Intramyocardial Cell Delivery: Observations in Murine Hearts

Summary

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.

Abstract

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.

Introduction

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 delivery^1,2, 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 c....

Protocol

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. Cell-specific protocols must be employed for growing and eventually differentiating pluripotent or adult stem cells into specific cell lineages.

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Results

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.......

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Isolator	Pfi systems		Quotation needed
Heating Pad	Vet Tech Solutions	HE006	For small animals
Medetomidine	National Veterinary Service		Veterinary prescription is necessary
Ketamine hydrochloride	National Veterinary Service		Veterinary prescription is necessary
Atipamezole	National Veterinary Service		Veterinary prescription is necessary
Hair removal cream	commercial shops
Buprenorphine	NVS		Veterinary prescription is necessary
Leica MZFLIII microscope	Leica	Model S6E	With swing arm stand TS0
Hamamatsu Nanozoomer digital slide scanner	Hamamatsu	RS series
Scanning Electron Microscope	Jeol	JSM-6610
Blunt scissors	FST	14084-09
Minivent	Harvard Apparatus	73-0043	Including small Y adapter (73-0027) and intubation cannula (73-2844)
Forceps	FST	11052-10
Retraction system	FST	18200-20	Kit for animals up to 200 g
30 G 12 mm; ½ inch	BBraun	A210	Fine yellow
Microliter syringe	ESSLAB	81201	Also include a Hamilton repeating dispenser PB 600-1 Catalog number 83700
6-0 Silk suture	Ethicon	W1614T