., similar cardiac electrophysiology compared to humans and other large animals), whilst retaining a good cost-effective balance. Here, we demonstrate how to perform a percutaneous contrast echocardiography-guided intramyocardial injection (IMI) technique, which is minimally invasive, safe, well tolerated, and very effective in the targeted delivery of injectates, including cells, into several locations within the myocardium of a rabbit model. For the implementation of this technique, we also have taken advantage of a widely available clinical echocardiography system. After putting in practice the protocol described here, a researcher with basic ultrasound knowledge will become competent in the performance of this versatile and minimally invasive technique for routine use in experiments, aimed at hypothesis testing of the capabilities of cardiac regenerative therapeutics in the rabbit model. Once competency is achieved, the whole procedure can be performed within 25 min after anaesthetizing the rabbit.

Novel therapeutic strategies in cardiac regenerative medicine require extensive and detailed studies in large preclinical animal models before they can be considered for use in humans. Here, we demonstrate a percutaneous contrast echocardiography-guided intramyocardial injection technique in rabbits, which is valuable for hypothesis testing the efficacy of such novel therapies.

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection ...

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.

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 ...

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 ...