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Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues

Published: February 18th, 2021



1Institute of Biomedical Engineering, University of Toronto, 2Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 3Department of Cell and Systems Biology, University of Toronto

This manuscript describes a detailed protocol to produce arrays of 3D human skeletal muscle microtissues and minimally invasive downstream in situ assays of function, including contractile force and calcium handling analyses.

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study skeletal muscle reformation and function in a scalable format that is amenable to experimental manipulations. 3D muscle culture systems are desirable as they enable scientists to study skeletal muscle ex vivo in the context of human cells. 3D in vitro models closely mimic aspects of the native tissue structure of adult skeletal muscle. However, their universal application is limited by the availability of platforms that are simple to fabricate, cost and user-friendly, and yield relatively high quantities of human skeletal muscle tissues. Additionally, since skeletal muscle plays an important functional role that is impaired over time in many disease states, an experimental platform for microtissue studies is most practical when minimally invasive calcium transient and contractile force measurements can be conducted directly within the platform itself. In this protocol, the fabrication of a 96-well platform known as 'MyoTACTIC', and en masse production of 3D human skeletal muscle microtissues (hMMTs) is described. In addition, the methods for a minimally invasive application of electrical stimulation that enables repeated measurements of skeletal muscle force and calcium handling of each microtissue over time are reported.

Skeletal muscle is one of the most abundant tissues in the human body and supports key body functions such as locomotion, heat homeostasis and metabolism1. Historically, animal models and two-dimensional (2D) cell culture systems have been used to study biological processes and disease pathogenesis, as well as for testing pharmacological compounds in the treatment of skeletal muscle diseases2,3. While animal models have greatly improved our knowledge of skeletal muscle in health and in disease, their translational impact has been hampered by high costs, ethical considerations and inters....

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1. PDMS MyoTACTIC plate fabrication

NOTE: PDMS MyoTACTIC plate fabrication requires a PU negative mold, which can be manufactured as previously described30. The computer-aided design (CAD) SolidWorks file for the MyoTACTIC plate design has been made available on GitHub (

  1. Prepare ~ 110 g of PDMS polymer solution in a disposable plastic cup at a 1:15 ratio of monomer to curing agent using the.......

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Described herein are methods to cast a 96-well PDMS-based MyoTACTIC culture platform from a PU mold, to fabricate arrays of hMMT replica tissues, and to analyze two aspects of hMMT function within the culture device-force generation and calcium handling. Figure 1 offers a schematic overview of the preparation of MyoTACTIC culture wells before hMMT seeding. PDMS is a widely used silicone-based polymer, that can be easily molded to create complex devices32. A PDMS-based.......

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This manuscript describes methods to fabricate and analyze a 3D hMMT culture model that can be applied to studies of basic muscle biology, disease modeling, or for candidate molecule testing. The MyoTACTIC platform is cost-friendly, easy to manufacture, and requires a relatively small number of cells to produce skeletal muscle microtissues. hMMTs formed within the MyoTACTIC culture platform are comprised of aligned, multinucleated, and striated myotubes, and respond to electrical stimuli by initiating calcium transients .......

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We would like to thank Mohammad Afshar, Haben Abraha, Mohsen Afshar-Bakooshli, and Sadegh Davoudi for contributing to the invention of the MyoTACTIC culture platform and establishing the fabrication and analysis methods described herein. HL received funding from a Natural Sciences and Engineering Research Council (NSERC) Training Program in Organ-on-a-Chip Engineering and Entrepreneurship Scholarship and a University of Toronto Wildcat graduate scholarships. PMG is the Canada Research Chair in Endogenous Repair and received support for this study from the Ontario Institute for Regenerative Medicine, the Stem Cell Network, and from Medicine by Design, a Canada First Re....

