preparation of cardiomyocytes and setting-up the apparatus for the slow injection method

Un nuovo metodo di iniezione lenta minimamente invasivo per ridurre le lesioni tissutali

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 &#181;m/s)13. To the best of the author&#39;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.

Autore in primo piano: un nuovo metodo di iniezione cellulare con un'invasione minima  

Un nuovo metodo di iniezione cellulare con un'invasione minima

In this work, the purpose is to improve the efficacy of regenerative medicines, especially heart regenerative medicine using induced pluripotent stem cells. Heart regenerative medicine is our most contributed research field. My significant findings include a metabolic selection of cardiomyocytes, i.e.a simple and mass purification mass way for cardiomyocytes with cultural condition of withdrawn glucose and added lactate. Another finding includes the efficient engraftment method for cardiomyocytes by publication of cardiomyocyte poles. This transplantation method with cardiomyocyte poles confirmed the enhanced engraftments of cardiomyocyte poles.However, the lack of direct interaction between the host and graft cardiomyocytes was found. This technique offers fewer invasions to the tissues receiving the cell injections. As a result of this, direct interaction between the host and graft cardiomyocytes was facilitated.This technology can diminish injuries, inflammatory responses, and eventually fibrosis, i.e. scar formation. Hopefully, this technology will be applied to the next generation of regenerative medicines, including heart regenerative medicine using pluripotent stem cells.We have already found several ways to bring significant merit to heart regenerative medicine. These studies will be continued in the future.

Watch this Scientific Journal Video about Preparation of Cardiomyocytes and Setting-Up the Apparatus for the Slow Injection Method at JoVE.com

Preparation of Cardiomyocytes and Setting-Up the Apparatus for the Slow Injection Method  

Preparazione dei cardiomiociti e messa a punto dell'apparecchio per il metodo dell'iniezione lenta

Preparare la soluzione madre di gelatina al 10% pesando e sciogliendo la gelatina nel tampone ADS e sterilizzando la soluzione in autoclave. Riscaldare il brodo di gelatina al 10% a 37 gradi Celsius, fino a quando non diventa liquido. Quindi diluire il brodo di gelatina, utilizzando un tampone ADS preriscaldato, per ottenere la soluzione finale iniettabile di gelatina al 5%.Sospendere 96 sfere di cardiomiociti in 100 microlitri di soluzione iniettabile preriscaldata. Caricare la sospensione a sfera del cardiomiocita nella siringa per iniezione cellulare senza aspirare l'aria in eccesso. Quindi, tenga la siringa verticalmente, con l'ago rivolto verso l'alto, e picchietta la siringa per raccogliere le bolle sulla cresta superiore della siringa.Inoltre, osservare le sfere di cardiomiociti che si depositano gradualmente sulla guarnizione in gomma dello stantuffo. Mantenere la posizione verticale della siringa e spingere lentamente lo stantuffo per eliminare le bolle e la soluzione di sospensione cellulare in eccesso, fino a quando 20 microlitri della sospensione cellulare rimangono nella siringa e la sfera del cardiomiocita rimane appoggiata sulla guarnizione in gomma dello stantuffo. Una volta fatto, immergere la siringa con tappo in un bagno di ghiaccio per cinque minuti e fissare l'apparato della siringa di iniezione sul tavolino dell'animale prima di attivare la pompa della siringa principale durante il trapianto di cellule percutanee in un ratto anestetizzato.

preparation-cardiomyocytes-setting-up-apparatus-for-slow-injection

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JoVE Journal

Bioengineering

Preparation of Cardiomyocytes and Setting-Up the Apparatus for the Slow Injection Method

342 Views.  Kansai Medical University. Preparare la soluzione madre di gelatina al 10% pesando e sciogliendo la gelatina nel tampone ADS e sterilizzando la soluzione in autoclave. Riscaldare il brodo di gelatina al 10% a 37 gradi Celsius, fino a quando non diventa liquido. Quindi diluire il brodo di gelatina, utilizzando un tampone ADS preriscaldato, per ottenere la soluzione finale iniettabile di gelatina al 5%.Sospendere 96 sfere di cardiomiociti in 100 microlitri di soluzione iniettabile preriscaldata. Caricare la sospen...

