Magnetic Adjustment of Afterload in Engineered Heart Tissues

1. Preparation of the Afterload Tuning Platform

NOTE: The steps involved in this portion of the protocol are not time-sensitive.

1. Manufacturing the magnetically responsive silicone racks
   NOTE: These racks serve as the culture platform for EHTs. Each EHT is suspended between two silicone posts, which impart afterload to the tissue. The degree of afterload is directly related to the stiffness of these posts. To enable magnetic afterload tuning, some of the posts need to be magnetically responsive.
   1. Acquire 24-well plate compatible racks of silicone posts (dimensions given in Supplementary Figure 1). The racks used in this study were produced by a commercial silicone goods supplier according to these dimensions using silicone with a shore hardness of 40.
   2. Determine the polarity of the post magnets (e.g., d = 0.5 mm, h = 2.0 mm; see Table of Materials) by placing them on a larger permanent magnet.
   3. Keeping a fixed polarity, lubricate the magnets with water and insert them, one at a time, into the outermost posts of the silicone racks.
      NOTE: If you attempt to insert more than one magnet at a time, the additional resistance will make it more difficult to push the magnets to the bottom of the post.
   4. Use a blunted piece of stainless-steel dental wire (d ≈ 0.4 mm, see Table of Materials) to carefully push them to the bottom of the hollow post cavity. You can stack up to five magnets in each post.
   5. Use (round-nose) pliers to bend stainless steel dental wire (d ≈ 0.4 mm, see Table of Materials) into braces 11.25 mm wide and 15 mm long. Use wire cutters to cut the braces and a file to smooth the cutting surface. To ensure the correct dimensions are achieved, one can use a self-made jig to aid in the wire bending.
      NOTE: Post immobilization can also be achieved by braces made from other materials, as long as they are non-magnetic and rigid.
   6. Lubricate the braces with water and insert them into the silicone rack, fixing the second- and third- to outermost post in the process (see Figure 1 for complete silicone rack setup).
      NOTE: Optionally, in order to adapt the baseline stiffness of the control posts to match that of the magnetically responsive posts, fragments of styrene round rod (see Table of Materials) can be inserted into the empty posts (those without a brace or magnet).
   7. Let the racks stand for 1–2 days to allow for any remaining water to air dry.
   8. When the posts are dry, seal the holes at the top of the hollow silicone posts containing the magnets using a drop of silicone glue.
2. The afterload tuning device
   NOTE: As the magnets in the afterload tuning device are moved towards or away from those in the silicone posts, the attractive magnetic forces increase or decrease accordingly, resulting in altered stiffness of the silicone posts. This movement is achieved using a piezoelectric stage. Due to the prototypical nature of the afterload tuning device, detailed step-by-step instructions on how to replicate it will not be provided. Instead, generalized guidelines for constructing a similar afterload tuning device are detailed herein.
   1. Obtain a highly precise piezoelectric linear motor to enable the vertical translation of the magnet plate towards and away from the EHTs (see Table of Materials).
      NOTE: It is highly suggested that this motor be fitted with a linear encoder to correct for stage positioning.
   2. Position a set of permanent magnets within a non-magnetic holder such that they are axially aligned with the post magnets when placed directly below them. Here, large cylindrical magnets (d = 13 mm, h = 14 mm; see Table of Materials) were press-fit within an acrylic plastic plate (“magnet plate”).
   3. Attach the magnet holder to the piezoelectric stage using a non-magnetic material. This can be achieved using an L-shaped piece of aluminum (see Figure 2).
   4. Construct a frame that can house the components of the afterload tuning device. At a minimum, this structure should have a location on which to vertically mount the piezoelectric stage, as well as a rigid frame on which to place the 24-well plate.
      NOTE: It is suggested that the location of this mount be modifiable in the horizontal plane in order to allow for adjustments in axial alignment between the two sets of magnets. A system of mechanical drives was used to achieve this maneuverability in the presented system (Figure 3). The afterload tuning device described here was designed to be compatible with the EHT contractility analysis system (see Table of Materials). As such, its dimensions were restricted to 29 cm in width, 29 cm in depth, and 16 cm in height to fit within this system.
   5. To enable visual analysis of the tissues, install a light source within the afterload tuning device. Here, an array of LEDs was employed (Figure 4) to illuminate the EHTs from below (Figure 5).
3. Calibrating the afterload tuning system
   NOTE: In order to precisely augment EHT afterload to the desired value, the relationship between magnet spacing and the resulting post stiffness will need to be determined.
   1. Measure the closest (d_min) and farthest (d_max) magnet spacing possible in your setup. These distances will dictate the maximum and minimum achievable afterloads.
      NOTE: The bottom of the culture plate will prevent direct contact between the magnet plate and the magnetically responsive silicone posts.
   2. Produce a range of non-magnetic weights and mount them on string to serve as test loads.
   3. Determine the weights of the test loads using a fine scale and label them according to this weight. Here, six different acrylic glass weights ranging from 30 mg to 200 mg were used.
      NOTE: Select weights that are heavy enough to bend the post, but not so heavy that they bend the post more than a few millimeters. Using a larger number of test loads ensures that the calibration is more precise but it will also be more time-consuming.
   4. Mount one of the silicone racks vertically (using non-magnetic materials), such that the magnetically responsive silicone posts are oriented horizontally.
   5. Mount one of the plate magnets (the “calibration magnet”) on a horizontally traveling linear stage such that it is axially aligned with the magnetically responsive post.
   6. Position the calibration magnet a defined distance from the magnetically responsive silicone post using the horizontal stage (preferably, start at a distance equal to the maximum magnet spacing achievable by the afterload tuning device).
   7. Place a camera (for an example, see Table of Materials) to the side of this set-up in order to be able to optically record the post’s deflection under the influence of the test loads.
      NOTE: It is suggested that the user employ a camera with a resolution of at least 2 megapixels to ensure accurate determination of post deflection.
   8. Take a picture of the post in the absence of any weights to use as a reference for the post’s “neutral” position.
   9. Without changing the camera’s perspective, attach one of the loads to the very end of the silicone post and take a picture of the post bending under the influence of the weight.
   10. Repeat this measurement for all of the weights.
   11. Optically determine the deflection of the silicone post caused by the gravitational force of each weight.
   12. Graph the deflection of the silicone post (x, on the x-axis) against the gravitational force of each test weight (mg, on the y-axis). This should yield a linear relationship between force and deflection.
       NOTE: If the data is non-linear, this may indicate that the post is outside its linear range of deflection, i.e., the utilized weights were too heavy.
   13. Plot a linear regression function passing through (0,0) and the acquired data (see Figure 6A for examples). The slope of this function (mg = kx) is the stiffness k of the magnetically responsive silicone post at the tested magnet spacing.
   14. Repeat these steps at several spacings between d_max and d_min. Here, deflections at nine different magnet positions ranging from ~31 mm to ~5 mm were analyzed.
   15. Determine the base stiffness of the magnetically responsive silicone post in the absence of the calibration magnet using the same technique.
   16. Also determine the stiffness of a mobile, non-magnetically responsive control post using the same technique.
   17. Plot the resulting k values against the respective magnet distances. This should yield a negative exponential relationship.
   18. Plot a regression function through these values. For example, use Non-linear fit | One-phase decay function in the analysis software (see the Table of Materials). This regression function describes the relationship between magnet spacing and afterload (see Figure 6B for an example).

