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.

<ol>
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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

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...

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

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

3.9K Views.  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.

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

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

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.

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

Medicine

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

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which facilitate the functional assessment of the seeded cells. To that end, microscale cantilever technology offers a platform with which to measure the contractile functionality of a range of cell types, including skeletal, cardiac, and smooth muscle cells, through assessment of contraction induced substrate bending. Application of multiplexed cantilever arrays provides the means to develop moderate to high-throughput protocols for assessing drug efficacy and toxicity, disease phenotype and progression, as well as neuromuscular and other cell-cell interactions. This manuscript provides the details for fabricating reliable cantilever arrays for this purpose, and the methods required to successfully culture cells on these surfaces. Further description is provided on the steps necessary to perform functional analysis of contractile cell types maintained on such arrays using a novel laser and photo-detector system. The representative data provided highlights the precision and reproducible nature of the analysis of contractile function possible using this system, as well as the wide range of studies to which such technology can be applied. Successful widespread adoption of this system could provide investigators with the means to perform rapid, low cost functional studies in vitro, leading to more accurate predictions of tissue performance, disease development and response to novel therapeutic treatment.

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

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.

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which ...

Preclinical Drug Testing in Scalable 3D Engineered Muscle Tissues

Accurately modeling healthy and disease conditions in vitro is vital for the development of new treatment strategies and therapeutics. For cardiac and skeletal muscle diseases, contractile force and kinetics constitute key metrics for assessing muscle function. New and improved methods for generating engineered muscle tissues (EMTs) from induced pluripotent stem cells have made in vitro disease modeling more reliable for contractile tissues; however, reproducibly fabricating tissues from suspended cell cultures and measuring their contractility is challenging. Such techniques are often plagued with high failure rates and require complex instrumentation and customized data analysis routines. A new platform and device that utilizes 3D EMTs in conjunction with a label-free, highly-parallel, and automation-friendly contractility assay circumvent many of these obstacles. The platform enables facile and reproducible fabrication of 3D EMTs using virtually any cell source. Tissue contractility is then measured via an instrument that simultaneously measures 24 tissues without the need for complex software analysis routines. The instrument can reliably measure micronewton changes in force, allowing for dose-dependent compound screening to measure the effect of a drug or therapeutic on contractile output. Engineered tissues made with this device are fully functional, generating twitch and tetanic contractions upon electrical stimulation, and can be analyzed longitudinally in culture over weeks or months. Here, we show data from cardiac muscle EMTs under acute and chronic dosing with known toxicants, including a drug (BMS-986094) that was pulled from clinical trials after patient fatalities due to unanticipated cardiotoxicity. Altered skeletal muscle function in engineered tissues in response to treatment with a myosin inhibitor is also presented. This platform enables the researcher to integrate complex, information-rich bioengineered model systems into their drug discovery workflow with minimal additional training or skills required.

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.

Accurately modeling healthy and disease conditions in vitro is vital for the development of new treatment strategies and therapeutics. For cardiac and ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

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.

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...

Biology

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

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle biopsies are often collected for these endeavors. However, relatively few technical descriptions of biopsy procedures, outside of the commonly used musculus vastus lateralis, are available. Although the biopsy techniques are often adjusted to accommodate the characteristics of each muscle under study, few technical reports share these changes to the greater community. Thus, muscle tissue from human participants is often wasted as the operator reinvents the wheel. Expanding the available material on biopsies from a variety of muscles can reduce the incident of failed biopsies. This technical report describes a variation of the modified Bergstr&#246;m technique on the musculus tibialis anterior that limits fiber damage and provides fiber lengths adequate for mechanical evaluation. The surgery is an outpatient procedure that can be completed in an hour. The recovery period for this procedure is immediate for light activity (i.e., walking), up to three days for the resumption of normal physical activity, and about one week for wound care. The extracted tissue can be used for mechanical force experiments and here we present representative activation data. This protocol is appropriate for most collection purposes, potentially adaptable to other skeletal muscles, and may be improved by modifications to the collection needle.

This technical report describes a variation of the modified Bergstr&#246;m technique for the biopsy of the musculus tibialis anterior that limits fiber damage.

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle ...

