This research is supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by Ministry of Education (NRF-2018R1D1A1A02049346)

All animal research procedures were provided in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals and the Guidelines and Policies for Rodent Experiments provided by the Institutional Animal Care and Use Committee (IACUC) in the School of Medicine of The Catholic University of Korea.
1. Preparation of MSCs and injectable gelatin hydrogels
<ol>
	<li>Culture MSCs in a 100 mm culture dish at 37 &#176;C and 5% CO2. When MSCs growth reaches 80% confluence, wash the dish twice with DPBS and add 1 mL of trypsin-substitute at 37 &#176;C for 3 min. 
	NOTE: MSCs were isolated from murine bone marrow following conventional procedures16, cultured in Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotic&#8722;antimycotic solution, and used between passage 7&#8210;9 for this study.</li>
	<li>Add 9 mL of culture medium and centrifuge at 500 x g for 3 min. Next, discard the resulting supernatant, resuspend the cells in 1 mL of PBS, and maintain the cell suspension on ice.</li>
	<li>Dilute 10 &#181;L of cell suspension with 10 &#181;L of Trypan blue and obtain the cell concentration using an automated cell counter.</li>
	<li>Resuspend and transfer MSCs to a 1 mL tube at a density of 1 x 107 cells/mL.</li>
	<li>Prepare a 6.25 wt% of GH conjugate solution in PBS and separate into 2 vials. Next, mix the GH solutions with either 6 &#181;g/mL of HRP (GH solution A) or 0.07 wt% of H2O2 (GH solution B). 
	NOTE: Prepare gelatin-hydroxyphenyl propionic acid (GH) conjugates according to published protocols12,15.
	<ol>
		<li>Keep a 9:1 volumetric ratio of GH conjugate solution to HRP (GH solution A) and GH conjugate solution to H2O2 (GH solution B), respectively.</li>
	</ol>
	</li>
	<li>Prior to mixing the MSCs with GH solution A, briefly centrifuge the cell suspension at 1,000 x g and carefully aspirate the resulting supernatant. Subsequently, mix the pellet containing MSCs with GH solution A.</li>
</ol>
2. In situ MSC-loading and three-dimensional in vitro culture
<ol>
	<li>Load GH solution A (containing MSCs) and GH solution B into either side of a dual syringe. Plate 300 &#181;L of the combined GH solutions with MSCs at a final density of 5 x 106 cells/mL onto an eight-well chamber slide.</li>
	<li>After in situ hydrogel formation and subsequent MSC encapsulation via enzymatic cross-linking, add 700 &#181;L of DMEM containing 10% FBS and 1% antibiotic&#8722;antimycotic solution.</li>
	<li>Incubate the slide at 37 &#176;C and 5% CO2 and replace the culture medium every 2&#8210;3 days.</li>
</ol>
3. Confirmation of in vitro proliferation and survival of MSCs within GH hydrogels
<ol>
	<li>To determine the viability of 3D cultured MSCs within GH hydrogels, use a live/dead cell staining assay after the predetermined incubation time.</li>
	<li>Following incubation of the encapsulated MSCs in GH hydrogels for 3, 5, 7 or 14 days, aspirate the medium and wash the well twice with PBS.</li>
	<li>Prepare a staining solution containing 5 &#181;L of calcein AM and 20 &#181;L of ethidium homodimer-1 (EthD-1) in 10 mL of DPBS.</li>
	<li>Add 200 &#181;L of the staining solution to the well and incubate for 30 min in the dark at room temperature.</li>
	<li>Aspirate the staining solution and wash the well twice with PBS.</li>
	<li>Carefully separate the chamber from the slide and place a full coverslip over the GH hydrogels. Use a confocal microscopy to visualize the degree of proliferation and morphological changes of the encapsulated MSCs. 
	NOTE: Fluorescent images were acquired under 200x magnification and imaged at the excitation/emission wavelengths of 470/540 nm for calcein and 516/607 nm for EthD-1.</li>
</ol>
4. Induction of myocardial infarction in mice
<ol>
	<li>Anesthetize 7-week-old male C57BL/6 mice (20&#8210;22 g) with intraperitoneal injection of a mixture of Zoletil (30 mg/kg) and Rompun (10 mg/kg) in saline.</li>
	<li>Prior to surgery, depilate the mouse chest using hair removal cream and sterilize the skin with iodine.</li>
