This protocol describes a cell culture model to study the differentiation of fetal rat skeletal muscle cells. The model uses serum-free medium, a nonbiological substrate N-1[3(trimethoxysilyl)propyl] diethylenetriamine (DETA) and fabricated microcantilevers to promote the differentiation of dissociated rat myocytes into robust myotubes. In this protocol, we also describe how to characterize the myotubes on the basis of morphology, immunocytochemistry and electrophysiology. Here, four major techniques are employed: fabrication of cantilevers, surface modification of the glass and cantilever substrates with a DETA SAM, a serum-free medium and refined culture techniques. This culture system has potential applications in biocompatibility studies, bioartificial muscle engineering, skeletal muscle differentiation studies and for better understanding of myopathies and neuromuscular disorders. The model can be established in 26–33 d.
Differentiation of skeletal muscle is a highly controlled, multistep process, during which single muscle cells initially freely divide and then align and fuse to form multinucleated myotubes. This process of muscle differentiation in vivo is governed by a complex interplay of a wide range of growth and trophic factors. Several such factors have been discovered, which have been observed to promote muscle differentiation in vivo1,2,3. However, very little systematic research efforts have been undertaken to use this extensive in vivo knowledge of growth factors to develop a chemically defined medium that promotes muscle differentiation in vitro.
Most of the existing in vitro culture methods for studying skeletal muscle differentiation use serum-containing medium as well as a biological growth substrate4,5,6. The presence of many unknown components in serum-containing medium and the technical difficulties in creating reproducible biological substrates has led to extensive variations in the results from experiment to experiment. In order to remove the inherent drawback of utilizing serum-containing medium and biological substrates, a defined culture system consisting of a serum-free medium based on the extensive knowledge of growth factors from in vivo studies and a synthetic silane substrate to study skeletal muscle differentiation was developed7.
The system has three features that differentiate it from all other previously reported systems. First, a unique chemically defined serum-free medium supplemented with specific growth factors was developed to study the muscle differentiation process. Second, a synthetic, nonbiological, patternable8,9, cell growth promoting substrate, N-1[3(trimethoxysilyl)propyl] diethylenetriamine (DETA), coated on glass coverslips10,11,12, was used as a template to grow the skeletal muscle cells. Third, it was demonstrated that when the dissociated muscle cells were plated on fabricated microcantilevers, they aligned along the long axis of the cantilever to form aligned myotubes.
In this protocol, step-by-step instructions on how to fabricate and modify the cantilever and glass substrates and then grow the muscle cells on the chemically modified substrates using this serum-free medium, to form robust myotubes, is described. Figure 1 shows a diagram of the general outline of the whole procedure, while Figure 2 outlines the microcantilever manufacture process. Note that all fabrication procedures should be performed in a clean room. The protocol consists of two figures: Figure 3, which shows a microcantilever array micrograph, and Figure 4, which outlines the cell culture process; as well as substrate modification of either a cantilever or a glass substrate (Steps 1–22), preparation of the serum-free medium, as well as both intracellular and extracellular solutions (REAGENT SETUP), dissection of the skeletal muscle tissue (Steps 23–24), purification of the tissue and the culture of the myocytes to form myotubes (Steps 25–38), and characterization of the myotubes morphologically, immnunocytochemically and electrophysiologically (Steps 39–40A–C).