In vivo Measurement of Knee Extensor Muscle Function in Mice

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the clinic and the research laboratory, maximal muscular strength is widely measured longitudinally in humans, with knee extensor musculature the most reported functional outcome. Pathology of the knee extensor muscle complex is well documented in aging, orthopedic injury, disease, and disuse; knee extensor strength is closely related to functional capacity and injury risk, underscoring the importance of reliable measurement of knee extensor strength. Repeatable, in vivo assessment of knee extensor strength in pre-clinical rodent studies offers valuable functional endpoints for studies exploring osteoarthritis or knee injury. We report an in vivo and non-invasive protocol to repeatedly measure isometric peak tetanic torque of the knee extensors in mice across time. We demonstrate consistency using this novel method to measure knee extensor strength with repeated assessment in multiple mice producing similar results.

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.

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the ...

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

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

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.

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches involve either differentiating myogenic progenitor cells into immature myotubes or the short-term ex vivo culture of isolated individual muscle fibers. A key benefit of ex vivo culture over in vitro is the retention of the complex cellular architecture and contractile characteristics. Here, we detail an experimental protocol for the isolation of intact flexor digitorum brevis muscle fibers from mice and their subsequent ex vivo culture. In this protocol, muscle fibers are embedded in a fibrin-based and basement membrane matrix hydrogel to immobilize the fibers and maintain their contractile function. We then describe methods to assess the muscle fiber contractile function using an optics-based, high-throughput contractility system. The embedded muscle fibers are electrically stimulated to induce contractions, after which their functional properties, such as sarcomere shortening and contractile velocity, are assessed using optics-based quantification. Coupling muscle fiber culture with this&#160;system allows for high-throughput testing of the effects of pharmacological agents on contractile function and ex vivo studies of genetic muscle disorders. Finally, this protocol can also be adapted to study dynamic cellular processes in muscle fibers using live-cell microscopy.

Skeletal muscle function can be assessed by quantifying the contractility of isolated muscle fibers, traditionally using laborious, low-throughput approaches. Here, we describe an optics-based, high-throughput method to quantify the contractility of hydrogel-embedded muscle fibers. This approach has applications for drug screening and therapeutic development.

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches ...

Enzymatic Isolation and Characterization of Variable Length Mouse Muscle Fibers

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

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal with the short fibers of the flexor digitorum brevis (FDB), which restrains the scope of results dealing with fiber types, limits the amount of biological material available, and impedes a clear connection between cellular physiological phenomena and previous biochemical and dynamical knowledge obtained in other muscles.
This paper describes how to obtain intact fibers from six muscles with different fiber type profiles and lengths. Using C57BL/6 adult mice, we show the muscle dissection and fiber isolation protocol and demonstrate the suitability of the fibers for Ca2+ transient studies and their morphometric characterization. The fiber type composition of the muscles is also presented. When dissociated, all muscles rendered intact, living fibers that contract briskly for more than 24 h. FDB gave short (&#60;1 mm), peroneus digiti quarti (PDQA) and peroneus longus (PL) gave intermediate (1-3 mm), while extensor digitorum longus (EDL), extensor hallucis longus (EHL), and soleus muscles released long (3-6 mm) fibers.
When recorded with the fast dye Mag-Fluo-4, Ca2+ transients of PDQA, PL, and EHL fibers showed the fast, narrow kinetics reminiscent of the morphology type II (MT-II), known to correspond to type IIX and IIB fibers. This is consistent with the fact that these muscles have over 90% of type II fibers compared with FDB (~80%) and soleus (~65%). Moving beyond FDB, we demonstrate for the first time the dissociation of several muscles, which render fibers spanning a range of lengths between 1 and 6 mm. These fibers are viable and give fast Ca2+ transients, indicating that the MT-II can be generalized to IIX and IIB fast fibers, regardless of their muscle source. These results increase the availability of models for mature skeletal muscle studies.

Author Spotlight: Isolation of Long Muscle Fibers from Mouse Hindlimb Muscles for Studying Excitation-Contraction Coupling Across Fiber Types

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).

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal ...

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

Summary

Explore More Videos

Chapters in this video

Myo-mechanical Analysis of Isolated Skeletal Muscle

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

Preclinical Drug Testing in Scalable 3D Engineered Muscle Tissues

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

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

In vivo Measurement of Knee Extensor Muscle Function in Mice

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

High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System

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