Our platform uses 3D engineered muscle tissues and magnetic sensing hardware to measure contractility across 24 wells simultaneously. This provides a higher throughput method for assessing new therapeutics in vitro using human muscle tissues. Our casting method improves the consistency and reproducibility in fabricating 3D engineered muscle tissues.
We have designed a consumable 24-well casting plate equipped with magnetic sensing hardware for contractility measurements and analysis on our platform. This method can provide valuable insight into in vitro disease modeling, drug discovery, and gene therapy for any muscle disease that affects contractility. We are currently incorporating motor neurons into our 3D constructs to create a neuromuscular junction model as well.
To begin, place a pre-chilled tissue casting kit onto a cold block or ice inside the cell culture hood. Lay the casting plate flat on ice. Add thrombin to base medium to make the thrombin solution and move the post lattice from the casting plate to a new sterile 24-well plate.
Pipette 50 microliters of thrombin solution into each pre-chilled well of the casting plate and place the lattice back onto the kit. Set the casting kit aside on ice. Next, add 500 milliliters of RPMI medium, 10 milliliters of B27, and 2.5 grams of aminocaproic acid, or ACA, to prepare the cardiac EMT medium.
Take out the EMT medium that will be used to cast the tissues and add 10 micromolar ROCK inhibitor to it. Prepare the skeletal EMT medium by adding 50 milliliters of F10 medium and 0.25 grams of aminocaproic acid, or ACA, for primary myoblasts. Use 0.1 grams of ACA for IPSC-derived myoblasts.
Then, sterile filter the casting medium containing ACA. Warm the cell culture grade dissociation reagent and an equal volume of EMT medium to 37 degrees Celsius. This warmed EMT medium will be used to dilute the dissociation reagent.
Wash the cells with PBS and add the warmed dissociation reagent to lift the cells. Incubate at 37 degrees Celsius for five minutes or until the cells have lifted and check the cultures every two to three minutes by tapping the side of the plate. Once the cells have lifted, transfer them to a 50 milliliter conical tube.
Triturate with a P-1000 pipette to ensure a single cell suspension. Wash the plate and the flask with an additional EMT medium to collect the remaining cells and add them to the conical tube. Again, triturate the cells to ensure a single cell suspension.
Add EMT medium to terminate the dissociation process and then take samples of the cell suspensions for cell counts. Perform cell counts using an automated cell counter or a hemocytometer in trypan blue. Spin the cells at 200 G for four minutes, aspirate the supernatant, and re-suspend the cells in five milliliters of the EMT base medium to remove the residual dissociation reagent.
After centrifuging again for four minutes, aspirate the supernatant and prepare the cell suspensions at the appropriate densities. Calculate the volumes of each cell solution required to make up the desired number of tissues and pipette the calculated volumes of cells into a 15 milliliter conical tube. Add 10 microliters of fibrinogen per EMT to the cell suspension and place it on ice.
Ensure that each EMT has 90 microliters of cell suspension, 10 microliters of fibrinogen, and 50 microliters of thrombin solution in the final tissue construct. In a cell culture hood, remove the lid of the tissue casting kit and place the casting plate with the post lattice lying flat on the ice. Mix the cell fibrinogen mixture and draw up 100 microliters with a P-200 pipette.
Add 100 microliters of the mixture to wells prepared with 50 microliters of thrombin solution and triturate five times to mix well. Do not push the pipette past the first stop and remove the tip after trituration to avoid creating any bubbles. Mix the cell suspension in the conical tube before casting every tissue.
Hold both the lattice and casting well plate simultaneously using the pointer finger and thumb of one hand to avoid any movement of the lattice or leave the plate flat on ice and rock the ice bucket to see into the casting plate. Repeat with a fresh P-200 tip for each tissue until all the tissues are cast. Carefully transfer the seeded kit to the incubator and incubate at 37 degrees Celsius for 80 minutes.
Next, prepare a fresh 24-well plate with two milliliters per well of the EMT medium for cardiac tissues and incubate the plate at 37 degrees Celsius to warm the medium. After the 80 minute incubation, gently add one milliliter of the EMT medium to the edge of the casting wells and incubate for another 10 minutes at 37 degrees Celsius. After 10 minutes of incubation, carefully lift the post lattice and transfer the tissues from the casting plate to a prepared 24-well plate with the warmed medium.
Finally, return the plates with tissues to the cell culture incubator at 37 degrees Celsius. Unsuccessful EMT production can range from catastrophic failures such as tissue detachment from the posts, to more subtle structural flaws such as air bubbles and unequal tissue deposition around both posts. EMT construct one day after casting is shown here.
Skeletal muscle tissues were transferred into a differentiation medium beginning day zero of cell fusion and hydrogel compaction. From day seven through day 28, the same EMT displayed a slightly shorter overall length between the two posts and a smaller width when measured through the middle section of the EMT. Four plates of tissues were tracked over 21 days comparing EMT diameter throughout compaction.
Time points show consistent EMT size between plates. Maximal compaction is reached on day 21 as matrix remodeling is stabilized. The immunohistochemistry of the EMTs is depicted here.
EMTs were fixed on day 10 of culture and embedded in paraffin. Thin cross-sections were stained with antibodies against myosin heavy chain and dystrophin before imaging. These graphical images represent the average absolute twitch force measured from the cardiac EMTs and the skeletal EMTs over time.
Acute and chronic doxorubicin treatment in engineered heart tissues is shown here. Three different dose concentrations of Dox were delivered either in bolus or administered continuously to engineered heart tissues over the course of 27 days. The dose response to BDM in engineered skeletal muscle tissues is presented in this figure.
The cardiac beat rate or twitch frequency ceases in a dose-dependent manner in tandem with a cessation of contractile force indicating cardiotoxicity when EMTs are exposed to this compound. The most important things to remember when casting tissues are to keep everything cold at all times, including your cell-suspension and reagents and to keep the post lattice completely stationary once casting begins.
