A. Introduction

Over the last thirteen years, stem cell therapy has progressed significantly in an attempt to meet the critical need and demand to treat a variety of cardiovascular diseases including heart failure (1-8), myocardial infarction (9-11) , refractory angina (12-14) and peripheral arterial disease (15). Cells can be delivered by a variety of routes: intracoronary (11,16), intravenous (17,18), transendocardial (19-21) and transvenous (22). The survival and homing of cells in the infarcted myocardium after injection depend on the method of delivery, cell type and the clinical indication. For instance, Hofmann et al (23) showed that homing of cells to the myocardium was affected by both the cell type and route of injection. They injected either whole, unfractionated bone marrow cells (BMC) or an immunoselected CD34+ sub-fraction of BMC via an intracoronary or intravenous route within 5-10 days after myocardial infarction in patients. When either population of cells was given intravenously, there were no cells retained in the myocardium. However, when these cells were given via intracoronary injection, 1.3-2.6% were retained in the area of myocardial infarction and the border zone. Changing the cell type to CD34+ cells, increased the retention rate to 14-39%, but the cells localized only in the border zone. Therefore, it is essential with studies that involve cell delivery that the route, cell type and clinical indication be specified.

For the purposes of this chapter, we will focus on the route of delivery, and more specifically, on the catheters used and the advantages, disadvantages and potential complications of all. Five principal devices have been used for studies injecting cells directly into the myocardium. These devices include the Helix™ (Biocardia, South San Francisco, CA) (24,25), the Myocath™ (Bioheart, Sunrise, FL) (26,27), the Myostar™ (Biologics Delivery Systems, Diamond Bar, CA) (1,8,28) and the Stiletto™ (Boston Scientific, Natick, MA) (20). All all are designed to inject cells from within the ventricular chamber to the endocardium. Whereas the 5th device, the Trans Access Delivery System™ (Medtronic Vascular, Santa Rosa, CA) (22,25,29) uniquely delivers to the epicardial surface, entering via the cardiac venous system. The challenge is to identify and address the unique requirements associated with the cells to be used for the disease target such that therapeutic potential can be maximized.

B. Delivery via the Coronary Circulation

Standard balloon angioplasty catheter

Intracoronary infusion has been used to deliver various cell types in multiple clinical trials into the largest number of patients (10,11,16,30-38) than any other technique. This method for treatment of myocardial infarction has been used for over a decade and has utilized a standard balloon angioplasty procedure. The classical method is an over the wire catheter to inject cells, and a balloon to halt coronary flow as the cells are released. The intracoronary delivery of cells has been shown to be superior to intravenous administration of cells in regards to cell retention ( ≥ 5% intracoronary and < 0.5% for intravenous) (17,18,23,39,40). Typically, cell transplantation has been performed using multiple (Range 3 – 6) fractional infusions of 3 to 5 ml of cell suspension each containing 10 to 20 x10⁶ cells parallel to balloon inflation over 2 to 4 minutes (10,30-34,37,41). All cells were infused directly into the infarcted zone through the infarct- related artery via an angioplasty balloon catheter (see table 1 for examples of catheters), which was inflated at a low pressure (2 to 4 atm) and was located within the previously stented coronary segments. This prevented backflow of cells and produced stop flow beyond the site of balloon inflation to facilitate high-pressure infiltration of cells into the infarcted zone. Standard balloon intracoronary delivery of cells can result in a 5% risk of dissections, vessel trauma, periprocedural cardiac enzyme elevation and even death. Actual experience has demonstrated that in a series of 776 patients treated there was a 2% rate of coronary artery dissection, 2.4% troponin elevation and a 30 day mortality of 0.5% (42).