	<li>Place the mouse on an operating table and intubate by inserting a catheter into the trachea to provide supplemental oxygen via mechanical ventilation.</li>
	<li>Gently cut through the skin using surgical scissors and then penetrate the intercostal muscles by micro scissors. Separate the 2nd and 3rd left ribs using a 5-0 silk suture to maintain an open chest cavity.</li>
	<li>Carefully ligate the left anterior descending (LAD) coronary artery using a needle holder with an 8-0 polypropylene suture and cut the suture using electrocautery.</li>
	<li>Observe an immediate color change in the anterior left ventricular wall.</li>
</ol>
5. Intramyocardial transplantation of MSC-loading GH hydrogels
<ol>
	<li>After inducing the myocardial infarction by LAD ligation, inject 10 &#181;L of MSC-loading GH solutions into two different points at the infarct border zone (total: 2 x 105 MSCs/20 &#181;L) using a dual-syringe equipped with a 26G needle.
	<ol>
		<li>Following the same procedure described in Step 1, prepare and transfer MSC-loading GH solutions into a dual syringe. 
		NOTE: To assess the engraftment of MSC-loading GH hydrogels within the infarcted area, MSCs and GH conjugates were pre-labeled with PHK26 and fluorescein isothiocyanate (FITC), respectively.</li>
	</ol>
	</li>
	<li>Restore the opened chest cavity and close the muscles and skin using 5-0 sutures. 
	NOTE: Prior to chest closure, remove the air using a catheter syringe.</li>
	<li>Remove the tracheal tube and place the mouse in a cage under an infrared lamp during recovery.</li>
	<li>For post-operative analgesia,&#160;administer subcutaneous Ketoprofen injections (5 mg/kg per day) for a minimum of 72 h. All mice should be closely monitored for an appropriate time to ensure proper recovery after surgical procedures as well as adequate pain treatment.</li>
</ol>
6. Echocardiography
<ol>
	<li>Four weeks following transplantation, initially anesthetize the mouse with 5% isoflurane and then adjust the isoflurane concentration to 1%.</li>
	<li>Depilate the chest using hair removal cream and place the mouse on a heating pad. Apply ultrasound transducer gel onto the chest.</li>
	<li>Acquire two-dimensional parasternal short axis views and record M-mode tracings at the level of the papillary muscle. 
	NOTE: Place a linear array transducer (7&#8210;15 MHz) in the left parasternal line and view the anatomical structures.</li>
	<li>Measure corresponding lines for LVAW, LVID, and LVPW to obtain cardiac wall thickness, chamber dimension, and fractional shortening. 
	NOTE: Compare cardiac function including the ejection fraction (EF), fractional shortening (FS), and end-systolic volume (ESV) at the level of the papillary muscle to ensure proper assessment at the same anatomic location.</li>
</ol>
7. Histological evaluation
<ol>
	<li>At the predetermined time after transplantation of MSC-loading GH hydrogels into the infarcted heart, euthanize the mouse in a CO2 chamber and collect the heart for histological analysis15.</li>
	<li>For hematoxylin and eosin (H&#38;E) and Masson&#8217;s trichrome (MT) staining, fix the dissected heart tissues in 4% paraformaldehyde (PFA) and embed in paraffin. Next, cut paraffin-embedded heart blocks into 4 &#181;m serial sections using a microtome and stain the sections with MT stain according to standard protocols17.</li>
	<li>Acquire images on a slide scanner at 20x magnification and calculate the infarct size of the treatment groups. 
	Infarct size (%) = total infarct circumference / total LV circumference x 100</li>
	<li>Calculate both circumferences by midline length measurement. For LV midline circumferences, measure the centerline lengths between the endocardial and epicardial surfaces. For midline infarct circumferences, measure the lengths of infarct including more than 50 % of the whole thickness of myocardium18. 
	NOTE: All image analyses were performed using ImageJ software.</li>
	<li>Measure the wall thickness of the scar at the papillary muscle levels.</li>
	<li>Calculate the fraction of collagen area. 
	Collagen area (%) = total area of interstitial fibrosis/myocyte area x 100</li>
</ol>