Video: Preparation of Cardiomyocytes and Setting-Up the Apparatus for the Slow Injection Method  

A Novel Cell Injection Method with Minimum Invasion

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 &#176;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.

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 ...

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Preparation of the Neonatal Rat Cardiomyocyte Balls for the Slow Injection Method

Begin by measuring the neonatal Rat Ventricular Cardiomyocytes concentration using a hemocytometer. Dilute the cell suspension solution to 3, 000 cells per milliliter, with alpha MEM containing 10%heat inactivated FBS. Distribute 100 microliters of the diluted ventricular cardiomyocyte in each well of the cell's non-adhesive 96 well plates.Centrifuge the cells for five minutes at 100 G.And culture them for two to three days in the cell culture incubator, with 5%carbon dioxide at 37 degrees Celsius. After incubation, aspirate the culture medium from the Rat Cardiomyocyte balls seeded in the non-adhesive 96 well plate. Then, transfer the cardiomyocyte balls from each well to a 15 milliliter tube.Centrifuge the cardiomyocyte balls and aspirate the supernatant. Then, wash them using an ADS buffer. To facilitate the tracking of the cardiomyocyte cells in the Rat after engraftment, stain the cells with PKH26.Before centrifuging the cells, track the development of bidim cardiomyocyte balls by observing them under the microscope regularly after seeding. Begin preparing the main injection apparatus for a neonatal cardiomyocyte ball injection using a 29 gauge, 50 millimeter long needle equipped with a syringe. Connect an 18 gauge one milliliter power transfer syringe needle to a thin polypropylene tube with low lumen expandability.Aspirate the water using a syringe, and remove all the bubbles from the syringe and the connected tubes. Repeat it for another 18 gauge needle. Then, connect the other side of the power transfer tube to the 18 gauge needle of the same one milliliter syringe.Confirm the successful power transition by pushing one syringe plunger down, and another syringe plunger up. Once done, set the syringe in a syringe pump. Next, set up the cooling system by attaching the tightly coiled copper tube to the cell containing part of the cell injection syringe.Leaving 10 millimeter of excess pipe at both ends. Connect the copper tube to the flexible plastic tubing before connecting the other ends of the plastic tubing to an external pump. Fill the line with cooling water, and immerse the excess copper tube into crushed ice to cool the water.This cooling system maintains the cell suspension solution in the cylinder at approximately 2 degrees Celsius.

Preparazione delle palline di cardiomiociti neonatali di ratto per il metodo di iniezione lenta

Prepare the 10%gelatin stock solution by weighing and dissolving the gelatin in the ADS buffer, and autoclaving the solution. Rewarm the 10%gelatin stock at 37 degrees Celsius, until it becomes liquid. Then dilute the gelatin stock, using a prewarmed ADS buffer, to obtain the final injectable 5%gelatin solution.Suspend 96 cardiomyocyte balls in 100 microliters of prewarmed injection solution. Load the cardiomyocyte ball suspension into the cell injection syringe without aspirating the excess air. Next, hold the syringe vertically, with the needle facing upward, and tap the syringe to collect the bubbles at the upper ridge of the syringe.Also, observe the cardiomyocyte balls gradually settling onto the rubber seal of the plunger. Maintain the vertical position of the syringe, and slowly push the plunger to discard the bubbles and excess cell suspension solution, until 20 microliters of the cell suspension remain in the syringe, and the cardiomyocyte ball remains resting on the rubber seal of the plunger. Once done, immerse the capped syringe in an ice bath for five minutes, and fix the injection syringe apparatus on the animal stage before activating the main syringe pump during percutaneous cell transplantation in an anesthetized rat.