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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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. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MSC-Encapsulating+in+situ+Cross-Linkable+Gelatin+Hydrogels+To+Promote+Myocardial+Repair.">MSC-Encapsulating in situ Cross-Linkable Gelatin Hydrogels To Promote Myocardial Repair.</a> ACS Applied Bio Materials. 3 (3), 1646-1655 (2020).</li><li>Meirelles Lda, S., Nardi, N. 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>

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 (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 ...

This method can help address key issues in current exacerbated cardiac therapy, such as low applicability to low retention and survival of transplanted cells in infarcted hearts. To address this issue, this method can provide a reliable technique to study intramyocardial transplantation with stem cells using injectable hydrogels in a murine model, which is an excellent platform to use for investigating cardiac tissue repair and regeneration after myocardial infraction. The main advantage of this technique is that it is a feasible method to improve the retention and survival of transplanted stem cells after intra-myocardial transplantation using in-situ cross-link cover injectable hydrogels.These hydrogels offer diverse opportunities for cardiac tissue engineering, such as delivering not only stem cells, but also growth factors, genetic materials and drugs to maximize their therapeutic effect in infarcted heart tissues. Demonstrating the procedure for in-vitro experiment will be Eunmi Lee, a research technologist from my laboratory. Eun-Hye Park and Eunhwa Seong research technologists will demonstrate the procedure for in-vivo experiments.Culture mesenchymal stem cells, or MSCs in a 100 millimeter culture dish at 37 degrees Celsius and 5%carbon dioxide. When the cells reach 80%confluence, wash the dish twice with DPBS and incubate them with one milliliter of trypsin substitute at 37 degrees Celsius for three minutes. Then add nine milliliters of culture medium to the cells and centrifuge them at 500 times G for three minutes.Discard the resulting supernatant. Resuspend the cells in one milliliter of PBS and maintain the cell suspension on ice. Dilute 10 microliters of cell suspension with 10 microliters of trypan blue and determine the cell concentration using an automated cell counter.Resuspend the MSCs to a density of one times 10 to the seventh cells per milliliter and transfer them to a one milliliter tube. Prepare a 6.25%GH conjugate solution in PBS and separate it into two vials. Next mix the GH solutions with either six micrograms per milliliter of HRP or 0.07%hydrogen peroxide.Briefly centrifuge the cell suspension at 1000 times G and carefully aspirate the resulting supernatant. Then mix the cell pellet with GH solution A.Load the MSCs in GH solution A and GH solution B into either side of a dual syringe. Plate 300 microliters of the combined GH solutions with MSCs at a final density of 5 million cells per milliliter onto an eight well chamber slide.After in situ hydrogel formation and subsequent MSC encapsulation via enzymatic cross-linking add 700 microliters of DMEM containing 10%FBS and 1%antibiotic antimycotic solution. Incubate the slide at 37 degrees Celsius and 5%carbon dioxide and replace the culture medium every two to three days. Prior to surgery, depolate the mouse chest using hair removal cream and sterilize the skin with iodine.Place the mouse on an operating table and intubate it by inserting a catheter into the trachea to provide supplemental oxygen via mechanical ventilation. Gently cut through the skin using surgical scissors. Then penetrate the intercostal muscles with micro scissors.Separate the second and third left ribs using a 5-O silk suture to maintain an open chest cavity. Carefully ligate the left anterior descending coronary artery using a needle holder with an 8-0 polypropylene suture and cut the suture using electrocautery. Observe an immediate color change in the anterior left ventricular wall.After inducing the myocardial infarction, inject 10 microliters of the MSC loading GH solutions into two different points at the infarct border zone. Restore the opened chest cavity and close the muscles and skin using 5-0 sutures. Remove the tracheal tube and place the mouse in a cage under an infrared lamp during recovery.Perform an echocardiography and measure corresponding lines for LV anterior wall, LV internal and LV posterior wall to obtain cardiac wall thickness, chamber dimension, and fractional shortening. Prior to in vivo transplantation, the proliferation and survival of MSCs in GH hydragels were confirmed by a 3D in vitro live dead cell staining assay. Representative images exhibited sufficient MSC proliferation showing branched networks within GH hydrogels.An extensive multicellular 3D structure of MSCs was clearly observed at day 14, indicating that GH hydrogels could provide a proper microenvironment for the encapsulated cells. After the induction of myocardial infarction, MSC loading GH hydrogels were intramyocardially transplanted into the peri-infarct areas. The MSCs and gel were appropriately sustained within the infarcted region.MSCs were well-integrated into GH hydrogels. To verify the therapeutic effects of the MSC loading GH hydrogels, the changes in cardiac function and structure were evaluated by echocardiography and histological analysis at day 28 post transplantation, and compared among the different treatment groups. The representative echocardiography showed improved cardiac functions, including fractional shortening, ejection fraction, and end systolic volume in the MSC gel treated group.In addition, histological analysis exhibited less fibrosis, thicker infarcted walls and a smaller infarct size in the MSC gel treated group than in the other groups indicating that this protocol significantly attenuated LV remodeling. Using this technique, we achieved long term stem cells in and proliferation and led to significant improvement in the cardiac structure and function following myocardial infarction in murine model. This technique can be extended for use in large animals and to clinical translation as a new strategy for prevention of a post infarct heart failure by providing cardiac tissue repair and regeneration.

intramyocardial-transplantation-msc-loading-injectable-hydrogels

Research

JoVE Journal

Medicine

4.3K Görüntüleme.  The Catholic University of Korea. This method can help address key issues in current exacerbated cardiac therapy, such as low applicability to low retention and survival of transplanted cells in infarcted hearts. To address this issue, this method can provide a reliable technique to study intramyocardial transplantation with stem cells using injectable hydrogels in a murine model, which is an excellent platform to use for investigating cardiac tissue repair and regeneration after myocardial infraction. The main advantage of t...