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The authors have no conflicts of interest to declare with this work.

Injectable GH hydrogels have great potential for in vivo applications because of their ability to homogenously incorporate diverse therapeutic agents in situ. Furthermore, their physical and biochemical properties can be easily manipulated based on disease-dependent requirements. In this respect, injectable hydrogels have been proposed to address the major limitations in current cardiac stem cell therapy hampered by poor survival and cell retention (i.e., &#60; 10% within 24 h post-transplantation) in the injured heart<s...

Cardiac stem cell therapy has emerged as a potential approach for myocardial repair and regeneration1,2. Despite the recent positive results in animal models and clinical trials, the application of stem cell-based therapy for myocardial repair is limited due to low retention and poor survival of injected cells at the infarcted heart tissues3,4. As a result, the use of cell-based tissue engineering, including injectable biomaterials5, cardiac patches6, and cell sheets7, has been intensively studied to improve cell retention and integration within the host myocardium.
Among the various potential approaches to bioengineered cardiac tissue repair, injectable hydrogels combined with appropriate cell types, such as mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs), are an attractive option to effectively deliver cells into myocardial regions8,9. Gelatin, a well-known natural polymer, can be used as an injectable matrix due to its great biocompatibility, considerable biodegradability, and reduced immunogenicity when compared with a wide range of biomaterials used in biomedical applications. Although gelatin-based injectable platforms have great potential, their applicability in vivo remains limited based on their low mechanical stiffness and easy degradability in the physiological environment.
To overcome these limitations, a novel and simple design of gelatin-based hydrogels consisting of hydroxyphenyl propionic acid has been proposed for in vivo applications. Gelatin-hydroxyphenyl propionic acid (GH) conjugates can be cross-linked in situ in the presence of an enzyme, horseradish peroxidase (HRP), and subsequently encapsulate various drugs, biomolecules, or cells within the hydrogel, suggesting great potential in tissue engineering applications10,11,12,13,14. In addition, we have recently investigated the therapeutic effects of GH hydrogels containing encapsulated MSCs and demonstrated their use in successful cardiac repair and regeneration after MI in a murine model15. In this protocol, we describe a simple technique for the encapsulation and in vitro three-dimensional (3D) proliferation of MSCs within GH hydrogels. We also introduce a surgical procedure designed to generate a murine MI model via coronary artery ligation and intramyocardial transplantation of MSC-loading GH hydrogels into the infarcted heart.

<table><tbody><tr><td>4 % paraformaldehyde (PFA)</td><td>Intron</td><td>IBS-BP031-2</td><td></td></tr><tr><td>5-0 silk suture</td><td>AILEE</td><td>SK534</td><td></td></tr><tr><td>8-0 polypropylene suture</td><td>ETHICON</td><td>M8732H</td><td></td></tr><tr><td>8-well chamber slide</td><td>Nunc LAB-TEK</td><td>154534</td><td></td></tr><tr><td>Angiocath Plus (22GA) catheter</td><td>BD Angiocath Plus</td><td>REF382423</td><td></td></tr><tr><td>Antibiotic-antimyocotic</td><td>Gibco</td><td>15240-062</td><td></td></tr><tr><td>Centrifuge</td><td>GYROGEN</td><td>1582MGR</td><td></td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td>LSM 510</td><td></td></tr><tr><td>Cover slipe</td><td>MARIENFELD</td><td>101242</td><td></td></tr><tr><td>Deluxe High Temperature Cautery kit</td><td>Bovie</td><td>QTY1</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>11995-065</td><td></td></tr><tr><td>DPBS</td><td>Gibco</td><td>14040-133</td><td></td></tr><tr><td>Dual-syringe</td><td></td><td></td><td></td></tr><tr><td>EOSIN</td><td>SIGMA-ALDRICH</td><td>HT110116</td><td></td></tr><tr><td>Ethanol</td><td>EMSURE</td><td>K49350783 739</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>Fechtner conjunctiva forceps titanium</td><td>WORLD PRECISISON INSTRUMENTS</td><td>WP1820</td><td></td></tr><tr><td>Fluorescein isothiocyanate isomer I (FITC)</td><td>SIGMA-ALDRICH</td><td>F7250</td><td></td></tr><tr><td>Forcep</td><td>HEBU</td><td>HB0458</td><td></td></tr><tr><td>Hair removal cream</td><td>Ildong Pharmaceutical</td><td></td><td></td></tr><tr><td>Heating pad</td><td>Stoelting</td><td>50300</td><td>Homeothermic Blanket System</td></tr><tr><td></td><td></td><td>50301</td><td>Replacement Heating Pad for 50300 (10 X 12.5cm)</td></tr><tr><td>Hematoxylin</td><td>SIGMA-ALDRICH</td><td>HHS80</td><td></td></tr><tr><td>Horseradish peroxide (HRP; 250-330 U/mg)</td><td>SIGMA-ALDRICH</td><td>P8375</td><td></td></tr><tr><td>Hydrogen peroxide (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;; 30 wt % in H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>SIGMA-ALDRICH</td><td>216763</td><td></td></tr><tr><td>Iodine</td><td>Green Pharmaceutical</td><td></td><td></td></tr><tr><td>LIVE/DEAD cell staining kit</td><td>Thermo Fisher</td><td>R37601</td><td></td></tr><tr><td>Mechanical ventilator</td><td>Harvard Apparatus</td><td></td><td></td></tr><tr><td>Micro centrifuge</td><td>HANIL</td><td>Micro 12</td><td></td></tr><tr><td>Micro needle holder</td><td>KASCO</td><td>37-1452</td><td></td></tr><tr><td>Micro scissor</td><td>HEBU</td><td>HB7381</td><td></td></tr><tr><td>Microscope</td><td>OLYMPUS</td><td>SZ61</td><td></td></tr><tr><td>MT staining kit</td><td>SIGMA-ALDRICH</td><td>HT1079-1SET</td><td>Weigert&amp;rsquo;s iron hematoxylin solution</td></tr><tr><td></td><td></td><td>HT15-1KT</td><td>Trichrome Stain (Masson) Kit</td></tr><tr><td>Paraffin</td><td>LK LABKOREA</td><td>H06-660-107</td><td></td></tr><tr><td>PBS buffer</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>PHK26 staining kit</td><td>SIGMA-ALDRICH</td><td>MINI26</td><td></td></tr><tr><td>Slide scanner</td><td>Leica</td><td>SCN400</td><td></td></tr><tr><td>Surgical scissor</td><td>HEBU</td><td>HB7454</td><td></td></tr><tr><td>Surgical tape</td><td>3M micopore</td><td>1530-1</td><td></td></tr><tr><td>Tissue cassette</td><td>Scilab Korea</td><td>Cas3003</td><td></td></tr><tr><td>Transducer gel</td><td>SUNGHEUNG</td><td>SH102</td><td></td></tr><tr><td>Trout-Barraquer needle holder curved</td><td>KASCO</td><td>50-3710c</td><td></td></tr><tr><td>Ultrasound system</td><td>Philips</td><td>Affiniti 50</td><td></td></tr><tr><td>Xylene</td><td>JUNSEI</td><td>25175-0430</td><td></td></tr></tbody></table>

