We would like to thank Sarah Dick for providing critical reading and comments. M.A.R. holds the Canada Research Chair in Molecular Genetics and is an International Research Scholar of the Howard Hughes Medical Institute. This work was supported by grants to MAR from the National Institutes of Health, the Howard Hughes Medical Institute, the Canadian Institutes of Health Research, the Muscular Dystrophy Association and the Canada Research Chair Program.

All experiments were handled according to the University of Ottawa regulations for animal care and handling.
See Table 1 for an overview of the protocol.
1. Before Starting the Isolation
<ol>
 <li>Prepare the following solutions: 
 0.2% Collagenase type I in DMEM (Dulbecco's modified Eagle's medium; high glucose, L-glutamine with 110 mg/ml sodium pyruvate). For two EDL muscles prepare 2 ml of 0.2% Collagenease in DMEM. Filter the solution through 0.22 &mu;m filter. 10 min before the isolation, prewarm at 37 &deg;C in a water bath. Additional aliquots of undiluted collagenase can be frozen at -20 &deg;C for later use. 
 Note: use DMEM with sodium pyruvate throughout all of the procedure. Fibers do not survive in sodium pyruvate-free medium.
 <ul>
 <li>Washing media (use to perform all the washes). Supplement DMEM with 1% Penicillin/Streptomycin. Filter through 0.22 &mu;m filter before use.</li>
 <li>Myofiber culture media. Supplement DMEM with 20% FBS, 1% Chicken Embryo extract and 1% Penicillin/Streptomycin. Filter through 0.22 &mu;m filter before use.</li>
 <li>Matrigel coated dishes. To culture fibers for long period of time, we recommend using Matrigel as coating substrate. To prepare Matrigel coated dishes, thaw an aliquot of Matrigel at 4 &deg;C overnight. The day after, dilute Matrigel 1:10 in DMEM. Keep Matrigel at 4 &deg;C at all times and avoid abrupt temperature changes as this will create microscopic crystals within the Matrigel solution resulting in uneven coating. Place dishes to be coated on ice. Coat dishes with just enough volume to cover the surface. Let it sit for 1 min. Completely remove the leftover Matrigel. Let dishes dry at 37 &deg;C for at least 3 hr prior to use or overnight.</li>
 </ul>
 
 Note: aliquots of diluted Matrigel can be re-used multiple times for coating purpose if stored at 4 &deg;C.</li>
 <li>Lastly, be sure to clean the microscope station and dissecting tools with 70% ethanol.</li>
 <li>For two EDL isolations (one mouse) prepare five plastic Petri dishes (60*15mm) as follows:
 <ul>
 <li>Four dishes for the isolation (1 for muscle dissociation, 3 for serial washes). All dishes must be coated with horse serum (HS) to prevent myofibers from attaching to plastic. To coat, pipette 3 ml of horse serum into each dish, swirl to allow even coating, remove the horse serum and let the dish dry for at least 30 min. Add 4 ml of DMEM to each dish. Keep dishes at 37 &deg;C in a 5% CO2 incubator prior to use.</li>
 </ul>
 