A limitation to intracoronary delivery is the inability to get cells to the infarct except during the immediate post-infarct period. Infarction is accompanied by tissue destruction, vascular breakdown and inflammation that triggers selectin expression which targets cells to the infarct borders (43). The subsequent migration of cells is from peri-infarct distributions of cells delivered through the remaining vasculature surrounding the infarct. The immediate post-infarct zone is a harsh environment for cells – reduced oxygen, high levels of inflammatory factors and large numbers of macrophages followed by fibroblasts depositing collagen fibrils necessary to consolidate and buttress the infarct zone (43-47). Consequently, cells collect and remain predominantly within the peri-infarct zone and show little migration into the core of the infarct (23,48-50). Data to the contrary, have been limited by the low resolution of in vivo visualization techniques, with markers carried by injected cells often taken up by host macrophages, which are then mistaken for the transplanted cells (51,52). A fact little appreciated is the exquisite sensitivity to and elimination of foreign (non-self) cells within the myocardium (45,47,53-55). Non-autologous cells are immediately and vigorously rejected within the myocardium (56). Cells like mesenchymal stem cells (MSCs), shown to suppress local immune cell activity in vitro, are rejected within the myocardium (45,47,53-55). The rejection of non-autologous cells coupled with the limited distribution of cells post infusion makes interpretation of improvements difficult to reconcile with current proposed mechanisms for stem cell function. Designing well controlled studies that show unequivocal improvement and favorable safety profiles are critical if the stem cell field is to successfully advance and effectively treat heart damage.

The intracoronary delivery of cells with existing catheters is an added area for concern as these catheters were not designed for cell delivery, and although, they are composed of inert materials there may be aspects of the design or composition of these catheters that could compromise cell viability or delivery. Some unforeseen complications have already been experienced. Cells within current catheters can become clumped, and this has been shown to cause embolization, compromising cardiac blood flow (39,57) sometimes resulting in death. Mortality rates as high as 63% were observed in one pre-clinical study in pigs. In this particular example, the cell type used was bone marrow derived MSCs and the high mortality was in the group that received intermittent over the balloon infusions. Continuous flow cell delivery methods (either high rate (10 ml at 20ml/min) or very high rate (10 ml at 60ml/min)) resulted in low (very high rate group) or no (high flow rate group) mortality. No observed difference was seen in the percent of cells retained at the infarct site (57). The high mortality was also associated with observed reduction in blood flow due to the blockage created by the MSCs. Thus, certain cells will require infusion techniques and perhaps catheters that minimize cell clumping and possible embolization.

A study delivering neural stem cells via intra-arterial injection illustrates how catheter design can influence cell clumping and subsequent embolization (58). Laser Doppler measurements showed that cerebral blood flow (CBF) was unaffected when cells were injected to the common carotid artery through a microneedle. In contrast, when cells were injected through a standard catheter there was a 65% decrease in CBF during the first minute of cell injection that partially recovered to 60% to 70% of baseline after 15 minutes (58). Both the initial drop and the overall CBF values in the catheter method were significantly decreased compared with the microneedle method. The mortality in the catheter method was 35% at 24 hours, whereas no deaths were recorded using the microneedle technique. Clearly, the design of the needle had an impact on the results of the cell delivery.

ND Infusion catheter

New catheters are being developed that will minimize some of the observed problems with current intracoronary devices. The ND infusion catheter, developed by the author, is an arterial catheter designed to decrease cell clumping and potential embolization. It is a 3Fr monorail catheter with six micro-channels and a separate guide wire lumen. The cross-sectional area of each micro-channel is seven-fold less than that of a typical coronary over the wire (OTW) balloon catheter (0.018 mm² vs. 0.129mm²) (see Table 1). The cross sectional area of each channel is currently 6 fold smaller than any other single lumen catheter on the market.

The 6 small channels will not allow for large clumps of cells to exit the catheter. During infusion, arterial blood flow can be regulated by volume-adjusted inflation of the balloon, accommodating a range of vessel diameters up to 4.5mm. Consequently one catheter can be used for different vessel diameters. The balloon is less traumatic than the standard balloon, designed to be a “hand volume dependent inflation” with a short balloon measuring 3.25 mm in length (Fig. 1-5).

Figure 2. Schematics of the ND Infusion Catheter

Figure 3. Representative images of the ND Infusion catheter

Figures 4 and 5. Representative images of the ND Infusion catheter. Fig. 4 (left) depicts the microchannels. Fig. 5 (right) is a representative image of the fluid exiting the catheter in spray form.

