Name: intramyocardial cell delivery: observations in murine hearts
Uploaded: 2014-01-24T15:00:00
Description: Watch this Scientific Journal Video about Intramyocardial Cell Delivery: Observations in Murine Hearts at JoVE.com

We thank the Magdi Yacoub Institute (MYI) for supporting microscopy analysis and the projects involving cardiac repair, the Technicians and the Manager of our animal facility. This work has been supported by the British Heart Foundation (BHF), Project grant PG/10/019. MPS is supported by the MYI and BHF. TP is a BHF-Research Excellence Fellow. NR is an NH&amp;MRC Australia Fellow.

All animal studies were performed in compliance with international (Directive 2010/63/EU of the European Parliament) and national (UK Home Office, Act 1986) regulations. The procedures herein described are part of our plan of work under UK license authorities and have not been undertaken for the purpose of recording.
1. Preparation of Cells
This protocol describes the preparation of a specific cell line (Human Embryonic Kidney, HEK293 cells) for demonstration purposes...

In this manuscript, we have shown how to perform intramyocardial injection of cells in murine hearts. As a proof of this methodology, we have used HEK293 cells. It is important to emphasize that HEK293 cells are not used in any cell therapy study and therefore the findings of this manuscript are not appropriate for direct translation to a therapeutic approach. However, the fact that HEK293 cells are not contractile cells and do not transdifferentiate in other cell types focuses attention on technical aspects such as the ...

Various cell delivery protocols have been tested in murine and rat models of cardiovascular diseases with the aim of translating the efficiency, efficacy and safety of this experimental procedure in human patients. In small rodent hearts, intramyocardial cell delivery is the most feasible method of cell delivery1,2, whereas in the rat heart antegrade3 and retrograde4 intracoronary cell infusion can also be used. Both methods have limits and advantages. Cell delivery via the intracoronary route has theoretical advantages over direct intramuscular injection in promoting global cell dissemination3, but it also has the risk of causing coronary embolism3,5. Limitations on intramyocardial delivery are associated with mechanical injury, acute inflammation, and myocardial damage6,7. In humans, cells for cardiac repair are delivered by intramyocardial injection through an endocardial or surgical epicardial approach or by intracoronary arterial route8. Injection by transvascular routes is suited in patients with acute infarct and reperfused myocardium, but may not be possible in case of total occlusions or poor flow within the vessels of the effected territory9. Direct injection into the ventricular wall by transendocardial or transepicardial injection is technically feasible depending on the patient health status. Indeed, it has been shown that this technique is safe10,11, although for transepicardial injection an open chest surgery is necessary and for transendocardial approaches an electrophysiological mapping for each patient is required to differentiate sites of viable ischemic or scarred myocardium9.
Importantly, in cell therapy studies the choice of the best cell to be transplanted is still under investigation. Short-term analyses (4 weeks) showed that injection of cardiac stem cells defined as cardiospheres12 or side population cells from bone marrow13 induced cardiac functional recovery in murine14 and rat15 models of myocardial infarct by decreasing scar size and cell death. Allogeneic transplantation of cardiospheres in a rat myocardial infarct model without immunosuppression was found to be safe, promoted cardiac regeneration, and improved heart function through stimulation of endogenous repair mechanisms15. Lin-/c-kit+ adult progenitor cells in the heart were shown to be self-renewing, clonogenic, and multipotent in vitro and in vivo, and when injected into an ischemic rat heart reconstituted large portions of the injured myocardial wall16 and had the ability to form conductive and intermediate-sized coronary arteries17. These promising data fuelled phase I and II clinical trials in humans: injection of autologous and allogeneic mesenchymal stem cells (MSC)18, cardiospheres19, or c-Kit positive cardiac stem cells (CSC)20 in ischemic human hearts each showed beneficial effects in cardiac function in long term studies. Nevertheless, extensive long-term follow-up and retrospective meta-analysis demonstrated that stem cell therapy provides significant benefit to some patients, but not in others with a range of unpredictable outcomes21. It is possible that these limitations will necessitate the design of specific protocols of cell delivery for each individual and each disease.
In mouse and rat models, long-term studies revealed that cell injection did not further improve cardiac function (12 months). Indeed, grafts of human embryonic stem cell-derived cardiomyocytes (hESC-CM) were largely isolated from the host myocardium by a layer of fibrotic tissue22,23. Similar results have been observed after intramyocardial transplantation of skeletal myoblasts into the heart of infarcted mice24. Furthermore, the long-term ability of allogeneic MSCs to preserve function in the infarcted heart has been limited by transition from an immunoprivileged to an immunogenic state after differentiation25.
Taking into consideration the challenges and prospects outlined above, we show here how to deliver cells by intramyocardial injection in mice. We observe that cells without cardiomyocyte contractile properties are not connected with the host myocardium and form a cohesive mass with a thin fibrotic barrier. Although in some cases this outcome may be advantageous, the following analysis may be useful to understand how cell engraftment may be modulated to generate functionally connected myocardial structures as well.