 Note: Horse serum can be re-used multiple times, if kept sterile. Alternatively, a solution of 10% HS in DMEM can be used to coat dishes. Use HS coated dishes throughout the whole protocol and when culturing myofibers on floating conditions.
 <ul>
 <li>One dish will be used to culture myofibers after the isolation. Alternative dish sizes or formats can be used for the final culture of myofibers depending on downstream applications. However, we do not suggest dish sizes larger than 60*15mm. Myofibers can be kept in suspension for no longer than 96 hr before hyper contraction occurs. For longer culture times, we recommend using Matrigel coated dishes or plates.</li>
 </ul>
 </li>
 <li>For one fiber isolation (2 EDL), prepare two sterile Pasteur pipettes: one large bore pipette for muscle handling and one small bore pipette for myofiber manipulation. Use a diamond pen to cut each glass pipette to the desired length and heat polish to smooth pipette's edges. By using the flame, curve the tip of the small bore pipette. This will help handling single fibers. Flame to sterilize. Coat each pipette with HS before use.</li>
</ol>
2. Muscle Dissection and Digestion
<ol>
 <li>For the purpose of these experiments, 8 week old SV129, Pax7 CreERCklr;Rosa26TdTomato (Pax7Cre-TdTomato) and Myf5-Cre;Rosa26YFP were used.</li>
 <li>Spray hind limbs with 70% ethanol. Pin the animal (face up) to a support board to have a better grasp of the hind limb during the procedure.</li>
 <li>With the help of scissors, cut through the entire length of the limb and expose the underlying muscle. Remove the skin as well as any hair or fur (Figure 1A).</li>
 <li>With a fine scissor, cut through the thin fascia without damaging the underlying muscles. Visually localize the EDL. The EDL is found in the anterior compartment of the hind limb just underneath the Tibialis Anterior (TA) muscle.</li>
 <li>With the help of two forceps, expose the distal tendons.</li>
 <li>Cut the distal tendons (of both the TA and EDL) with sharp Cohann-Vannas spring scissors.</li>
 <li>With the help of forceps hold both TA and EDL muscles by their tendons and delicately pull the muscles up towards the proximal end. At this point you should be able to clearly see the EDL muscle just underneath the TA muscle. Now, separate the EDL from the TA muscle by pulling the two tendons in opposite directions (Figure 1B). Avoid stretching the EDL muscle while performing this operation as this will damage the myofibers.</li>
 <li>Expose the EDL tendon. To better visualize the proximal tendon it may help at this point to remove the TA muscle. It may also help to cut off some the connective tissue around the knee (Figure 1C).</li>
 <li>Cut the proximal tendon and gently remove the EDL (Figure 1D).</li>
 <li>By holding the muscle through its tendons, transfer it to 2 ml of previously prepared collagenase solution. Incubate at 37 &deg;C in a water bath. 
 Note: for successful fiber isolation, it is important to isolate the EDL from tendon to tendon so that the myofiber integrity is maintained. Steps 2.3 to 2.9 can be performed under a dissecting microscope. Alternatively, the use of a magnifying glass may also help to have a better view of the muscles.</li>
 <li>Repeat steps 2.3 to 2.9 to isolate the second EDL. Transfer the second EDL in the same tube. To avoid uneven digestion, isolation of the second EDL should be completed no more than 5 min after the first. 
 Note 1: incubation time may need to be adjusted depending on collagenase activity. Longer or shorter incubation might be required depending on the size, age and/or muscle condition (e.g. fibrotic muscles need longer digestion time). 
 Note 2: If examining satellite cells behavior under quiescence conditions, agitation during muscle digestion time may activate satellite cells 10.</li>
 <li>During the digestion time, regularly check the muscle to avoid over-digestion. Stop the digestion when muscles start to loosen up and myofibers are visible. To stop digestion, carefully transfer both muscles to a prewarmed Petri dish with 4 ml of DMEM (dissociation dish). Use the large bore size pipette to perform this operation. 
 Note: avoid muscle overdigestion as this inevitably results in the isolation of hyper contracted myofibers.</li>
</ol>
3. Single Myofiber Dissociation and Culture
<ol>
 <li>To release myofibers, use the large bore glass pipette to flush the muscle with warm medium until fibers naturally start being released. Do not triturate the muscle as this will inevitably result in damaging fibers. Perform this and the following steps under a dissecting microscope.</li>
 <li>Continue releasing myofibers until the desired number is reached. If the dish is required at room temperature for more than 10 min allow a 5 min (minimum) incubation at 37 &deg;C, 5% CO2 to re-equilibrate the medium. 
 Note: if medium reaches temperatures below physiological (37 &deg;C) for an extended time myofibers will die.</li>