Charles River or Sprauge Dawley, fetal rats age E18
Hydrofluoric acid (HF) (Sigma)
Diethylenetriamine trichmethoxysilane (United Chemical Technologies)
AZ 5214E photoresist (Clariant Corporation)
AZ 9245E photoresist (Clariant Corporation)
AZ 400K photoresist developer (Clariant Corporation)
Deep reactive ion etcher (PlasmaTherm)
Leibovitz medium (L15) (Invitrogen, cat. no. 11415064)
Medium 199 (Invitrogen, cat. no. 11150059)
B27 supplement (50×) (Invitrogen, cat. no. 17504044)
Basic fibroblast growth factor (b-FGF; Invitrogen, cat. no. 13256029)
Brain-derived neurotrophic factor (BDNF; Invitrogen, cat. no. 10908019)
Glial-derived neurotrophic factor (GDNF; Invitrogen, cat. no. 10907012)
Cardiotrophin-1 (CT-1; Cell sciences, cat. no. CRC700B)
Sodium bicarbonate (Fisher, cat. no. 5233500)
Embryonic myosin heavy chain (MHC) (F1.652, IgG; Developmental Studies Hybridoma Bank)
Primary antibody (mouse anti-α-actin, Sigma, cat. no. A2172)
Secondary antibody (Alexa Fluor 488-conjugated donkey antimouse; Molecular Probes, cat. no. A21202)
Citiflour-mounting solution (Ted Pella)
Clean glass coverslips 22 × 22 mm2 no. 1 (Thomas Scientific, cat. no. 6661F52)
O2 plasma cleaner (Harrick PDC-32G; Harrick)
DETA (United Chemical Technologies Inc., cat. no. T2910KG)
0.1% (vol/vol) mixture of organosilane in freshly distilled toluene (Fisher, cat. no. T2904)
PBS (calcium- and magnesium-free) (GIBCO, cat. no. 14200075)
Trypsin–EDTA (GIBCO, cat. no. 25300054)
L15 medium (Invitrogen, cat. no. 41300021)
BSA (Sigma, cat. no. A3059)
Alexa Fluor 488-conjugated donkey antimouse antibody (Molecular Probes, cat. no. A21202), for immunocytochemistry only
Mouse anti-α-actin (Sigma, cat. no. A2172), for immunocytochemistry only
HEPES (Fisher, cat. no. BP310), for electrophysiology only
HEPES–sodium (Fisher, cat. no. BP410), for electrophysiology only
EGTA (Fisher, cat. no. O2783), for electrophysiology only
K-gluconate (Sigma, cat. no. G4500), for electrophysiology only
MgCl2 (Fisher, cat. no. S320), for electrophysiology only
Na2ATP (Fisher, cat. no. BP413), for electrophysiology only
Antibody against embryonic MHC (F1.652, IgG, Developmental Studies Hybridoma Bank)
Serum-free medium (see REAGENT SETUP)
Intracellular solution (see REAGENT SETUP)
Extracellular solution (see REAGENT SETUP)
Saponin (permeabilization solution)
Fetal calf serum
Pyrex bottles (VWR)
Pyrex beakers (VWR)
4-inch silicon-on-insulator (SOI) wafers (MXF)
Ceramic racks (VWR)
Glove box (MBraun)
Microscope—regular and confocal
Carbon dioxide incubator
Contact angle goniometer (KSV Instruments, Cam 200 (or similar instrument))
X-ray Photoelectron Spectrometer (Kratos Axis 165 (or similar instrument))
Axioscope 2 FS plus upright microscope (Zeiss, Jena, Germany), for electrophysiology only
PCS-5000 piezoelectric three-dimensional (3D) micromanipulator (Burleigh EXFO, Quebec, Canada), for electrophysiology only
Gibraltar platform (Burleigh EXFO, Quebec, Canada), for electrophysiology only
Vibration isolation table (TMC, Peabody, MA), for electrophysiology only
Multiclamp 700B patch clamp amplifier (Axon/Molecular Devices, Sunnyvale, CA), for electrophysiology only
Digidata 1320 A/D converter (Axon/Molecular Devices, Sunnyvale, CA), for electrophysiology only
pClamp 9.0 software (Axon/Molecular Devices Sunnyvale, CA), for electrophysiology only
P97 pipette puller (Sutter, Novato, CA), for electrophysiology only
Inline solution heater (self-made but available from Warner Instruments, Hamden, CT), for electrophysiology only
Osmometer (Fiske), for electrophysiology only
Suss ACS200 wafer coater
Suss MA6 aligner PlasmaTherm 770 DRIE etcher
Metroline M4L plasma resist etcher
Nitto Model 3195V thermal release tape
Zeiss LSM 510 confocal microscope
Borosilicate glass pipettes
Laminar flow hood
Serum-free medium Combine the 375 ml Leibovitz medium (L15), 125-ml medium 199, 10 ml B27 Supplement (50×), 10 ng ml−1 of b-FGF, 1 ng ml−1 of BDNF, 1 ng ml−1 of glial GDNF, 10 ng ml−1 of CT-1 and 0.70 g sodium bicarbonate. Adjust the pH of the medium to 7.3 with 1 N NaOH and the osmolarity to 320–325 mOsm with distilled water.
