This system can be used to expose engineered heart tissues to varying regimens of afterload, in order to study the effects of these stimuli on tissue force development, remodeling and maturation. Our technique allows one to precisely subject beating tissues to a broad range of customizable afterload routines over prolonged periods of culture without even needing to open the cell culture plate. Our method could be modified to control afterload in other muscle tissue culture system, such as skeletal muscle, smooth muscle or excised papillary muscle.
Since our systems approach is rather unique and features technical aspects that many scientists are not familiar with, visual demonstration may aid in recreating our setup. To manufacture the magnetically responsive silicone racks, acquire the 24 well plate can paddle racks of silicone posts described in the text protocol. Using a fixed polarity, lubricate the magnets with water and insert them one at a time into the outermost posts of the silicone racks.
Use a blended piece of stainless steel dental wire to carefully push them to the bottom of the hollow post cavity. Up to five magnets can be stacked in each post. Use round nose pliers to bend stainless steel dental wire into braces 11.25 millimeters wide and 15 millimeters long.
To ensure the correct dimensions are achieved, one can use a self made jig to aid in the wire bending, then use wire cutters to cut the braces and the file to smooth the cutting surface. Lubricate the braces or posts with water and insert them into the silicone rack fixing the second and third to the outermost post in the process. Begin preparing the afterload the tuning device as described in the text protocol, attach to the magnet holder to the piezoelectric stage using a non magnetic material, this can be achieved using an L shaped piece of aluminum.
To enable visual analysis of the tissues, install a light source within the afterload tuning device. Here an array of LEDs was employed to illuminate the engineer to heart tissues from below. To calibrate the afterload tuning system, mount one of the silicone racks and vertically using non magnetic materials, such that the magnetically responsive silicone posts are oriented horizontally.
Now mount one of the plate magnets on a horizontally traveling linear stage such that it is axially aligned with the magnetically responsive post. Position the calibration magnet a defined distance from magnetically responsive silicone post using the horizontal stage. Place a camera to the side of this setup in order to be able to optically record the posts deflection under the influence of the test loads.
Make sure that there is enough space below the post for the attached loads to hang freely. Take a picture of the post in the absence of any weights to use as a reference for the posts neutral position. Without changing the camera's perspective, attach one of the loads to the very end of the silicone post, then take a picture of the post bending under the influence of the weight.
Now graph the deflection of the silicone post on the x-axis against the gravitational force of each test weight on the y-axis. This should yield a linear relationship between force and deflection. Plot a linear regression function passing through 00 and the acquire data.
The slope of this function is the stiffness, k of the magnetically responsive silicone post at the tested magnet spacing. Repeat these steps at several spacings between dmax and a dmin. Here deflections at eight different magnet positions, ranging from about 31 millimeters to about five millimeters were analyzed.
A lot of regression function through these values. For example, use nonlinear fit, one phase decay function in the analysis software. This regression function describes the relationship between magnet spacing and afterload.
To prepare the afterload tuning device for experiments, connect the piezoelectric stage motor to the motion controller and connect the motion controller to the computer. Make sure the motion controller is also connected to a power source. Then start the motion controller platform software, connect the software to the piezo stage motor by selecting the port designated as the stage board during installation of the motion control software and then click the open port button.
Go to the system panel, select open loop, in the loop drop down menu. Manually move the magnet plate to its highest position, the closest possible magnet spacing dmin. The magnet plate should make contact with the culture plate mount.
Now go to the Motion Panel, like the zero button to reset the current position of the piezo stage to zero millimeters. Manually move the magnet blade to its lowest possible position, write down the encoder position to determine the range of motion for the piezoelectric stage motor. Set the travel limits in the system panel to values within the range of motion determined in the previous step.
This prevents the magnet blade from bumping into the culture blade or the bottom of the afterload tuning device. Once again, move the magnet blade to its highest position and click the zero button. Go to the system panel and change the feedback loop mode to closed loop, doing this ensures that the stage will correct for any errors in its positioning.
Click the save button in the store parameters box to store these settings in the system. Now place the 24 well culture plate containing engineered heart tissues on magnetically responsive silicone racks on the culture plate mount. To calculate the magnet spacing necessary to achieve a desired afterload, solve the nonlinear regression function determined earlier for d.
Subtract dmin from the calculated magnet spacing, d. The result is the distance the magnet plate has to travel from its zero position to achieve the desired afterload. Type this value into the Target Position one input field in the Motion panel and click Go to adjust the engineered heart tissue's afterload to the calculated value.
Control an MREHTs produced from rat hearts were cultured in the absence of magnetic afterload until a plateau in contractile force was reached. On this day, MREHTs and controlled EHTs had similar mean forces. Over the next week, the afterload exerted on MREHTs was incrementally increased from point nine one to six point eight five millinewton's per millimeter, while afterload for controlling EHTs remained constant.
Mean contractile force increased with increasing afterload up to point nine five millinewton, which marks more than a three fold increase in force compared to the average value measured for controlled EHTs. Post deflection on the other hand decreased compared to control tissues. On the last day of culture the main deflection measured for MREHTs was only point one one millimeters, compared to point four eight millimeters for control EHT's.
Rad EHTs on magnetically responsive silicone posts were cultured at a minimal afterload of point nine one millinewton per millimeter until a plateau and contractile force was reached. From this day onwards, MREHTs underwent a seven day afterload regimen which expose the EHTs to cycles of afterload alternating between point nine one and six point eight five millinewton per millimeter. The afterload of control EHT's was kept constant at point six zero millinewton per millimeter over the entire duration of culture.
The observed differences were not statistically significant. Combined with optical contractility analysis, this method enables the real time measurement of short term contractile response to fluctuating magnitudes of afterload, which could be useful for investigating physiological muscle properties. Strong magnets may suddenly cling to each other, potentially causing injury to the user and damaging the magnets themselves, to avoid this, keep the magnet separated at a safe distance.
