Our protocol describes detailed methods to fabricate and analyze a 3D human skeletal muscle microtissue culture. Muscle microtissues can be applied to studies of basic muscle biology, disease modeling, or for candidate molecule testing. The main advantage of this technique is that it permits in-situ assessment of contractile force and calcium transients.
Because of this, you can conduct longitudinal studies of muscle tissue function. Demonstrating the procedure will be Brennen Musgrave and Heta Lad, graduate students from my laboratory. Two to three hours before cell seeding, place a six well mild tactic plate portion into a 10 centimeter cell culture dish.
Prepare each individual myotactic culture well by adding 100 microliters of a 5%pluronic F-127 solution. Use the lid to cover the wells, and apply a paraffin film to seal the dish. Centrifuge the dish at 1, 550 times G for one minute in a centrifuge outfitted with a plate spinner adapter.
Store the culture dish at four degrees Celsius until the cells are ready for seeding. Then slowly thaw 150 microliter aliquot of basement membrane extract, and 110 microliter aliquot of thrombin on ice in the culture hood. Working in the cell culture hood, add 700 microliters of 0.9%saline solution to a 1.5 milliliter micro centrifuge tube containing seven milligrams of powdered fibrinogen.
Place the tube in a 37 degrees Celsius cell culture incubator for three to five minutes without vortexing. Remove and flick the tube gently, then pulse spin the dissolved solution in a micro centrifuge before returning it to the culture hood. Filter the fibrinogen solution using a one milliliter syringe outfitted with a 0.22 micrometer syringe filter.
Then transfer the dissolved fibrinogen solution to ice alongside the basement membrane extract and thrombin aliquots. Prepare and pre-warm the tissue growth media at 37 degrees Celsius, which will be introduced to the culture wells after seeding the tissues. Remove the cell culture plates from the incubator and aspirate the culture media.
Then wash the cells once by adding five milliliters of DPBS into each culture plate. Aspirate the DPBS and detach the cells by adding one milliliter of 0.25%trypsin EDTA into each culture dish. Place the plate in the cell culture incubator for three minutes, hold the trypsin by adding three milliliters of wash medium to the culture dish, then transfer the cells to an appropriately sized conical tube.
Pellet the cells by centrifuging at 400 times G for 10 minutes. Aspirate the media carefully without damaging the cell pellet, then re-suspend the cells in one milliliter of the wash medium. Count the cells using a hemocytometer and trypan blue dye under bright field microscopy.
To seed six tissues, prepare enough cells and extracellular matrix for eight tissues, thus accounting for losses and bubble formation. Transfer 1.2 million cells to a new conical tube, then increase the volume to 10 millimeters with wash medium. Pellet the cells by centrifuging at 400 times G for 10 minutes.
Prepare 150 microliters of ECM mixture in a 1.5 milliliter micro centrifuge tube, and store the mixture on ice until use. Aspirate the media from the conical tube containing the cells, taking care to avoid the cell pellet. Vigorously flick the end of the tube with a gloved finger until the pellet appears as a cell slurry.
Transfer 120 microliters of the ECM solution to the tube containing the cell slurry, and carefully pipette up and down to completely re-suspend the cells within the ECM to generate a single cell suspension. Avoid creating bubbles, then place the cell ECM suspension on ice until use. Place the refrigerated 10 centimeter dish containing the myotactic wells on top of an ice pack inside of the cell culture hood, and aspirate the pluronic F-127 solution from each well.
Allow residual pluronic F-127 solution to release from the porous PDMS, and settle to the bottom of the well by letting the wells sit for five minutes on ice, then aspirate again. Pipette the cell ECM suspension carefully to re-suspend the cells, then transfer 105 microliters of the suspension into a fresh, pre-chilled 1.5 milliliter tube by grasping it close to the top. Add 0.84 microliters of 100 units per milliliter thrombin stock solution to the 105 microliters of cell ECM suspension.
Pipette rapidly and thoroughly to mix, avoiding the introduction of bubbles. Add 15 microliters of the cell ECM mixture to each well without pressing the pipette into the bottom of the well. With two light motions, spread the cell suspension behind each post in the well.
Place the lid on the 10 centimeter culture plate and transfer it to a 37 degrees Celsius tissue culture incubator for approximately five minutes. Add 200 microliters of pre-warmed tissue growth media to each myotactic well after the cell ECM mixture has polymerized. Replace the lid on the 10 centimeter dish and keep it in the incubator until the differentiation.
During a 14 day culture period, myoblasts displayed the aspects of native tissue by self-organizing in the mild tactic well to form a 3D microtissue with multi-nucleated striated myotubes. Myotubes have sarcomere striations, which were visualized by immunostaining for sarcomeric alpha actinin. To achieve well-aligned myotubes, technical errors such as bubble formation while pipetting, or damaging pluronic coating during aspiration, should be avoided.
A representative displacement of the myotactic well plate post in response to low and high frequency electrical stimulation, is shown here, indicating that myotubes responds to varying electrical stimuli and contract accordingly. Quantification of the post displacement allows for conversion to absolute contractile force of the HMMTs. Calcium transience on HMMTs made from GCaMP6-transduced myoblasts were also analyzed in response to low and high frequency stimulation.
Quantification of fluorescent intensity can be used as measurement for the calcium handling properties of HMMTs. Most people who perform this tissue seeding for the first time, struggle with adding the cell extracellular matrix to the wells, and with bubble formation. We recommend practicing the technique on some spare microtissue culture wells with a very simple viscous solution before the first attempt with cells.