Note: The components can be added all at once, in no particular order.
Intracellular solution Combine 0.15 ml HEPES–sodium salt (from a 1 M solution), 11.4 mg EGTA, 983.6 mg K-gluconate, 0.3 ml MgCl2 (from a 200 mM stock solution), 30.7 mg Na2ATP and 30 ml deionized water. Adjust the pH to 7.2 with HEPES, and adjust the osmolarity to 276 mOsm, with distilled water.
Extracellular solution Take 50 ml of the Leibovitz medium (L15), check the pH and adjust to 7.34 with HEPES, also adjust the osmolarity to 320 mOsm with distilled water.
Microcantilever fabrication and surface modification of microcantilevers and glass coverslips
Timing: 2–3 weeks
Fabrication of microcantilevers (Figs. 2 and 3). Start procedure with a 4-inch double-sided polished SOI wafers that contain a 5-μm thick top layer of crystalline silicon bonded onto a 500-μm thick silicon dioxide layer.
Perform photoresist spinning and developing in a Suss ACS200 wafer coater/developer (or similar instrument). First, prime the device layer (5 μm silicon) with a 100 Å layer of hexamethyldisilazaine (HMDS). Then place the wafer, silicon layer up, on a resist spinner vacuum holder. Center the wafer and apply the AZ5214e photoresist to the center of the wafer. Begin spinning the wafer to spread the resist to a final thickness of 2.1 μm (spin at 1,900 r.p.m.).
Bake the resist at 110 °C for 50 min.
Expose the wafer on a Suss MA6 aligner to the photomask containing the pattern for the cantilever. After exposure, develop the resist in a 1:1 mixture of AZ320 and dH2O.
Perform a deep reactive ion etching (DRIE) in a PlasmaTherm 770 DRIE etcher (or similar instrument) at a rate of 4 μm min−1. Etch the 5-μm device layer through to the silicon dioxide layer. Remove the photoresist using a Metroline M4L plasma resist etcher (or a similar instrument).
Flip the wafer over and prime the silicon dioxide layer with a 100 Å layer of HMDS. Next, coat with AZ9245 photoresist to a final thickness of 10 μm (1,000 r.p.m.) and softbake at 100 °C for 2 min. Place the coated wafer in a Suss MA6 aligner (or similar instrument) and expose the resist to the mask containing the pattern for the back side pattern.
Note: The Suss MA6 aligner is capable of front/back alignment. For this process it is necessary to incorporate the alignment marks in the photomasks.
After exposure, immerse the wafer in AZ 400K developer to develop the patterned resist. This is followed by a hard bake at 120 °C for 30 min. Etch until approximately 50 μm etch depth is left.
Mount the wafer on a dummy wafer using Nitto Model 3195V thermal release tape. Complete the DRIE.
Demount the wafer by heating to 170 °C on a hotplate. Remove the photoresist by plasma etching in a Metroline M4L plasma resist etcher (or similar instrument). Perform the final etch in 49% HF for 10 min to remove the buried oxide layer.
Surface modification of the cantilevers with DETA
Timing: 2–3 d
Immerse the cantilever die in a 1:1 solution of absolute MeOH and concentrated HCl for 15–30 min, followed by a 3× wash in dH2O. Next, place the die in a bath of concentrated sulfuric acid for 30–45 min. Note that, at this point you need to ensure that the die are not touching each other as they may stick together during this step.
Gently rinse the cantilever die in dH2O. Finally, boil the cantilever die in deionized water for 30 min. Then place the cantilevers in a 120 °C oven overnight.
Before the surface modifications, prepare all glassware and reagents for the reaction. Clean all glassware in a base bath (saturated KOH in 100% methanol) overnight and then rinse in dH2O (18 MΩ resistance). Bake the glassware overnight in a 120 °C oven. Distill toluene over metallic sodium in order to ensure minimal water content.
Transfer the desired volume of toluene to a clean/dry (see Step 2) Pyrex bottle. Before sealing the bottle, blow dry nitrogen into the unused volume in order to minimize the gaseous oxygen. Seal the bottle and place in the antechamber of the glove box. Evacuate the antechamber for 10 min and then vent with dry nitrogen, repeat three times. Transfer the toluene to the main chamber of the glove box.