 <li>Using the small size bore pipette, transfer live single myofibers to a new prewarmed dish (first of three consecutive washes). Handle each myofiber individually instead of transferring bulk of myofibers all at once. If necessary, incubate at 37 &deg;C, 5% CO2 for 10-15 min to re-equilibrate the medium.</li>
 <li>Repeat step 3.3 for 2 more times or until all dead myofibers and debris are removed. We recommend at least three consecutive washes for proper clean-up. 
 Note: dead myofibers will appear as short and hyper contracted under the microscope light.</li>
 <li>Incubate single myofibers at 37 &deg;C, 5% CO2 in the last wash dish (DMEM only) for at least one hr prior to switching to culture medium. This allows myofibers to adjust to the in vitro conditions in the absence of serum. We found that immediate culture of myofibers in serum rich medium increases fiber shrinking.</li>
 <li>After one hr, transfer myofibers to a new prewarmed dish or to the appropriate culture format depending on the downstream application. Culture fibers in high serum medium to allow satellite cell activation. Alternatively, different concentrations of serum or chicken embryo extract can also be used. Change medium every other day.</li>
</ol>
4. Downstream Applications
<ol>
 <li>Immunostaining. Live myofibers can be fixed and stained at any time point during the isolation. Immunofluorescence can be performed on both floating and substrate-attached myofibers. If staining floating myofibers, use a small bore glass pipette to transfer myofibers from one solution to another. Alternatively, it is possible to keep myofibers in the same well throughout all the procedure and add/remove solutions using a glass pipette. Avoid standard aspiration as this will result in the removal of the myofibers as well. Briefly, completely remove culture medium; fix myofibers in prewarmed 4% paraformaldehyde (PFA) for 5 min. Extensively wash in PBS several times. Incubate myofibers in 1% glycine in PBS or other standard quenching solutions to minimize PFA background staining. If necessary, permeabilize myofibers with 0.1% Triton X-100 in PBS for 10 min followed by a 5 min wash in PBS. Incubate fibers in blocking solution (10% horse serum, 0.1% Triton X-100, 1% NaN) for 1 hr at room temperature or preferably overnight at 4 &deg;C. Alternative blocking solutions may be used, depending on the antibody of interest. Wash once in PBS for 5 min. Incubate with the appropriate primary antibody diluted in blocking solution for 1 hr at room temperature or overnight at 4 &deg;C. Wash fibers 3 times at 5 min per wash in PBS to remove any unbound antibody. Incubate with the appropriate secondary antibody for 45 min to 1 hr at room temperature. Wash 3 times at 5 min per wash in PBS. Counterstain nuclei with 1 mg/ml DAPI. If staining floating fibers, transfer each fiber to a glass slide suitable for microscopy. Remove any excess of PBS or medium. Apply mounting medium and then add coverslip. Proceed to visualize myofibers under a fluorescence microscope. See Figure 4 for representative immunofluorescence on EDL myofibers. Alternative staining procedures using stronger fixatives are described in Verma, M. et al. 11 and Wosniak, A.C. et al. 12.</li>
 <li>Oligonucleotide or plasmid transient transfection. Live myofibers can be transfected with plasmid or siRNA for specific gene/s of interest. When using siRNA, double transfection is recommended for efficient gene knockdown. We suggest performing the first transfection after 8 hr from the isolation. Six hr after the transfection, replace with fresh medium. For siRNA transfection, we suggest starting by using a final concentration of 50 nM. Gene expression knockdown can be analyzed by RNA extraction or preferably by immunostaining.</li>
 <li>Viral infection. Infection of live myofibers is possible although the incidence of cell death is higher than with oligo transfection and the efficiency of infection may be variable depending on muscle conditions and age. For instance, intact adult muscle myofibers are particularly non responsive to viral infection due to the presence of the basal lamina which has been shown to provide a protective barrier against host infection 13, 14. Lentiviral vectors are preferred over retroviral vectors because they can infect quiescent (non mitotic) cells. For viral infection, it is advisable to culture fibers on a Matrigel coated dish or plate and let myofibers adjust to media conditions for the first 24 hr.</li>
 <li>Live Imaging. Live imaging of myofibers is particularly time consuming and necessitates a microscope equipped with a 37 &deg;C, 5% CO2 chamber. It is useful when assessing the behavior of single satellite cells. Work done using this technique has been instrumental for the discovery of satellite cell heterogeneity and to study their behavior on fibers (15 and 16).</li>
</ol>

