Name: Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues (Video) | JoVE Journal
Uploaded: 2021-02-18T15:00:00.000+00:00
Duration: 9 min 30 s
Description: Learn to fabricate and analyze 3D human skeletal muscle microtissues using the MyoTACTIC platform. This method enables scalable production and repeated assessment of contractile force and calcium transients for longitudinal muscle studies.

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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 Research Excellence Program. Schematic diagrams were created with BioRender.com.

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</ol>

The authors have no conflicts of interest to declare.

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 ...

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 interspecies differences2,4. In turning to human cell-based systems to study skeletal muscle, 2D cell culture systems are favorable due to their simplicity. However, there is a limitation. This format often fails to recapitulate the cell-cell and cell-extracellular matrix interactions that occur naturally within the body5,6. Over the last several years, three dimensional (3D) skeletal muscle models have emerged as a powerful alternative to whole animal models and conventional 2D culture systems by allowing the modeling of physiologically and pathologically relevant processes ex vivo7,8. Indeed, a plethora of studies have reported strategies to model human skeletal muscle in a bioartificial 3D culture format1. One limitation for many of these studies is that active force is quantified following the removal of the muscle tissues from the culture platforms and attachment to a force transducer, which is destructive and hence, limited to serving as an endpoint assay9,10,11,12,13,14,15,16,17,18,19,20,21. Others have designed culture systems that allow for non-invasive methods of measuring active force, but not all are amenable to high content molecule testing applications7,8,9,10,14,18,22,23,24,25,26,27,28,29.
This protocol describes a detailed method to fabricate human muscle microtissues (hMMTs) in the skeletal muscle (Myo) microTissue Array deviCe To Investigate forCe (MyoTACTIC) platform; a 96 well plate device that supports the bulk production of 3D skeletal muscle microtissues30. The MyoTACTIC plate fabrication method enables generation of a 96 well polydimethylsiloxane (PDMS) culture plate and all corresponding well features in a single casting step, whereby each well requires a relatively small number of cells for microtissue formation. Microtissues formed within MyoTACTIC contain aligned, striated, and multinucleated myotubes that are reproducible from well to well of the device, and upon maturation, can respond to chemical and electrical stimuli in situ30. Herein, the technique to manufacture a PDMS MyoTACTIC culture plate device from a polyurethane (PU) replica, an optimized method to implement immortalized human myogenic progenitor cells to fabricate hMMTs, and the functional assessment of engineered hMMT force generation and calcium handling properties are outlined and discussed.