<table border="0" cellpadding="0" cellspacing="0" width="560">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:103px;">Isolator</td>
			<td style="width:115px;">Pfi systems</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;">Quotation needed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">Heating Pad</td>
			<td style="width:115px;">Vet Tech Solutions</td>
			<td style="width:88px;">HE006</td>
			<td style="width:255px;">For small animals</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:103px;">medetomidine</td>
			<td style="width:115px;">National veterinary Service</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;">Veterinary prescription is necessary</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:103px;">ketamine hydrochloride</td>
			<td style="width:115px;">National veterinary Service</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;">Veterinary prescription is necessary</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:103px;">atipamezole</td>
			<td style="width:115px;">National veterinary Service</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;">Veterinary prescription is necessary</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">Hair removal cream</td>
			<td style="width:115px;">Commercial shops</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:103px;">buprenorphine</td>
			<td style="width:115px;">NVS</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;">Veterinary prescription is necessary</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">Leica MZFLIII microscope</td>
			<td style="width:115px;">Leica</td>
			<td style="width:88px;">Model S6E</td>
			<td style="width:255px;">With swing arm stand TS0</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:103px;">Hamamatsu Nanozoomer digital slide scanner</td>
			<td style="width:115px;">Hamamatsu</td>
			<td style="width:88px;">RS series</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:103px;">Scanning Electron Microscope</td>
			<td style="width:115px;">Jeol</td>
			<td style="width:88px;">JSM-6610</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:103px;">Blunt scissors</td>
			<td style="width:115px;">FST</td>
			<td style="width:88px;">14084-09</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">Minivent</td>
			<td style="width:115px;">Harvard apparatus</td>
			<td style="width:88px;">73-0043</td>
			<td style="width:255px;">Including small Y adapter (73-0027) and intubation cannula (73-2844)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:103px;">Forceps</td>
			<td style="width:115px;">FST</td>
			<td style="width:88px;">11052-10</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">Retraction system</td>
			<td style="width:115px;">FST</td>
			<td style="width:88px;">18200-20</td>
			<td style="width:255px;">Kit for animals up to 200grams</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">30G 12mm; &#189; inch</td>
			<td style="width:115px;">BBraun</td>
			<td style="width:88px;">A210</td>
			<td style="width:255px;">Fine yellow</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:103px;">microliter syringe</td>
			<td style="width:115px;">ESSLAB</td>
			<td style="width:88px;">81201</td>
			<td style="width:255px;">Also include a Hamilton repeating dispenser PB 600-1 Catalogue number 83700</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:103px;">6-0 silk suture</td>
			<td style="width:115px;">Ethicon</td>
			<td style="width:88px;">W1614T</td>
			<td style="width:255px;"></td>
		</tr>
	</tbody>
</table>

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.

We injected HEK293 cells, which are distinguishable from the heart cells by their different morphology (Figure 1), with a cobblestone shape compared to the elongated cardiomyocytes (Figure 1A). HEK293 cells were more reactive to hematoxylin dye (blue color) compared to cardiomyocytes (pink color), likely because of their increased nuclear content (Figure 1A). To further distinguish the injected cells from the host tissue, HEK293 cells were labeled with DAPI 2 hours prior...

Watch this Scientific Journal Video about Intramyocardial Cell Delivery: Observations in Murine Hearts at JoVE.com

Intramyocardial Cell Delivery: Observations in Murine Hearts

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 ...

Research

JoVE Journal

Medicine

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 ...

Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR

Injection of  Algisyl-LVR, a treatment under clinical development, is intended to treat  patients with dilated cardiomyopathy. This treatment was recently ...

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

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

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

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 ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Slicing and Culturing Pig Hearts under Physiological Conditions

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly ...

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, ...

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 ...