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Name Company Catalog Number Comments
0.9% Saline Solution, Sterile House Brand 1010 10 mL aliquots of the solution are made and stored at 4°C
25G Needle BD, Medstore, University of Toronto 2548-CABD305127
6-Aminocaproic Acid, ≥99% (titration), Powder Sigma - Aldrich A2504-100G A 50 mg / mL stock solution is generated by dissolving 5 mg of 6-aminocaproic acid powder in 100 mL of autoclaved, distilled water. The solution is vaccum filtered and 10 mL aliquots are stored at 4°C
6.35 mm ID Tubing VWR 60985-528
AB1167 Myoblast Cell Line Institut de Myologie (Paris, France)
Arbitrary Waveform Generator Rigol DG1022Z
Basement Membrane Extract (Geltrex) Thermo Fisher Scientific A14132-02 Stored as aliquots of 50 µL or 100 µL at -80°C
Benchtop Vacuum Chamber Sigma - Aldrich D2672
BNC to Aligator Clip Cable Ordered from Amazon
Culture Plastics Sarstedt Includes culture plates, serological pipettes, etc
Dimethyl Sulfoxide Sigma - Aldrich D8418-250ML
DPBS, Powder, No Calcium, No Magnesium Thermo Fisher Scientific 21600069
Dulbecco's Modified Eagle Medium (DMEM) (1X) Gibco 11995-065 This is a high glucose DMEM with L-glutamine and sodium pyruvate
Fetal Bovine Serum Fisher Scientific 10437028
Fibrinogen from Bovine Plasma Sigma - Aldrich F8630-5G Aliquots ranging from 7 - 10 mg of fibrinogen powder are made and stored at -20°C
Filtropur Syringe Filter, 0.22um Pore Size Sarstedt 83.1826.001
Horse Serum Gibco 16050-122
Human Recombinant Insulin Sigma - Aldrich 91077C Stock solution is 100X and made by dissolving 1 mg of human recombinant insulin in 1 mL of DMEM and 1 µL of NaOH 10N. Solution is filtered and stored as 1 mL aliquots at 4°C
Image Acquisition Software Olympus cellSens Dimension
Image Processing Software National Institutes of Health ImageJ
Isotemp Oven Thermo Fisher Scientific 201
Microscope Olympus IX83
Microscope - Camera Mount Labcam Labcam for iPhone Ordered from Amazon
Penicillin-Streptomycin (10,000 U/mL) Gibco 15140-122
Plastic Disposable Syringes, 1cc BD 2606-309659
Plastic Disposable Syringes, 50cc BD 2612-309653
Pluronic F-127, Powder, BioReagent Sigma - Aldrich P2443-250G A 5% stock solution of pluronic acid is made by dissolving 5 g of pluronic acid powder in 100 mL of chilled, autoclaved, distilled water. The solution is vaccum filtered and 10 mL aliquots are stored at 4°C
Polydimethylsiloxane (Sylgard 184 Silicone Elastomer Kit) Dow 4019862 Kits are also available at Thermo Fisher Scientific, Sigma - Aldrich, etc.
Polyurethane Negative Mold In House
Release Agent Mann Release Technologies 200
Rotary Vane Vacuum Pump Edwards A65401906
Scalpel Almedic, Medstore, University of Toronto 2586-M36-0100
Single Edge Razor Blade VWR 55411-050
Skeletal Muscle Cell Basal Medium Promocell C-23260 30 mL aliquotes are generated and at stored at 4°C.
Skeletal Muscle Cell Growth Medium (Ready-to-use) Promocell C-23060 42 mL aliquots are generated and stored at 4°C.
Smartphone (iPhone) Apple SE
Standard Duty Dry Vacuum Pump Welch 2546B-01
Sterilization Bag Alliance 211-SCM2
Thimble Igege Ordered from Amazon
Thrombin from human plasma Sigma - Aldrich T6884-250UN 100 units of thrombin is dissolved in 1 mL of a 0.1% BSA solution. 10 µL aliquots are prepared and stored at - 20°C.
Tin coated copper wire Arco B8871K48 Ordered from Amazon
Trypan Blue Solution, 0.4% Thermo Scientific 15250061
Trypsin-EDTA, 0.25% Thermo FIsher Scientific 25200072
Vacuum Chamber 2 SP Bel-Art F42027-0000

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