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No competing interests.

The isolation and culture of single myofibers from intact muscles provides an excellent in vitro model to study the process of muscle regeneration. A unique feature of this system is the preservation of satellite cells in their physiological environment beneath the basal lamina. Most importantly, the technique can be used to investigate muscle stem cell behavior in both quiescence and activated states. Over the past 20 years, the myofiber culture system has provided meaningful insights into the biology of the satellite cell population with respect to both intrinsic and extrinsic determinants. By culturing individual myofibers, satellite cell heterogeneity with respect to myofiber, muscle type or regenerative potential has been addressed 15. Elaborate studies using live imaging of single cultured fibers allowed for the gaining information on satellite cell migration pattern 16. The acquisition of quantitative data is also possible. Although time consuming, it provides significant information on the distribution and occurrence of specific events (stem cells asymmetric division, proliferation and differentiation ratio, etc.). Together with previously described applications, protein or RNA extraction from single fibers is also possible, although isolation of pure population of satellite cells by FACS or other means may provide a better platform to investigate protein or gene expression changes at a molecular level. In our experience, the most critical step for successful fiber isolation is the "tendon to tendon" isolation (steps 2.5 to 2.9 in the protocol text). This guarantees that after muscle digestion, fibers are released from the EDL with minimal or no damage thus increasing their performance in the following steps.

<table><tbody><tr><td>Collagenase</td><td>Sigma-Aldrich</td><td>C0130-1g</td><td>Prepare fresh all times for optimal results. Additional aliquots of undiluted collagenase can be stored at -20 &amp;deg;C.</td></tr><tr><td>DMEM High Glucose + NaPyr </td><td>Invitrogen</td><td>11995073</td><td>Addition of Pen/Strep is necessary to minimize air contaminations</td></tr><tr><td>Characterized Fetal Bovine Serum</td><td>Hyclone</td><td>SH30396.03</td><td>Lot #KUJ35152</td></tr><tr><td>Horse Serum</td><td>Hyclone</td><td>SH300.74.03</td><td>Lot #AVJ82494</td></tr><tr><td>Chicken Embryo Extract</td><td>Accurate Chemical</td><td>CE-650-T, Batch B7035010</td><td>Avoid freezing-thawing cycles. </td></tr><tr><td>Matrigel</td><td>BD</td><td></td><td>Store aliquots at -20 &amp;deg;C. Avoid freezing-thawing cycles. Store diluted aliquots at 4 &amp;deg;C.</td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Cohann-Vannas Spring Scissor 6mm Blades-Straight-Sharp</td><td>Fine Science Tools</td><td>15000-02</td><td></td></tr><tr><td>Extra Thin Iris Scissors- 10.5cm</td><td>Fine Science Tools</td><td>14088-10</td><td></td></tr><tr><td>Moria-Iris Forceps Curved</td><td>Fine Science Tools</td><td>11373-12</td><td></td></tr><tr><td>Pasteur Pipettes</td><td>VWR</td><td>14672-380</td><td>14672-200</td></tr><tr><td>Diamond Pen</td><td>VWR</td><td>52865-005</td><td></td></tr><tr><td>60*15mm Petri Dish</td><td>VWR</td><td>25384-092</td><td></td></tr><tr><td>Acrodisc 0.22 &amp;mu;m Filter</td><td>VWR</td><td>CA28-145-477</td><td></td></tr><tr><td>Dissecting Microscope StemiV6</td><td>Zeiss</td><td></td><td>An additional light source may be required to have a better focus view</td></tr></tbody></table>

