This method can help address key issues in current exacerbated cardiac therapy, such as low applicability to low retention and survival of transplanted cells in infarcted hearts. To address this issue, this method can provide a reliable technique to study intramyocardial transplantation with stem cells using injectable hydrogels in a murine model, which is an excellent platform to use for investigating cardiac tissue repair and regeneration after myocardial infraction. The main advantage of this technique is that it is a feasible method to improve the retention and survival of transplanted stem cells after intra-myocardial transplantation using in-situ cross-link cover injectable hydrogels.
These hydrogels offer diverse opportunities for cardiac tissue engineering, such as delivering not only stem cells, but also growth factors, genetic materials and drugs to maximize their therapeutic effect in infarcted heart tissues. Demonstrating the procedure for in-vitro experiment will be Eunmi Lee, a research technologist from my laboratory. Eun-Hye Park and Eunhwa Seong research technologists will demonstrate the procedure for in-vivo experiments.
Culture mesenchymal stem cells, or MSCs in a 100 millimeter culture dish at 37 degrees Celsius and 5%carbon dioxide. When the cells reach 80%confluence, wash the dish twice with DPBS and incubate them with one milliliter of trypsin substitute at 37 degrees Celsius for three minutes. Then add nine milliliters of culture medium to the cells and centrifuge them at 500 times G for three minutes.
Discard the resulting supernatant. Resuspend the cells in one milliliter of PBS and maintain the cell suspension on ice. Dilute 10 microliters of cell suspension with 10 microliters of trypan blue and determine the cell concentration using an automated cell counter.
Resuspend the MSCs to a density of one times 10 to the seventh cells per milliliter and transfer them to a one milliliter tube. Prepare a 6.25%GH conjugate solution in PBS and separate it into two vials. Next mix the GH solutions with either six micrograms per milliliter of HRP or 0.07%hydrogen peroxide.
Briefly centrifuge the cell suspension at 1000 times G and carefully aspirate the resulting supernatant. Then mix the cell pellet with GH solution A.Load the MSCs in GH solution A and GH solution B into either side of a dual syringe. Plate 300 microliters of the combined GH solutions with MSCs at a final density of 5 million cells per milliliter onto an eight well chamber slide.
After in situ hydrogel formation and subsequent MSC encapsulation via enzymatic cross-linking add 700 microliters of DMEM containing 10%FBS and 1%antibiotic antimycotic solution. Incubate the slide at 37 degrees Celsius and 5%carbon dioxide and replace the culture medium every two to three days. Prior to surgery, depolate the mouse chest using hair removal cream and sterilize the skin with iodine.
Place the mouse on an operating table and intubate it by inserting a catheter into the trachea to provide supplemental oxygen via mechanical ventilation. Gently cut through the skin using surgical scissors. Then penetrate the intercostal muscles with micro scissors.
Separate the second and third left ribs using a 5-O silk suture to maintain an open chest cavity. Carefully ligate the left anterior descending coronary artery using a needle holder with an 8-0 polypropylene suture and cut the suture using electrocautery. Observe an immediate color change in the anterior left ventricular wall.
After inducing the myocardial infarction, inject 10 microliters of the MSC loading GH solutions into two different points at the infarct border zone. Restore the opened chest cavity and close the muscles and skin using 5-0 sutures. Remove the tracheal tube and place the mouse in a cage under an infrared lamp during recovery.
Perform an echocardiography and measure corresponding lines for LV anterior wall, LV internal and LV posterior wall to obtain cardiac wall thickness, chamber dimension, and fractional shortening. Prior to in vivo transplantation, the proliferation and survival of MSCs in GH hydragels were confirmed by a 3D in vitro live dead cell staining assay. Representative images exhibited sufficient MSC proliferation showing branched networks within GH hydrogels.
An extensive multicellular 3D structure of MSCs was clearly observed at day 14, indicating that GH hydrogels could provide a proper microenvironment for the encapsulated cells. After the induction of myocardial infarction, MSC loading GH hydrogels were intramyocardially transplanted into the peri-infarct areas. The MSCs and gel were appropriately sustained within the infarcted region.
MSCs were well-integrated into GH hydrogels. To verify the therapeutic effects of the MSC loading GH hydrogels, the changes in cardiac function and structure were evaluated by echocardiography and histological analysis at day 28 post transplantation, and compared among the different treatment groups. The representative echocardiography showed improved cardiac functions, including fractional shortening, ejection fraction, and end systolic volume in the MSC gel treated group.
In addition, histological analysis exhibited less fibrosis, thicker infarcted walls and a smaller infarct size in the MSC gel treated group than in the other groups indicating that this protocol significantly attenuated LV remodeling. Using this technique, we achieved long term stem cells in and proliferation and led to significant improvement in the cardiac structure and function following myocardial infarction in murine model. This technique can be extended for use in large animals and to clinical translation as a new strategy for prevention of a post infarct heart failure by providing cardiac tissue repair and regeneration.