intramyocardial transplantation of msc-loading injectable hydrogels after myocardial infarction in a murine model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

To effectively deliver MSCs to the infarcted myocardium, MSC-loading in situ cross-linkable hydrogels described in Figure 1 were used in this protocol. Prior to in vivo transplantation, the proliferation and survival of MSCs in GH hydrogels were confirmed by a 3D in vitro live/dead cell staining assay (live: green; dead: red). As shown in Figure 2, representative images exhibited sufficient MSCs proliferation, showing branched networks within GH hydrogels. In ad...

Watch this Scientific Journal Video about Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model at JoVE.com

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...

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JoVE Journal

Medicine

4.4K Views.  The Catholic University of Korea. Stamcelgebaseerde therapie is naar voren gekomen als een efficiënte strategie om gewonde hartweefsels te herstellen na een myocardinfarct. Wij bieden een optimale in vivo toepassing voor stamceltransplantatie met behulp van gelatinehydrogels die enzymatisch met elkaar verbonden kunnen zijn.

Video: Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

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 ...

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. Y-27632 is a highly potent inhibitor of Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) and is used to prevent dissociation-induced cell apoptosis (anoikis). We demonstrate that Y-27632 pretreatment for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs+RI) prior to implantation results in a cell engraftment rate improvement in a mouse model of acute myocardial infarction (MI). Here, we describe a complete procedure of hiPSC-CMs differentiation, purification, and cell pretreatment with Y-27632, as well as the resulting cell contraction, calcium transient measurements, and transplantation into mouse MI models. The proposed method provides a simple, safe, effective, and low-cost method which significantly increases the cell engraftment rate. This method cannot only be used in conjunction with other methods to further enhance the cell transplantation efficiency but also provides a favorable basis for the study of the mechanisms of other cardiac diseases.

In this protocol, we demonstrate and elaborate on how to use human induced pluripotent stem cells for cardiomyocyte differentiation and purification, and further, on how to improve its transplantation efficiency with Rho-associated protein kinase inhibitor pretreatment in a mouse myocardial infarction model.

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. ...

Biochemistry

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

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

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 ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

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) ...

Bioengineering

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 ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

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