Compared to the standard balloon, the ND Infusion catheter has several advantages:

1. Cell separation: where cell concentrations of 30 million cells/ml could be injected over 1 minute through both the single lumen standard balloon catheter or the Multichannel ND infusion catheter, cell separation was much cleaner with the ND infusion catheter (Fig. 6). There is an excellent safety margin with cell infusion; one can infuse up to 25 million MSCs/ml over one minute without affecting cell viability (Fig. 7).
2. The fluid exiting the catheter exits in a spray fashion that is designed to enhance mixing of the cell solution with the blood in a more homogeneous fashion and potentially enhance the distribution of cells to the intended myocardium (Figs. 4 and 5)
3. A compliant balloon uses volume dependent hand inflation and can increase up to 4.5 mm in diameter and provide occlusion to a variety of vessel sizes (Figs. 5,6,8 and 9).
4. The catheter is a monorail system (Figs. 1-2) with no need to remove the wire during cell delivery, thus providing stability of the catheter during infusion.

Figure 6. Cells before and after infusion through the ND infusion catheter (A and C) or infusion through the Balloon Catheter (B and D).

Figure 7. Cell viability measurements after passage through the ND catheter system.

Figure 8. ND Infusion catheter can be inflated to varying diameters.

Figure 9. ND Infusion catheter can be used to accommodate a variety of vessel diameters. Depicted are balloon occlusions to A) Left Circumflex Artery, B) Carotid Artery, C) Renal and D) Iliac artery in an animal model.

Intra-arterial adventitial catheter

A recently devised method used with significant success is the adventitial delivery of cells with the Cricket™ and Bullfrog® Micro-Infusion Catheters (Mercator Medsystems, San Leandro, CA). These are the first vascular-access systems enabling injection into the coronary adventitial layer (Fig. 10). The adventitial layer of coronary arteries is surrounded by loose connective tissue that extends along the arteries and their branches. Extravascular injection into this compartment provides an efficient depot for cell delivery and results in transmural vascular delivery. This catheter contains a single microneedle mounted within a noncompliant balloon. Upon inflation, the balloon exposes the needle and directs it along a perpendicular trajectory into the vessel wall. Needle length is preset to penetrate the external elastic lamina.

Figure 10. Cricket micro-infusion catheter.

The micro-infusion catheter was used to deliver cells to patients in a Phase I study with promising early results. Nineteen patients received escalating doses of MultiStem, a proprietary population of bone marrow stem cells derived from single donors and expanded in vitro. Two to 5 days after MI, cells were delivered to the adventitia of the infarct-related vessel in patients with first-time STEMI. At 4 months post treatment, patients receiving > 50 million cells showed a 10-13% absolute increase in ejection fraction (EF) versus a 0.9% increase in control patients not-receiving cells. As there were no complications with the injection of cells and evidence of significant improvement in cardiac function, this technique may prove to be a solution to some of the limitations of standard intracoronary delivery. The caveats are that allogeneic cells were used, and as no survival of non-autologous cells has been observed in the heart, the benefits would have to be related to a paracrine mechanism.

C. Delivery

Epicardial Surgical Injection

It is obvious that myocardial injection under direct visualization is an easy technique for the cardiac surgeon, and it might be an attractive approach for cell delivery during cardiac surgery; whether during bypass or insertion of a left ventricular assist device.

This approach clearly is effective as demonstrated by cell survival and retention.

When 300 million myoblast cells were injected at the time of implant of a left ventricular assist in patients who were candidates awaiting heart transplantation, the cells survived and formed myotubes and myofibers (as evident from histological and chemical staining using skeletal muscle specific antibodies (Fig. 11)) (7).

Figure 11. Direct injection of myoblast cell. A and B are trichrome staining. C depicts myosin staining and D depicts myosin 32 beta staining.

In a separate trial 2, myoblast cells were surgically injected in patients undergoing bypass surgery and were shown on positron emission tomography (PET) to have superior survival and viability of the cells. The previously non-metabolizing scar was converted into living tissue as demonstrated by PET glucose utilization (Fig. 12). Although the most effective means of cell delivery, surgical injection to the epicardium is impractical for the majority of potential patients and a less invasive method is preferred.