<table><tbody><tr><td>0.9% Saline Solution, Sterile</td><td>House Brand</td><td>1010</td><td>10 mL aliquots of the solution are made and stored at 4&amp;deg;C</td></tr><tr><td>25G Needle</td><td>BD, Medstore, University of Toronto</td><td>2548-CABD305127</td><td></td></tr><tr><td>6-Aminocaproic Acid, &amp;ge;99% (titration), Powder</td><td>Sigma - Aldrich</td><td>A2504-100G</td><td>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&amp;deg;C</td></tr><tr><td>6.35 mm ID Tubing</td><td>VWR</td><td>60985-528</td><td></td></tr><tr><td>AB1167 Myoblast Cell Line</td><td>Institut de Myologie (Paris, France)</td><td></td><td></td></tr><tr><td>Arbitrary Waveform Generator</td><td>Rigol</td><td>DG1022Z</td><td></td></tr><tr><td>Basement Membrane Extract (Geltrex)</td><td>Thermo Fisher Scientific</td><td>A14132-02</td><td>Stored as aliquots of 50 &amp;micro;L or 100 &amp;micro;L at -80&amp;deg;C</td></tr><tr><td>Benchtop Vacuum Chamber</td><td>Sigma - Aldrich</td><td>D2672</td><td></td></tr><tr><td>BNC to Aligator Clip Cable</td><td></td><td></td><td>Ordered from Amazon</td></tr><tr><td>Culture Plastics</td><td>Sarstedt</td><td></td><td>Includes culture plates, serological pipettes, etc</td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma - Aldrich</td><td>D8418-250ML</td><td></td></tr><tr><td>DPBS, Powder, No Calcium, No Magnesium</td><td>Thermo Fisher Scientific</td><td>21600069</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM) (1X)</td><td>Gibco</td><td>11995-065</td><td>This is a high glucose DMEM with L-glutamine and sodium pyruvate</td></tr><tr><td>Fetal Bovine Serum</td><td>Fisher Scientific</td><td>10437028</td><td></td></tr><tr><td>Fibrinogen from Bovine Plasma</td><td>Sigma - Aldrich</td><td>F8630-5G</td><td>Aliquots ranging from 7 - 10 mg of fibrinogen powder are made and stored at -20&amp;deg;C</td></tr><tr><td>Filtropur Syringe Filter, 0.22um Pore Size</td><td>Sarstedt</td><td>83.1826.001</td><td></td></tr><tr><td>Horse Serum</td><td>Gibco</td><td>16050-122</td><td></td></tr><tr><td>Human Recombinant Insulin</td><td>Sigma - Aldrich</td><td>91077C</td><td>Stock solution is 100X and made by dissolving 1 mg of human recombinant insulin in 1 mL of DMEM and 1 &amp;micro;L of NaOH 10N. Solution is filtered and stored as 1 mL aliquots at 4&amp;deg;C</td></tr><tr><td>Image Acquisition Software</td><td>Olympus</td><td>cellSens Dimension</td><td></td></tr><tr><td>Image Processing Software</td><td>National Institutes of Health</td><td>ImageJ</td><td></td></tr><tr><td>Isotemp Oven</td><td>Thermo Fisher Scientific</td><td>201</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>IX83</td><td></td></tr><tr><td>Microscope - Camera Mount</td><td>Labcam</td><td>Labcam for iPhone</td><td>Ordered from Amazon</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Plastic Disposable Syringes, 1cc</td><td>BD</td><td>2606-309659</td><td></td></tr><tr><td>Plastic Disposable Syringes, 50cc</td><td>BD</td><td>2612-309653</td><td></td></tr><tr><td>Pluronic F-127, Powder, BioReagent</td><td>Sigma - Aldrich</td><td>P2443-250G</td><td>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&amp;deg;C</td></tr><tr><td>Polydimethylsiloxane (Sylgard 184 Silicone Elastomer Kit)</td><td>Dow</td><td>4019862</td><td>Kits are also available at Thermo Fisher Scientific, Sigma - Aldrich, etc.</td></tr><tr><td>Polyurethane Negative Mold</td><td>In House</td><td></td><td></td></tr><tr><td>Release Agent</td><td>Mann Release Technologies</td><td>200</td><td></td></tr><tr><td>Rotary Vane Vacuum Pump</td><td>Edwards</td><td>A65401906</td><td></td></tr><tr><td>Scalpel</td><td>Almedic, Medstore, University of Toronto</td><td>2586-M36-0100</td><td></td></tr><tr><td>Single Edge Razor Blade</td><td>VWR</td><td>55411-050</td><td></td></tr><tr><td>Skeletal Muscle Cell Basal Medium</td><td>Promocell</td><td>C-23260</td><td>30 mL aliquotes are generated and at stored at 4&amp;deg;C.</td></tr><tr><td>Skeletal Muscle Cell Growth Medium (Ready-to-use)</td><td>Promocell</td><td>C-23060</td><td>42 mL aliquots are generated and stored at 4&amp;deg;C.</td></tr><tr><td>Smartphone (iPhone)</td><td>Apple</td><td>SE</td><td></td></tr><tr><td>Standard Duty Dry Vacuum Pump</td><td>Welch</td><td>2546B-01</td><td></td></tr><tr><td>Sterilization Bag</td><td>Alliance</td><td>211-SCM2</td><td></td></tr><tr><td>Thimble</td><td>Igege</td><td></td><td>Ordered from Amazon</td></tr><tr><td>Thrombin from human plasma</td><td>Sigma - Aldrich</td><td>T6884-250UN</td><td>100 units of thrombin is dissolved in 1 mL of a 0.1% BSA solution. 10 &amp;micro;L aliquots are prepared and stored at - 20&amp;deg;C.</td></tr><tr><td>Tin coated copper wire</td><td>Arco</td><td>B8871K48</td><td>Ordered from Amazon</td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Thermo Scientific</td><td>15250061</td><td></td></tr><tr><td>Trypsin-EDTA, 0.25%</td><td>Thermo FIsher Scientific</td><td>25200072</td><td></td></tr><tr><td>Vacuum Chamber 2</td><td>SP Bel-Art</td><td>F42027-0000</td><td></td></tr></tbody></table>

