電気刺激を使用してBiowires中のヒト幹細胞由来の心筋細胞の成熟

The overall goal of this procedure is to utilize controlled three dimensional cell cultivation methods in electrical stimulation to promote the maturation of human embryonic and induced pluripotent stem cells derived cardiomyocytes. This method can help answer key questions in the cardiac tissue engineering field including how different electrical stimulation parameters or length of time affect cardiac cell function and maturation state. The end results are engineered human cardiac tissues of improved maturity.

The implications of this technique extend toward diagnosis applications such as testing the safety and efficacy of new drugs because cardiomyocyte with mature characteristics such as electrical progress are most likely to serve as more accurate models of human cardiac cells in drug screening studies. Combine the PDMS monomer and curing agent at a 10:1 ratio and mix well. Let it set for five minutes to degas.

Then load a Petri dish containing the master for the biowire template with the PDMS mixture. Try to minimize air bubble formation. Next cure the PDMS at 70 degree Celsius for two hours.

Then recover the solidified mold by first using a sharp blade around the edges and then peeling it away. Then trim the excess PDMS and collect all the templates in a sterilization pouch. Steam auto clave the templates at 121 degree Celsius for 20 minutes.

And then load each template into a sterile Petri dish under the ventilation of a biosafety cabinet. Now for each template use tweezers to mount a piece of sterile 6.0 silk suture into the groove spanning the entire channel. The templates are then ready for cell seeding.

The suture will serve as a template for the formation of 3D constructs that align along the suture axis mimicking the shape of cardiac fibers. For each chamber, make a pair of rectangular polycarbonate pieces to serve as the frame for the electrical stimulation chamber. Along the center line of each piece, drill two three millimeter wide holes, two centimeters apart.

For each chamber prepare two 1.5 centimeter long carbon rods. About 3 millimeters from the end of each rod drill a one millimeter wide hole. Then thread each hole with a platinum wire and wrap one end of the wire around the rod.

Next, for each chamber insert two rods between two polycarbonate pieces making a frame. Then pour about two milliliters of PDMS into a well of a six well plate and place the frame into the well with PDMS, submerging the bottom of the frames. Cure the PDMS at 70 degree Celsius for two hours so the PDMS secures the frame.

Then remove the completed chamber from the plate with the PDMS attached. To test the function of an assembled chamber connect the platinum wires to a battery and use a voltmeter to check that electricity is being passed through the rods. Now transfer the functional chambers into a sterilization pouch and use 20 minutes of steam sterilization at 121 degree celsius to prepare them for cell culture.

To prepare the gel, precool all the gel components on ice before mixing them in a sterile tube. Make sure to add the collagen and extra cellular matrix last and mix the components using pipetting. For the cells have cardiomyocytes differentiated from human pluripotent stem cells.

Collect the cells and for each biowire preparation plant prepare 3.5 microliters of collagen gel containing half a million cells. Now use a P10 pipette tip to load approximately four milliliters of suspension into the 0.5 centimeter long channel template in each dish. Adjust the position of the suture using sterile forceps as needed so it is fully stretched.

Once seeding the cells, make sure to always resuspend the gel cell mixture before taking autoclave in order to have consistent results. After loading the channels, cover the bottom of the dish with sterile PBS making sure not to touch the gel. This will ensure that the biowires do not dehydrate during polymerization.

After incubating the biowires at 37 degree celsius for 30 minutes, replace the PBS with enough culture medium to cover the cells. 12 milliliters is usually enough. Then culture the dish for a week replacing the medium every other day.

In a bio safety cabinet add two milliliters of PBS to each well of a six well plate that will house an electrical stimulator chamber. Then use sterile tweezers to gently place the autoclave devices into the wells. Once in position, discard the PBS and replace it with five milliliters of warm medium which should completely cover the carbon rods.

Before culturing the cells for 24 hours, observe the cardiomyocytes contractility in response to electrical stimulation to ensure that the cells are capturing the electrical stimulus and responding with contractions. Next, place the biowires into the electrical stimulator chamber and orient the channels perpendicularly to the carbon rods. Extend the platinum wires away from the plate and then cover the plates.

Connect the wires to an electrical stimulator. Set the stimulator to deliver biphasic one millisecond pulses at one hertz and at three volts per centimeter. Then transfer the preparations in the electrical stimulator to an incubator at 37 degree celsius.

Increase stimulation rate incrementally every 24 hours from one hertz at day zero to six hertz at day six. During this week of culture, change the medium every other day. And observe the cardiomyocytes contractility on a daily basis.

After seven days of preculture in the biowire cells remolded the gel around the suture. The cells assembled along the axis of the suture to form aligned cardiac tissue. The cells expressed cardiac contractile proteins showed sarcomeric banding in the contractile apparatus and their myofibrils were aligned along the axis of the suture.

Cells in the biowire exhibited lower proliferation rates than cells in embryoid bodies. Calcium handling was significantly improved in the cardiomyocytes cultivated in biowires. Treatment with caffeine induced an abrupt release of calcium from the sacroplasmic reticulum.

Such transients were found only in stimulated cells. There were also significantly higher stimulation frequency depending calcium release amplitudes. In addition, cardiomyocytes cultured in biowires showed improved hERG and inward rectifier current densities compared to cultivation in embryoid bodies which was further enhanced by electrical stimulation.

The improved inward rectifier current was consistent with increased expression of the potassium inwardly rectifying channel jean, KCN J2.Automaticity the ability to beat spontaneously was significantly higher in embryoid body derived cardiomyocytes compared to controlled biowires. Control showed automaticity comparable to biowire preparation subjected to the six hertz regimen. Once mastered the setup of the biowires can be done in half a day.

The setup of electrical stimulation will take a couple of hours and the final results can be reached in two weeks. While attempting this procedure, it is important to remember that cardiomyocytes differentiated from different cell lines or by different protocols can vary slightly in their maturity and purity. Therefore optimizing the experimental parameters such as the stimulation rate to your cells might be required.

Following this procedure, other conditions including variation of the cellular composition and the utilization of mutant cardiomyocytes can be optimized to answer a bevy of additional questions.