isolation and culture of individual myofibers and their satellite cells from adult skeletal muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Here we described the isolation of single myofibers from the EDL muscle of adult mice. Successful myofibers yield depend on several factors, such as collagenase activity or muscle conditions or age. Most importantly, the isolation of the muscle from tendon to tendon results in long, intact myofibers from EDL muscle. Figure 1 shows a step by step graphical representation of the "tendon to tendon" isolation. Once the muscle is fully digested, myofibers are released by applying gentle pressure to the muscle. Figure 2A shows a representative picture of a myofiber isolation experiment after the first wash. At this point the culture contains a mixture of bundles of single myofibers which appear as long and shining tubular structures, hyper contracted myofibers which are dark and short and debris from the digestion process. By the end of at least three consecutive washes, only single live myofibers should remain in the dish for culture or downstream analysis (Figure 2B). Figure 2C shows a long, live fiber as compared to a hyper contracted fiber (C'). At the time of the isolation, satellite cells appear as tiny protuberances on the myofiber surface (Figure 3A). If maintained in serum rich medium, satellite cells activate, proliferate, migrate and eventually fuse into myotubes (B). After 15 days in culture, all myotubes express the Myf5 reporter YFP (C) suggesting that muscle regeneration in the adult is performed by cells that at some point during their development had expressed the myogenic determinant factor Myf5. All satellite cells express the paired box transcription factor Pax7 17. The reporter mouse line Pax7Cre-TdTomato can be used to trace satellite cells via the expression of the TdTomato fluorescent signal (Figures 3D and E). Figure 4 shows a representative immunofluorescence of double Pax7+/Myod satellite cells. Classically double Pax7+/Myod+ satellite cells are considered proliferative committed muscle progenitors which will either complete the differentiation program by down regulating Pax7 while maintaining Myod expression or return to quiescence by down regulating Myod and maintaining Pax7 expression 18
<img src="/files/ftp_upload/50074/50074fig1.jpg" alt="Dissection diagram showing mouse hind limb muscle anatomy: TA, EDL, distal and proximal points." data-altupdatedat="1747911167000" data-alt="Figure 1"> 
 Figure 1. EDL muscle isolation from mouse hindlimb. A . Hindlimb of an 8 week old adult mouse. Arrows indicates the distal (knee) tendon and the proximal (foot) tendon. B. Anatomical position of the Tibialis Anterior (TA) muscle and the Extensor Digitorum Longus (EDL) muscle. C. By holding the EDL through the proximal tendon, the muscle is pulled toward the knee to expose the distal tendon. D. The distal tendon is cut and the EDL is released.
<img src="/files/ftp_upload/50074/50074fig2.jpg" alt="Microscope image of muscle fibers: live fiber, hyper-contracted fiber, and debris in tissue analysis." data-altupdatedat="1747911167000" data-alt="Figure 2"> 
 Figure 2. Representative results of a single myofiber isolation experiment. A . Bright field picture of a myofiber isolation experiment at the first wash step. Red arrow indicates live myofibers, white arrow indicates hyper contracted myofibers and yellow arrow indicates cells, myofiber or ECM debris. B. After consecutive washes in DMEM, only live myofibers remain. C and C'. Image represents a long intact live myofiber (C) and a short hyper contracted myofiber (C'). Bar 10 μm. Pictures were taken with a Zeiss Axio Observer Z1 microscope equipped with AxioCam HR.
<img src="/files/ftp_upload/50074/50074fig3.jpg" alt="Microscopy analysis of cell structures; brightfield and fluorescence images highlight cell morphology." data-altupdatedat="1747911167000" data-alt="Figure 3"> 
 Figure 3. Representative results of a single myofiber culture experiment. A . Bright field picture of a single, live myofiber with its associated satellite cell (arrow) right after the isolation (Time 0). B. Single myofibers were plated on Matrigel and cultured in serum rich medium for 15 days. White arrow indicates differentiated myotubes as compared to single cells (yellow arrow) C. Single myofibers from a Myf5Cre;RosaYFP mouse were cultured as in B. Arrow indicates Myf5 derived myotubes. D and E. Single myofibers from Pax7Cre;TdTomato were isolated and fixed right after. Arrows indicate Pax7 positive quiescent satellite cell (E, red). Nuclei were counterstained with DAPI (D). Bar: 50 μm. Pictures were taken with a Zeiss Axio Observer Z1 microscope equipped with AxioCam HR.
<img src="/files/ftp_upload/50074/50074fig4.jpg" alt="Fluorescence microscopy diagram showing DAPI, Pax7, Myod staining for cellular analysis." data-altupdatedat="1747911167000" data-alt="Figure 4"> 
 Figure 4. Example of immunofluorescence staining of satellite cells on myofibers. Single myofibers were isolated and cultured for 72 hr in floating conditions. Satellite cells on myofibers were stained for the satellite cell specific marker Pax7 (B, red) and the myogenic regulatory factor Myod (C, green). Nuclei were counterstained with DAPI (A). Bars: 50 μm. Pictures were taken with a Zeiss Axio Observer Z1 microscope equipped with AxioCam HR.
<table border="1" fo:keep-together.within-page="always">
 <tbody>
 <tr>
 <td>Muscle Dissection (5 min each muscle)</td>
 <td><ul>
 <li>Tendon to tendon isolation</li>
 <li>Sharp tools</li>
 <li>Minimal muscle damage</li>
 </ul></td>
 </tr>
 <tr>
 <td>Muscle Digestion (30-45 min)</td>
 <td><ul>
 <li>Temperature sensitive</li>
 <li>Avoid overdigestion</li>
 </ul></td>
 </tr>
 <tr>
 <td>Fiber isolation</td>
 <td><ul>
 <li>Avoid over manipulation of fibers</li>
 <li>Eliminate debris with several washes</li>
 </ul></td>
 </tr>
 <tr>
 <td>Fiber Culture (Up to 3-4 weeks)</td>
 <td><ul>
 <li>Format: any</li>
 <li>Coating: horse serum, Matrigel</li>
 <li>Medium: basal or high serum</li>
 </ul></td>
 </tr>
 </tbody>
</table>
Table 1. Overview of the myofiber isolation protocol. The myofiber isolation protocol consists of 4 major steps. For each step, the approximate time and major critical points are discussed. For detailed discussion of each step, refer to the protocol text.