assessing functional metrics of skeletal muscle health in human skeletal muscle microtissues

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

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&#160;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 &#39;MyoTACTIC&#39;, 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.

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...

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 ...

assessing-functional-metrics-skeletal-muscle-health-human-skeletal

Research

JoVE Journal

Bioengineering

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

Myo-mechanical Analysis of Isolated Skeletal Muscle

This protocol describes the use of microscale silicon cantilevers as pliable culture surfaces for measuring the contractility of muscle cells in vitro. Cellular contraction causes cantilever bending, which can be measured, recorded, and converted into readouts of force, providing a non-invasive and scalable system for measuring contractile function in vitro.

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

This technical report describes a variation of the modified Bergström technique for the biopsy of the musculus tibialis anterior that limits fiber damage.

Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation

Quantification of knee extensor maximal strength is imperative to understand functional adaptations to aging, disease, injury, and rehabilitation. We present a novel method to repeatedly measure in vivo knee extension isometric peak tetanic torque.

In vivo Measurement of Knee Extensor Muscle Function in Mice

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

This protocol provides methods for generating 3D engineered cardiac and skeletal muscle tissues and describes their use in preclinical drug screening modalities. The described methods utilize a magnetic sensing system to facilitate the simultaneous assessment of 24 tissues in parallel.

Preclinical Drug Testing in Scalable 3D Engineered Muscle Tissues

We describe a protocol to obtain enzymatically dissociated fibers of different lengths and types from six muscles of adult mice: three of them already described (flexor digitorum brevis, extensor digitorum longus, soleus) and three of them successfully dissociated for the first time (extensor hallucis longus, peroneus longus, peroneus digiti quarti).

Intact Short, Intermediate, and Long Skeletal Muscle Fibers Obtained by Enzymatic Dissociation of Six Hindlimb Muscles of Mice: Beyond Flexor Digitorum Brevis

Here, we describe a comprehensive method for measuring mitochondrial oxidative phosphorylation in fresh permeabilized skeletal muscle fibers from either human or mouse muscle. This method allows for the real-time quantification of mitochondrial respiration and the assessment of fuel preference and metabolic flexibility while preserving existing mitochondrial networks and membrane integrity.

Measurement of Mitochondrial Respiration in Human and Mouse Skeletal Muscle Fibers by High-Resolution Respirometry

This protocol uses micro-computed tomography to enable the cost-effective quantification of lean mass, including skeletal muscle and visceral tissue, adipose tissue, and skeletal tissue in small animals. It distinguishes between lean and adipose tissue, which offers significant advantages for biomedical research, particularly in translational research in small animals.

Non-invasive Skeletal Muscle Quantification in Small Animals Using Micro-computed Tomography

Learn to fabricate and analyze 3D human skeletal muscle microtissues using the MyoTACTIC platform. This method enables scalable production and repeated assessment of contractile force and calcium transients for longitudinal muscle studies.

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

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