This method eliminates any major invasion during cell injections caused by the cell suspension solution.
Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient amount of suspension solution to allow the cells to enter the tissue. The volume of the suspension solution affects the tissue, and this can cause major invasive injury as a result of the cell injection. This paper reports on a novel cell injection method, called slow injection, that aims to avoid this injury. However, pushing out the cells from the needle tip requires a sufficiently high injection speed according to Newton’s law of shear force. To solve the above contradiction, a non-Newtonian fluid, such as gelatin solution, was used as the cell suspension solution in this work. Gelatins solution have temperature sensitivity, as their form changes from gel to sol at approximately 20 °C. Therefore, to maintain the cell suspension solution in the gel form, the syringe was kept cooled in this protocol; however, once the solution was injected into the body, the body temperature converted it to a sol. The interstitial tissue fluid flow can absorb excess solution. In this work, the slow injection technique allowed cardiomyocyte balls to enter the host myocardium and engraft without surrounding fibrosis. This study employed a slow injection method to inject purified and ball-formed neonatal rat cardiomyocytes into a remote area of myocardial infarction in the adult rat heart. At 2 months following the injection, the hearts of the transplanted groups showed significantly improved contractile function. Furthermore, histological analyses of the slow-injected hearts revealed seamless connections between the host and graft cardiomyocytes via intercalated disks containing gap junction connections. This method could contribute to next-generation cell therapies, particularly in cardiac regenerative medicine.
Cell administration and replacement are promising novel therapeutic strategies for heavily damaged organs. Among these novel therapeutic strategies, cardiac regenerative medicine has attracted considerable attention. However, the inflammation caused by injuries mediates scar formation in several organs1,2,3,4. The human heart consists of approximately 1010 cardiomyocytes; therefore, theoretically5,6, it must be treated with more than 109 cardiomyocytes. Administering a large number of cardiomyocytes via traditional injection methods may lead to significant tissue injuries7. This method provides a novel cell injection method with minimal tissue invasion.
Cell administration into the organ parenchyma requires injection(s). However, a discrepancy exists in that the injection itself may lead to tissue injury. Tissue injury causes local inflammation and incurable scarring in organs and tissues, as well as impaired regenerative ability8,9,10. The mammalian heart has an extremely high propensity to develop scars instead of regenerating because it requires immediate injury repair in order to endure the high blood pressure caused by its continuous pumping function11. Ablation therapy utilizes this high propensity toward scar formation and blocks the circuit likely to undergo scar formation using arrhythmia12. In a previous study, it was observed that the scar tissue isolated the injected cardiomyocytes in the host myocardium. Thus, this represents the next target issue that needs to be overcome to obtain improved therapeutic efficacy in cardiac regenerative medicine.
Tissue interstitial fluid flow plays a vital role in conveying oxygen and nutrients to cells and removing the excreted waste from cells. The physiological speed of interstitial fluid flow in each tissue/organ is different (the range is 0.01-10 µm/s)13. To the best of the author's knowledge, there are no data regarding the capacity of individual tissues/organs to support extra amounts of fluid without pathological edema; however, this experiment attempts to use a slow injection speed to possibly reduce tissue injury, and the results can be used to determine the practicality of this concept.
The animal experiments were conducted according to the Kansai-Medical University ethical guidelines for animal experiments and were approved by the ethical committees (approval number: 23-104). All the animals were raised under a constant light-dark cycle in a specific pathogen-free environment. All the sterilized surgical tools, such as scissors, forceps, and retractors, were autoclaved and dried thoroughly.
1. Preparation of the neonatal rat cardiomyocyte balls
2. Preparations for the slow injection method
3. Development of a rat myocardial infarction model by obtuse coronary artery occlusion
4. Echo-guided percutaneous cell transplantation using the slow injection method
5. Evaluation of cardiac function
6. Immunohistochemistry
Effects of the slow injection on cell survival and collagen deposition
Neonatal rat cardiomyocyte balls labeled with PKH26 were injected into normal nude rat myocardium using a normal or slow injection method. The results showed that the slow injection method significantly increased the engrafted cell volume (Figure 3A) and significantly decreased on-site type I collagen deposition (Figure 3B).
Effects of slow injection on treatment efficacy in a rat infarction model
The echocardiograph-guided slow injection method was used to inject neonatal rat cardiomyocyte balls or PBS(−) into the infarcted hearts of model rats. The cell-injected group alone exhibited significant improvement in heart contraction function after 2 months (Figure 4A). Immunohistochemical analyses revealed a seamless connection between the engrafted cells and host myocytes via intercalated disks containing gap junctions (Figure 4B).