Watch this Scientific Journal Video about Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle at JoVE.com

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

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Biology

62.8K Views.  Ottawa Hospital Research Institute. Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Video: Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Developmental Biology

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

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

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting 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.

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for muscle regeneration. Upon injury, which occurs frequently in skeletal muscles, SCs are activated. They proliferate as myoblasts and differentiate to repair muscle lesions. Among many events that take place during muscle differentiation, cytosolic Ca2+ signals are of great importance. These Ca2+ signals arise from Ca2+ release from internal Ca2+ stores, as well as from Ca2+ entry from the extracellular space, particularly the store-operated Ca2+ entry (SOCE). This paper describes a methodology used to obtain a pure population of human myoblasts from muscle samples collected after orthopedic surgery. The tissue is mechanically and enzymatically digested, and the cells are amplified and then sorted by flow cytometry according to the presence of specific membrane markers. Once obtained, human myoblasts are expanded and committed to differentiate by removing growth factors from the culture medium. The expression levels of specific transcription factors and in vitro immunofluorescence are used to assess the myogenic differentiation process in control conditions and after silencing proteins involved in Ca2+ signaling. Finally, we detail the use of Fura-2 as a ratiometric Ca2+ probe that provides reliable and reproducible measurements of SOCE.

Here, we describe a methodology to obtain a pure population of human myoblasts from adult muscle tissue. These cells are used to study in vitro skeletal muscle differentiation and, in particular, to study proteins involved in Ca2+ signaling.

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

Medicine

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 ...

Harvesting Mouse Skeletal Muscle to Culture Stem Cells and Their Niche

Unfractionated Bulk Culture of Mouse Skeletal Muscle to Recapitulate Niche and Stem Cell Quiescence

Skeletal muscle is the largest tissue of the body and performs multiple functions, from locomotion to body temperature control. Its functionality and recovery from injuries depend on a multitude of cell types and on molecular signals between the core muscle cells (myofibers, muscle stem cells) and their niche. Most experimental settings do not preserve this complex physiological microenvironment, and neither do they allow the ex vivo study of muscle stem cells in quiescence, a cell state that is crucial for them. Here, a protocol is outlined for the ex vivo culture of muscle stem cells with cellular components of their niche. Through the mechanical and enzymatic breakdown of muscles, a mixture of cell types is obtained, which is put in 2D culture. Immunostaining shows that within 1 week, multiple niche cells are present in culture alongside myofibers and, importantly, Pax7-positive cells that display the characteristics of quiescent muscle stem cells. These unique properties make this protocol a powerful tool for cell amplification and the generation of quiescent-like stem cells that can be used to address fundamental and translational questions.

Author Spotlight: Ex Vivo Protocol for Culturing Quiescent Muscle Stem Cells with Niche Components 

Skeletal muscle comprises multiple cell types, including resident stem cells, each with a special contribution to muscle homeostasis and regeneration. Here, the 2D culture of muscle stem cells and the muscle cell niche in an ex vivo setting that preserves many of the physiological, in vivo, and environmental characteristics are described.

Skeletal muscle is the largest tissue of the body and performs multiple functions, from locomotion to body temperature control. Its functionality and ...