Figure 12. Positron Emission Tomography showing new tissue formation within establish myocardial infarcts in 3 patients.

Endocardial Delivery

Transmyocardial injection of cells has several advantages over surgical epicardial delivery. It can be done in very sick patients with low ejection fraction. The procedure can be repeated if treatment is beneficial, and it is the only method by which cells can be delivered to the septum. It might be helpful to categorize guidance of endocardial delivery methods into 2-D fluoroscopic [the Helix™ (Fig. 13), the Myocath™ and the Stiletto™] or magnetic resonance imaging (MRI), The Stiletto™ versus 3-D guidance (the Myostar™) that utilizes electromechanical recordings from a mapping catheter (the NOGA mapping system) to create a 3-D map for targeting and injection (Figs. 14, 15 and 16. The advantages of 3-D are multiple:

1. Areas of infarct, border-zone, and normal tissue can be accurately identified and mapped for better targeting of injections (Fig. 11).
2. Able to localize the mitral valve area (Fig. 16).
3. Identify the left bundle branch block (LBBB) zone (Fig. 16).
4. The 3D-guidance has the disadvantage that it is a longer, more expensive procedure that depends on highly skilled operators.

Figure 13. The Helix™ catheter system.

Figure 15. NOGA 3D map and direction of cells to the border zone of a myocardial infarct. Injection sites are marked in black that were injected to the border zone of an MI (MI=red).

Figure 16. NOGA 3D map with examples of injection sites to avoid. Injections should be avoided in the apex, LBBB, and mitral valve.

Transendocardial delivery

Catheter-based direct intramyocardial injection approaches the myocardium from the endocardial surface. The demands of this task have led to the development of devices constructed of multiple components. The Helix™ catheter (Biocardia) has a helical tipped needle that is rotated into tissue to provide active fixation during drug delivery similar to the active fixation electrodes used in cardiac pacing (Fig. 13). The helical design, based on pacemaker lead technology, may be advantageous with regard to stability of the needle tip during injection. This catheter is under investigational use in multiple ongoing clinical trials (25,59). This system provides a means for fixation to the beating heart wall, uses simplified fluoroscopic imaging, crosses the aortic arch and valve safely over a guide wire with BioCardia’s unique steerable guide, and allows 3 degrees of freedom to maximize operator control. The Morph Deflectable Guide Catheter can be used to steer a guidewire across the aortic valve for transendocardial delivery from within the left ventricle. For transendocardial delivery, the Morph Deflectable Guide Catheter is advanced over the wire, and the wire is removed to allow for advancement and navigation of the Helical Infusion Catheter. The Stiletto™ catheter is one with a spring-loaded needle that is set to a fixed depth (3.5 mm) and may be more able to penetrate fibrotic tissue. The Stiletto™ has been used in patients for injection of gene therapy agents, but not for use with cells. With gene therapy, there was a high incidence of perforations, most likely related the relatively large volume injected per site.

The Myostar Injection Catheter (Biosense Webster) is a multi-electrode, percutaneous catheter with a deflectable tip and injection needle used to inject cells transendocardially into the myocardium (19,60). The tip of the injection catheter is equipped with a location sensor and a retractable, hollow, 27-gauge nitinol needle for fluid and/or cell delivery. Tip deflection is controlled at the proximal end by a tubular hand piece holding a piston and pull-wire mechanism. The high-torque shaft allows for controlled rotation of the curved tip to facilitate accurate positioning toward the desired site for agent injection (19). A second handle located at the proximal end of the catheter allows for controlled needle extension from the distal tip. The extended length of the needle is adjustable from 2 to 10 mm. The handle has a standard luer lock fitting for a syringe connection. The catheter interfaces with the NOGA (Biosense Webster) 3D electromagnetic cardiac mapping system for navigated local agent delivery into the myocardium. The NOGA map is a 3D reconstruction of the LV from points obtained by the mapping catheter. The NOGA map discriminates between areas of MI, the border zone, and normal myocardium (Figs. 14-16). With the combination of echocardiography and low voltage, one can identify the area of MI and thin wall.

