Our invitro model of three-dimensional stem cell derived human heart muscle improves the biofidelity of traditional two-dimensional cell culture while eliminating the interspecies differences associated with laboratory animal testing. Our multi-tissue bioreactor design with stable post trackers maximizes tissue success and improves the accuracy of in-depth characterization of engineered cardiac tissue function. As heart failure continues to be the leading cause of death worldwide, our engineered cardiac tissues enable researchers to model human cardiac diseases, as well as screen potential therapies.
To begin, position four aluminum negative master casts in the cast holder so that the post holes align with the dead space opposite the triangle shelves. Place the apparatus between two parallel brackets and clamp the sides together using a rectangular piece of 0.5 millimeter thick silicone sheeting as a gasket to prevent leakage of the liquid PDMS. In a shallow container, mix 0.5 milliliters of PDMS curing agent with five milliliters of PDMS elastomer base at a one to 10 ratio and stir vigorously for five minutes.
Degas the PDMS mixture in a vacuum chamber under a strong vacuum until the bubbles disappear. Next, pour the PDMS mixture onto the casting apparatus, overfilling to ensure complete coverage of each slot. Add small colored glass beads to the body of the PDMS racks opposite to the side with the posts for the unique identification of each rack.
Place the casting apparatus leveled horizontally into the vacuum chamber under a strong vacuum for at least 12 hours. Allow the PDMS to cure at room temperature away from dust for 48 hours to enable complete curing and maximum strength of the delicate posts. Remove the clamp, brackets, and silicone sheeting from the casting apparatus.
Using a stainless steel razor blade, trim away the PDMS film on the top of the casting apparatus and frame supports. Use fingers to separate the PDMS racks from the sides of the cast holder. Insert a blunted stainless steel razor blade into the dead space between the cast and the cast holder and pry them apart, ensuring that the PDMS filling the dead space remains with the cast holder.
Then, using a sharp stainless steel blade, cut away the remaining PDMS films and the dead space PDMS from the tips of the posts. Using the fingers, slowly separate the PDMS rack from the cast on the side opposite the posts. Continue to alternate sides until the posts are free of the master casts.
Free all the PDMS racks and the posts as previously demonstrated, and use a sharp razor blade to trim away any remaining excess PDMS from the racks. Using a thermoplastic fused deposition modeling 3D printer, print the components of the stable post tracker, or SPoT, casting apparatus. Ensure a secure press fit between the 3D printed pieces and between the PDMS racks and the three-pronged jig.
Also confirm that the PDMS racks fit snugly with the posts just reaching the bottom of the wells without being bent. Mix 0.5 milliliters of black PDMS part A to 0.5 milliliters of part B in a small weighing boat, or a shallow container until the solution is uniform in color. Degas the mixed black PDMS in a vacuum chamber under a strong vacuum for 20 minutes.
Pour the degassed black PDMS onto the 3D printed base to fill the holes and tap to ensure no bubbles remain. Scrape as much excess PDMS off the base as possible. Snap the three-pronged piece onto the base and place the PDMS racks in the grooves on the three-pronged jig, ensuring that the ends of the posts dip into the black PDMS in the circular wells.
Allow the black PDMS to cure at room temperature away from dust for 48 hours. Slide out the three-pronged piece, minimizing tension on the posts. Using small forceps, scrape away the thin black PDMS film surrounding each SPoT, then insert fine tipped, bent forceps into the SPoT well to free it from the 3D printed base.
Inspect the SPoTs and trim any remaining black PDMS film from the casting process using fine vannas scissors. Ensure that the finished posts are of the correct length by fitting the PDMS racks onto the polys cell phone frame, and then sliding this onto the black base plate. After autoclaving the bioreactor and preparing the cell mixture, proceed to fabricate the hECTs.
Wearing sterile gloves, remove the black base plate from the autoclaved bag containing the bioreactor parts and place it in a 60 milliliter dish with the wells facing up. Pipette 44 microliters of the cell mixture into each well slowly to avoid introducing bubbles. Wearing a fresh pair of sterile gloves, remove the polysulfone frame with the PDMS racks from the autoclave bag.