2. EHT Generation and Culture

NOTE: EHT generation and culture have been described in great detail in another article²⁵. Therefore, we will only cover these aspects briefly in our protocol. Please carry out the following steps under sterile conditions, adhering to good cell culture practices.

1. EHT generation
   1. Immerse the previously prepared silicone racks in a container filled with 70% ethanol for at least 20 min.
      CAUTION: Do not autoclave the afterload-adjustable silicone posts to sterilize them as high temperatures can damage the permanent magnets.
   2. Bring this container into the biosafety cabinet, rinse the racks 2x with sterile water, and let them air dry.
      NOTE: To reduce the likelihood of contamination, this process should be carried out in the same biosafety cabinet that will later be used to cast the EHTs.
   3. Acquire (and thaw if necessary) neonatal rat heart ventricular cells or hiPSC (human induced pluripotent stem cell)-derived cardiomyocytes (also commercially available) and prepare the EHT reconstitution mix according to Table 1.
   4. Pipet 1.5 mL of warm 2% agarose solution into the leftmost 4 wells of a 24-well culture plate and immediately insert a polytetrafluoroethylene (PTFE) spacer (see Table of Materials) into the liquid agarose solution.
   5. Repeat the previous step for the remaining 20 wells within the culture plate.
   6. After allowing the agarose to solidify for ~10 min, carefully remove the PTFE spacers.
      NOTE: The agarose turns turbid when it has solidified.
   7. Insert the posts of the magnetically sensitive silicone racks into the agarose voids produced by the PTFE spacers.
   8. Pipet 100 μL of the reconstitution mix into a 3 μL aliquot of 100 mU/L thrombin solution. Pipette up and down twice to mix and quickly transfer the mixture into the void within the first agarose mold on the culture plate.
   9. Repeat the previous step for the remaining 23 molds, using a new pipet tip for every EHT.
      NOTE: Gently mix the constitution mix every 6–8 EHTs in order to prevent cell sedimentation.
   10. Store the 24-well plate in an incubator (37 °C, 7% CO₂, 40% O₂) for 90 min. In the meantime, prepare the EHT medium by supplementing Dulbecco's modified Eagle medium (DMEM) with 10% horse serum, 1% penicillin/streptomycin, 10 μg/mL insulin and 33 µg/mL aprotinin.
   11. Add 500 μL of warm EHT medium to each well.
   12. Store the 24-well plate in an incubator (37 °C, 7% CO₂, 40% O₂) for 30 min. During this time, prepare a second 24-well plate with 1.5 mL of EHT medium in each well and place it in the incubator.
   13. Carefully remove the magnet-sensitive silicone racks with the freshly cast EHTs on them from the agarose molds and transfer them to the second 24-well plate.
2. EHT culture
   NOTE: Following tissue casting, change medium three times per week: on Monday, Wednesdays, and Fridays.
   1. For a medium change, pipet 1.5 mL of fresh EHT medium per well into a new 24-well culture plate and place this plate within the incubator at 37 °C, 7% CO₂, and 40% O₂ for at least 30 min.
   2. Transfer the silicone racks from the old 24-well plate to the new plate under a cell culture hood.
   3. Store the closed EHT plates in an incubator at 37 °C, 7% CO₂, and 40% O_2.