Figure 1: Schematic of the whole injection system. (A) Main injection apparatus. (B) Injection syringe cooling system. Please click here to view a larger version of this figure.
Figure 2: Echocardiography-guided percutaneous slow injection. (A) Setting of the animal, echo probe, and injection apparatus. (B) Echocardiographic view of the injecting syringe and heart. Note that the left and the right pictures are the same, but a yellow line has been added to indicate the needle position. Please click here to view a larger version of this figure.
Figure 3: Effect of the slow injection method on the engrafted cell volume and collagen deposition. (A) The engrafted cell volumes (N = 3) were calculated from serial sections. The error bars indicate standard deviations. *P < 0.01 in a non-paired t-test. (B) Immunohistochemical staining for type I collagen. The scale bar indicates 200 µm. Please click here to view a larger version of this figure.
Figure 4: Improvements in heart function and histological integration with cardiomyocyte balls engrafted by the slow injection method. (A) Representative echocardiograph M-mode views. The graph shows the transition of fraction shortenings in the cardiomyocyte ball-transplanted group (solid red line; N = 4) and vehicle (cell suspension solution for the slow injection method) group (dotted blue line; N = 3). Abbreviations: MI = myocardial infarction; Cx43 = connexin 43; DAPI = 4',6-diamidino-2-phenylindole; PKH26 = red fluorescent cell membrane label. The error bars indicate the standard deviations. *P < 0.01 in a paired t-test. (B) Immunohistochemical analyses of the relationship between the engrafted cardiomyocyte balls and host cardiomyocytes. The conventional laser microscopic observations using a 2x objective lens are shown in the left column. Zoomed-in versions (using a 20x objective lens) of two areas shown in the box, labeled with * and #, are presented below. Scale bars: top image = 300 µm; * and # = 30 µm. Confocal laser microscopic images using a 20x objective lens are shown along for comaparison. Three positions are shown. In the merged images, the arrowheads indicate the existence of gap junctions (Cx43) directly connecting the graft and host cardiomyocytes. The scale bar indicates 30 µm. Please click here to view a larger version of this figure.
Supplementary Video 1: Echo-guided slow injection method. The B-mode echocardiogram in the frontal view shows the injection needle tip advanced into the myocardium. Please click here to download this File.
One of the critical points in the successful performance of the slow injection method is the preparation of an effective injection system using a powerful syringe pump and a strong pressure transfer tube. A high-pressure system is required to push gel out from the tip of a fine needle. The second critical point is the stabilization of the heart. The beating of the heart against an injection needle advanced into the myocardium can injure the tissue. In this study, an echo-guided injection was conducted to avoid the animals undergoing a second open chest injury and to administer the cell injection in a stabilized heart with the lungs inflated. Moreover, in some applications for larger animals or humans, some injection devices attached onto the heart should be considered as part of the strategic design of the application. For open chest injections into the hearts of small animals, the use of a long, flexible needle is recommended given their higher heart rates.
In this work, the slow injection method significantly increased the surviving cardiomyocyte volume compared to the normal injection method. The normal injection causes cell damage via shear stress15. In contrast, the slow injection method does not cause such stress theoretically because it uses a non-Newtonian solution in addition to the slow injection.
In terms of local fibrosis, the interstitial space around the normally injected surviving cardiomyocytes showed strong and widespread type I collagen deposition. In contrast, the type I collagen signals around the engrafted cardiomyocytes grafted using the slow injection method were much weaker and more limited. This suggests that the slow injection method caused significantly less damage. The slow injection of neonatal cardiomyocytes into the adult myocardium significantly improved the contractile function of the infarcted heart. The histological analyses suggested that grafting the cardiomyocytes using the slow injection method resulted in direct connections and functional coupling with the host cardiomyocytes. This phenomenon explains the mechanism of the functional recovery of the host myocardium. To the best of our knowledge, this is the first report of engrafted neonatal cardiomyocytes with large-scale seamless connections to the host adult cardiomyocytes. The functional connections with the host myocardium via electrical and mechanical coupling may make the engrafted cardiomyocytes mature and allow them to act as functional myocytes that contribute to the host heart function. Long-term physical force interactions between the host and graft cardiomyocytes are crucial for full maturation. Therefore, 2 months might be required after the injection for the functional recovery of the infarcted heart. The time-dependent recovery of the patient's heart function may be an expected phenomenon in therapeutic applications, and this can be a hallmark of the successful establishment of de novo functional coupling and integration between the host and grafted cardiomyocytes.