Lower the frame onto the base plate such that the ends of the frame fit into the grooves at the end of the base plate. Ensuring that the posts are all straight and the frame is not tilted, place the bioreactor into a 60 millimeter dish. Add one milliliter of 10%FBS in cardiomyocyte maintenance medium to the 60 milliliter dish to increase the humidity as the hECTs solidify.
Place the dish without a lid into a high profile 100-millimeter dish and cover it with a 100-millimeter dish lid, then return the bioreactor to a 37 degrees Celsius, 5%carbon dioxide incubator to allow the collagen to form a gel with the cells in suspension. After two hours, remove the dish from the incubator. Add 13 milliliters of 10%FBS in the cardiomyocyte maintenance medium, tilting the dish to allow the medium to flow between the PTFE base plate and the PDMS racks.
Inspect the bioreactor from the side for no air bubbles and return the dish to the incubator. If air is trapped, tilt the bioreactor out of the medium to let the bubble break and slowly lower it again, or use a micro pipette with a gel loading tip to siphon the air without disturbing the posts. Inspect the hECT compaction through the window in the frame.
Over 24 to 96 hours, the hECTs compact and become opaquer. Remove the base plate when the hECTs are compacted by at least 30%compared to the original diameter. Fill the 60 millimeter dish containing the bioreactor with cardiomyocyte maintenance medium until the liquid flushes with the lip of the dish and add 14 milliliters to a new 60-millimeter dish.
While wearing sterile gloves, flip the bioreactor over in its dish so that the base plate is on top. After inspecting for trapped air bubbles, slowly lift the base plate, keeping it level. Ensure the post tips are in focus.
Turn on the thresholding switch and adjust the slider until the SPoTs are nicely demarcated and do not change shape as the hECT contracts. Use the rectangle tool to draw a rectangle around one of the SPoTs, and click on the set one button inside the post boundaries box to set the rectangle position around the SPoT, ensuring that the SPoT always remains within the boundary of the rectangle. Repeat the process to the other post and record it under set two.
Adjust the object size settings to prevent the program from tracking smaller objects, and ensure that the number of objects tracked in each rectangle remains constant. The interface shows the measured distance between the tracked objects in real time. Use this graph to monitor the noise.
Under the pacing frequency hertz header, indicate the range of desired frequencies and the desired interval for stepping from min to max. In the boxes to the right, choose the desired setting time to allow the hECT to adjust to the new pacing frequency, then specify the recording time and pacing voltage. Begin the program by clicking on the start program button.
After data from one frequency has been recorded, increase the stimulator frequency by the desired interval for a new recording until the max frequency has been reached. Representative images of hECTs are shown here as viewed from the bottom, created without SPoTs and with SPoTs. The SPoTs provided a single defined shape to track during the data acquisition.
In some extreme instances, the tracking object was even obscured. A more reliable shape for optical tracking and noise reduction enabled the measurement of weak tissues with a developed force as low as one micro Newton. The SPoTs provided a cap geometry that prevented the hECT loss.
The measured hECT function was stable over extended culture time in the current pacing setup with the heated stage. Compared to measurements at physiological temperature, hECTs showed altered contraction dynamics at room temperature, with slower rates of contraction and relaxation. At higher frequencies, the hECTs tended to be weaker at room temperature.
At lower frequencies, the hECTs tended to be stronger at room temperature. When paced at 36 degrees Celsius, the hECTs had a higher spontaneous beat rate and a more expansive range of capturing frequencies. The temperature controlled environment ensures the physiologic relevance of the measured hECTs function.
Additionally, the shared media bath of the multi-tissue bioreactor facilitates studies of paracrine signaling between hECTs of different cellular compositions. The addition of SPoTs to our multi tissue bioreactor allows researchers to efficiently study diseased myocardium with abnormally high or low tension, which would otherwise slip off the ends of uncapped posts.