3. Afterload Modification Experiments

NOTE: The following protocol steps are specific to the piezoelectric motor and optical contractility analysis platform listed in the Table of Materials.

1. Preparing the afterload tuning device for experiments
   1. To measure the effects of afterload manipulations on EHT contractility, disconnect and remove the lighting system from the innermost compartment of the optical contractility analysis platform and insert the afterload tuning device including the light source.
   2. Install the stage motion control software (see Table of Materials) on the computer that will be used to run the afterload tuning device.
   3. Connect the piezoelectric stage motor to the motion controller (see Table of Materials), and the motion controller to the computer. Make sure the motion controller is also connected to a power source.
      NOTE: There are two lights on the face of the motion controller. Upon connecting to power, both lights flash red for a few seconds. During operation, the upper light remains green while the lower one should only turn red if an error occurs.
   4. Place an empty 24-well culture plate on the plate mount at the top of the afterload tuning device.
   5. Optically align the empty culture plate with the magnet plate below using the XY mechanical drive system attached to the mount.
2. Operating the afterload-tuning device
   1. Start the motion controller platform software.
   2. Connect the software to the piezo stage motor by selecting the port designated as the stage port during installation of the motion control software and then click the open port button.
      NOTE: After completing this step, the port should be designated as “open” and appear in a green box.
   3. Go to the System panel. Select Open Loop in the Loop dropdown menu.
   4. Manually move the magnet plate to its highest position, i.e., the closest possible magnet spacing d_min. The magnet plate should make contact with the culture plate mount.
   5. Go to the Motion panel. Click the Zero button to reset the current position of the piezo stage to 0 mm.
   6. Manually move the magnet plate to its lowest possible position. Write down the encoder position (indicated in the Motion panel by Enc) to determine the range of motion for the piezoelectric stage motor.
   7. Set the Travel Limits in the System panel to values within the range of motion determined in the previous step. This prevents the magnet plate from bumping into the culture plate or the bottom of the afterload tuning device.
   8. Once again, move the magnet plate to its highest position and click the Zero button.
   9. 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.
   10. Click the Save button in the Save Parameters box to store these settings in the system.
   11. Place a 24-well culture plate containing EHTs on magnetically responsive silicone racks on the culture plate mount.
   12. To calculate the magnet spacing necessary to achieve a desired afterload, solve the nonlinear regression function from step 1.3.19 for the magnet spacing parameter d. For example, if the equation is: , d being the magnet spacing (in mm) necessary to achieve the desired afterload k in mN/mm, a magnet spacing of 12.12 mm would be necessary to achieve an afterload of 5 mN/mm.
   13. Subtract d_min 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.
   14. Type this value into the Target Position 1 input field in the Motion panel and click Go to adjust the EHTs’ afterload to the calculated value.
3. Optional: Programming the stage for an interval afterload regimen
   NOTE: The previous section describes how to program the stage to move to and stay at a single position. However, it is also possible to chain different commands together into a program to achieve an automatically executed sequence of motions, which may be repeated on a continuous loop. For more detailed instructions regarding the motion controller platform software, consult the operation manual provided by the stage manufacturer.
   1. After setting up the afterload tuning device as described in the previous section, open the Command panel. Type the command 1PGM1 and press Enter to start recording a program.
   2. To create a program which, for example, will cause the piezoelectric stage to move down 30 mm from the Zero position (away from the EHTs) and return after 40 s, enter the following chain of commands: 1MVA30 → 1WST → 1WTM40000 → 1MVA0 → 1WST
   3. Use the command 1END to conclude recording a program and save it.
   4. Use the command 1EXC1 to execute the recorded program.
   5. To keep the program running on a continuous loop, enter 1PGL1, followed by the 1EXC1 command.
   6. To terminate a looping program, enter the command 1EST.
      NOTE: Table 2 contains some useful commands for afterload modification experiments. A complete list of available commands for this system can be found in the reference manual for the modular motion control system.