Name: intramyocardial transplantation of msc-loading injectable hydrogels after myocardial infarction in a murine model
Uploaded: 2020-09-20T15:00:00
Description: Watch this Scientific Journal Video about Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model at JoVE.com

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...

<ol>
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N., Yan, W., Srivastava, A., Desiderio, V., Dhingra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+injectable+hydrogels+for+cardiac+stem+cell+therapy+and+tissue+engineering.">Application of injectable hydrogels for cardiac stem cell therapy and tissue engineering.</a> Reviews in Cardiovascular Medicine. 20 (4), 221-230 (2019).</li><li>Gaetani, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicardial+application+of+cardiac+progenitor+cells+in+a+3D-printed+gelatin/hyaluronic+acid+patch+preserves+cardiac+function+after+myocardial+infarction.">Epicardial application of cardiac progenitor cells in a 3D-printed gelatin/hyaluronic acid patch preserves cardiac function after myocardial infarction.</a> Biomaterials. 61, 339-348 (2015).</li><li>Gao, L., 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=Myocardial+Tissue+Engineering+With+Cells+Derived+From+Human-Induced+Pluripotent+Stem+Cells+and+a+Native-Like,+High-Resolution,+3-Dimensionally+Printed+Scaffold.">Myocardial Tissue Engineering With Cells Derived From Human-Induced Pluripotent Stem Cells and a Native-Like, High-Resolution, 3-Dimensionally Printed Scaffold.</a> Circualtion Research. 120 (8), 1318-1325 (2017).</li><li>Hasan, 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=Injectable+Hydrogels+for+Cardiac+Tissue+Repair+after+Myocardial+Infarction.">Injectable Hydrogels for Cardiac Tissue Repair after Myocardial Infarction.</a> Advanced Science. 2 (11), 1500122 (2015).</li><li>Wu, R., Hu, X., Wang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+Review:+Optimized+Strategies+for+Stem+Cell-Based+Therapy+in+Myocardial+Repair:+Clinical+Translatability+and+Potential+Limitation.">Concise Review: Optimized Strategies for Stem Cell-Based Therapy in Myocardial Repair: Clinical Translatability and Potential Limitation.</a> Stem Cells. 36 (4), 482-500 (2018).</li><li>Lee, 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=In+situ+forming+gelatin-based+tissue+adhesives+and+their+phenolic+content-driven+properties.">In situ forming gelatin-based tissue adhesives and their phenolic content-driven properties.</a> Journal of Materials Chemistry B. 1 (18), 2407-2414 (2013).</li><li>Lee, Y., Bae, J. W., Lee, J. W., Suh, W., Park, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzyme-catalyzed+in+situ+forming+gelatin+hydrogels+as+bioactive+wound+dressings:+effects+of+fibroblast+delivery+on+wound+healing+efficacy.">Enzyme-catalyzed in situ forming gelatin hydrogels as bioactive wound dressings: effects of fibroblast delivery on wound healing efficacy.</a> Journal of Materials Chemistry B. 2 (44), 7712-7718 (2014).</li><li>Lee, S. 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=In+situ+Crosslinkable+Gelatin+Hydrogels+for+Vasculogenic+Induction+and+Delivery+of+Mesenchymal+Stem+Cells.">In situ Crosslinkable Gelatin Hydrogels for Vasculogenic Induction and Delivery of Mesenchymal Stem Cells.</a> Advanced Functional Materials. 24 (43), 6771-6781 (2014).</li><li>Jung, B. 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=A+hydrogel+matrix+prolongs+persistence+and+promotes+specific+localization+of+an+oncolytic+adenovirus+in+a+tumor+by+restricting+nonspecific+shedding+and+an+antiviral+immune+response.">A hydrogel matrix prolongs persistence and promotes specific localization of an oncolytic adenovirus in a tumor by restricting nonspecific shedding and an antiviral immune response.</a> Biomaterials. 147, 26-38 (2017).</li><li>Kim, 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=Tonsil-derived+mesenchymal+stem+cell-embedded+in+situ+crosslinkable+gelatin+hydrogel+therapy+recovers+postmenopausal+osteoporosis+through+bone+regeneration.">Tonsil-derived mesenchymal stem cell-embedded in situ crosslinkable gelatin hydrogel therapy recovers postmenopausal osteoporosis through bone regeneration.</a> PLoS One. 13 (7), 0200111 (2018).</li><li>Kim, C. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+marrow-derived+mesenchymal+stem+cell:+isolation,+in+vitro+expansion,+and+characterization.">Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization.</a> Br J Haematol. 123 (4), 702-711 (2003).</li><li>Ojha, 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=Characterization+of+the+structural+and+functional+changes+in+the+myocardium+following+focal+ischemia-reperfusion+injury.">Characterization of the structural and functional changes in the myocardium following focal ischemia-reperfusion injury.</a> Am J Physiol Heart Circ Physiol. 294 (6), 2435-2443 (2008).</li><li>Takagawa, 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=Myocardial+infarct+size+measurement+in+the+mouse+chronic+infarction+model:+comparison+of+area-+and+length-based+approaches.">Myocardial infarct size measurement in the mouse chronic infarction model: comparison of area- and length-based approaches.</a> Journal of Applied Physiology. 102 (6), 2104-2111 (2007).</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> Journal of the American College of Cardiology. 54 (17), 1619-1626 (2009).</li><li>Dib, N., Khawaja, H., Varner, S., McCarthy, M., Campbell, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Therapy+for+Cardiovascular+Disease:+A+Comparison+of+Methods+of+Delivery.">Cell Therapy for Cardiovascular Disease: A Comparison of Methods of Delivery.</a> Journal of Cardiovascular Translational Research. 4 (2), 177-181 (2011).</li></ol>

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 (H2O2; 30 wt % in H2O)</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&#8217;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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