The slow injection method can be performed during open chest surgery. In addition, this method can be applied to mice. For future applications in human therapy, we still need to resolve several issues. The injection speed should be optimized by considering the buffer capacity of the interstitial fluid flow in each human target organ. Xeno-free materials, such as human gelatin or biodegradable synthetic materials, should be applied. Clinical GMP-grade slow injection apparatus, such as compact organ-specific disposable tools or a reusable wide-organ applicable apparatus, should be developed.
The authors have nothing to disclose.
This study was supported by a grant from JSPS KAKENHI (Grant No. 23390072 and 19K07335) and AMED (Grant No. A-149).
18-gauge needle & tuberculin, 1 mL | Terumo | NN1838R, SS-01T | |
29-gauge 50 mm-long needle | Ito Corporation, Tokyo, Japan | 14903 Type-A | |
A copper tube | General Suppliers | outer diameter, 1 mm; inner diameter, 0.3 mm; thickness, 0.35 mm | |
Ads Buffer | Each ingredient was purchased from Fuji Film Wako Chemical Inc., Miyazaki, Japan | Hand made, Composition: 116 mM NaCl, 20 mM HEPES, 12.5 mM NaH2PO4, 5.6 mM glucose, 5.4 mM KCl, 0.8 mM MgSO4, pH 7.35 | |
alpha-MEM | Fuji Film Wako Chemical Inc., Miyazaki, Japan | 051-07615 | |
Anti-collagen type I rabbit polyclonal antibody (H+L) | Proteintech | 14695-1-AP | using dilution 1:100 |
Anti-Connexin-43 rabbit polyclonal antibody (H+L) | Sigma Aldrich | C6219 | using dilution 1:100 |
Anti-rabbit IgG (H+L) donley polyclonal antibody-AlexaFluo488 | Thermo Scientific | A21206 | using dilution 1:300 |
blocking solution (Blocking One) | Nacalai Tesque, Kyoto, Japan | 03953-95 | |
collagenase | Fuji Film Wako Chemical Inc., Miyazaki, Japan | 034-22363 | |
confocal laser microscope | Carl Zeiss Inc., Oberkochen, Germany | LSM510 META | |
DNase I | Sigma-Aldrich | DN25 | |
FACS Aria III | Becton Dickinson, Franklin Lakes, NJ, USA | ||
fetal bovine serum | BioWest, FL, USA | S1820-500 | |
fine movement device (Micromanipulator) | Narishige Co., Ltd., Tokyo, Japan | M-44 | |
fluorescence microscope | Nikon Instruments, Tokyo, Japan | Eclipse Ti2 | |
gelatin from bovine skin | Sigma-Aldrich | G9382 | dissolving in PBS (-) to 10%, and autoclaving it |
Neonatal Sprague-Dawley (SD) rats | Japan SLC Inc., Shizuoka, Japan | 0–2 d after birth | |
non-adhesive 96-well plates (spheloid plate) | Sumitomo Bakelite, Tokyo, Japan | MS-0096S | |
Optimal Cutting Temperature (OCT) Compound | Sakura Finetek USA, Inc., CA, USA | Tissue-Tek OCT compound | |
peristaltic pump (for cooling system) | As One Co., Osaka, Japan | SMP-23AS | |
PKH26 | Sigma-Aldrich | PKH26GL | |
Stir Bar, Micro, Magnetic, PTFE, Length x Dia. in mm: 5 x 2 | Chemglass life sciences LLC, NJ, USA | CG-2003-120 | |
syringe | Ito Corporation, Tokyo, Japan | MS-N25 | |
syringe pump with remote controller | As One Co., Osaka, Japan | MR-1, CT-10 | |
tetramethylrhodamine methyl ester | Thermo Fisher Scientific, Waltham, MA, USA | T668 | |
trypsin | DIFCO, Becton Dickinson, Franklin Lakes, NJ, USA | 215240 | |
Tween-20 | Fuji Film Wako Chemical Inc., Miyazaki, Japan | 167-11515 | |
veterinarian ointment | Fujita Pharmaceutical Co., Ltd. | Hibikusu ointment #WAK-95832 | |
Vevo 2100 Imaging System | Fujifilm VisualSonics, Inc., Toronto, Canada | Vevo 2100 | |
Vevo 2100 Imaging System software version 1.0.0 | Fujifilm VisualSonics, Inc., Toronto, Canada | Vevo 2100 | |
Weakly curved needle with ophthalmic thread | Natsume Seisakusho Co., Ltd., Tokyo, Japan | C7-70 |