All of the intramyocardial injection systems carry common risks such as local tissue damage up to and including cardiac perforation that could lead to pericardial tamponade, inflammation leading to arrhythmia, peripheral embolization and stroke. 3D – mapping with NOGA has shown the lowest occurrence of any of these events, with no reports of perforations which were seen with 2D approaches61. The perforations could have been due to exaggerated or forceful injection, which is less likely with the NOGA system as the operator has direct feedback from the mapping catheter as to the placement of the needle relative to the tissue, and the length of the needle can be adjusted to avoid puncturing the thinnest areas of the infarct. Also, excessive injected volume can cause perforations. More injections can be performed with the 3D system as tracking of injections is accurately displayed on the 3D map (Figs. 14–16). Arrhythmias have been associated with both the 3D- mapping catheter and needle injections as these can cause irritation to the endocardium that then produces non sustained and sustained ventricular tachycardia (VT) and even ventricular fibrillation (V-Fib). Attention should be paid to VT and the catheter should be pulled back from the endocardium when non-sustained VT becomes prolonged to avoid sustained VT and V-Fib. Injecting the area of left branch bundle might lead to and to bradycardia, especially in patients with previous RBBB.

The 3D-NOGA mapping system provides feedback during injections as to the placement of the catheter on the endocardial surface, providing a stability measure that protects against cells being injected into the ventricular cavity resulting in possible embolization (Fig. 15). Careful attention to the position of the tip of the catheter during needle advancement is essential. In some cases advancement of the needle can push the catheter backward without needle penetration of the wall of the ventricle leading to cell leakage to the ventricular chamber. Too much pressure must be avoided as well. Perforation can occur by applying aggressive pressure since aggressive pressure can decrease the thickness of that point of the wall and consequently the needle rip through the stretched area (Fig. 18). The position and freedom of movement of the catheter must be guarded. It is recommended that the catheter be free and moving with the beating heart and not jammed between the walls of the left ventricle in a position where one wall can push the catheter toward the opposite wall, jamming and causing uncontrolled pressure generated by the heart contractions.

Figure 17. Example of needle advancement that caused a catheter to move backward.

Figure 18. Setting the needle length. Too much pressure can decrease the thickness of the myocardium, therefore, the needle length must always be set shorter than the depth of the injection.

Certain areas of the heart should always be avoided such as apex, as it is too thin and the risk of perforation too high. It is also recommended to avoid injection to the mitral valve areas to avoid any unnecessary consequences. The mitral valve areas can be seen as low voltage areas on the 3D-NOGA mapping (Fig. 14). Finally, it is recommended to avoid crossing severely calcified aortic valve.

Retrograde Coronary Sinus Epicardial Injection

Retrograde coronary sinus delivery has been compared with both intracoronary (IC) and intramyocardial (IM) delivery in preclinical models62. The technique seems to be very safe and has potential advantages for more homogenous delivery across the myocardium. Theoretically, it might be lower risk than IM delivery, with more uniform and higher rates of delivery. The coronary sinus approach would also be available for patients excluded from trials using percutaneous endomyocardial delivery, such as patients with severe aortic valve disease. Compared with IC delivery, it has the theoretical advantage of more homogeneous delivery, in particular, in patients with severe subtotal stenosis. The technique involves placement of a catheter into the coronary sinus via either the internal jugular or femoral vein, with the infusion catheter placed over a wire. The TransAccess MicroLume Intramyocardial Injection System (Medtronic, Inc., Santa Rosa, California) has been the system used for this delivery method.

D. Conclusions

Many exciting advances have occurred in the area of cell delivery to the infarcted myocardium, but many technical challenges remain. The success of any cell therapy for heart damage will depend predominantly on the efficacy of cell delivery. The notion that heart repair can be achieved without cell survival needs to be challenged and researchers need to refine the methods for cell delivery and choice of cells for repair. The data collected so far argue that there still need to be improvements in the injection devices used. The best route: intracoronary, intramyocardial or transvenous still need to be sorted out. The intravenous approach however can be removed as an alternative as it is simply too ineffective at delivering cells to the myocardium. As researchers learn more about the best type of cell for cardiac repair, it will be interesting to see which method and catheter system will prove the most effective.

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