Add stock DETA solution to the toluene for a final concentration of 0.1% (e.g., 0.2 ml in a final volume of 200 ml). Remove the 0.1% DETA solution from the glove box immediately and transfer into a glass beaker containing the cantilever die.
Place the reaction mixture on a hotplate and heat to approximately 80 °C for 30 min. After 30 min, remove the reaction mixture from the hotplate and cool for 30 min or until the reaction mixture can be handled with bare hands.
Rinse the die three times in clean/dry toluene. Transfer the cantilever die into a fresh beaker and cover with distilled toluene.
Place the beaker back onto the hotplate and heat to approximately 80 °C for 30 min. Finally, remove the cantilevers from the beaker and bake overnight at 120 °C.
Cleaning glass coverslips and subsequent surface modification with DETA
Clean glass coverslips (22 × 22 mm no. 1) using an O2 plasma cleaner for 20 min at 400 mTorr.
Form the DETA film by reacting the cleaned surface with a 0.1% (vol/vol) mixture of organosilane in freshly distilled toluene.
Heat the DETA coated coverslips to just below the boiling point of the toluene, rinse with toluene, reheat to just below the boiling temperature, and then dry in an oven.
Characterizing the glass coverslips using contact angle measurements and x-ray photoelectron spectroscopy (XPS)
Characterize surfaces by contact angle measurements using an optical contact angle goniometer.
Muscle cell isolation and culture: isolation of the myoblasts
Timing: 4–6 h
Dissect the skeletal muscle from the hind limb thighs of a rat fetus at fetal rat age of E18.
Collect the tissue samples in a sterile 15-ml centrifuge tube containing 1 ml of PBS (calcium- and magnesium-free).
Enzymatically dissociate the tissue samples using 3 ml of 0.05% of trypsin–EDTA solution for 60 min in a 37 °C water bath, with agitation of 100 r.p.m.
After 60 min, remove the trypsin solution and add 6 ml of L15 containing 10% FBS to terminate the trypsin action.
Mechanically triturate the tissue using a sterile narrow bore Pasteur pipette.
Allow the dissociated tissue to settle for 3 min.
Transfer the 6-ml supernatant to a 15-ml centrifuge tube.
Repeat the same process (Steps 26–29) twice by adding 6 ml of L15 + 10% FBS each time.
Suspend the 18 ml cell suspension obtained after mechanical trituration on a 6 ml, 4% wt/vol BSA (prepared in L15 medium) cushion and centrifuge at 300g for 10 min at 4 °C.
Resuspend the obtained pellet in 10 ml L15 + 10% FBS and plate in 100-mm uncoated dishes for 20–30 min depending on the amount of tissue. During this step, remove the contaminating fibroblasts. The fibroblasts should settle down and attach to the bottom of the 100 mm dish faster than the myocytes. The supernatant should contain pure myocytes.
Gently remove the nonattached cells that are present in the supernatant after 20–30 min by using a sterile Pasteur pipette.
Transfer the supernatant to the top of a 6 ml 4% BSA cushion, and centrifuge at 300g for 10 min at 4 °C.
Resuspend the pellet in 1 ml of serum-free medium.
The cells are now ready for plating. At this point, conduct a cell count and plate the cells at a density of 500–800 cells mm−2 on either coverslips or fabricated cantilevers.
Add the medium 1 h after plating. Maintain the cultures in a 5% CO2 incubator (relative humidity, 85%).
Change half of the medium after every 4 d.
Characterization of the myotubes: morphological quantification and fusion index
Quantify the myotube yield using the fusion index, which is defined as the number of nuclei contained in the myotubes divided by the total number of nuclei counted in a given microscope field.
Immunostaining myotubules for α-actin
Rinse coverslips in PBS, fix in cold ethanol (100%) for 20 min, and rinse again in PBS.
Block the cultures by incubating in 5% BSA in PBS for 2 h at 4 °C.
Add the primary antibody (mouse anti-α-actin, 1:800 dilution in blocking solution) and incubate for 12 h at 4 °C.
Then add the secondary antibody (Alexa Fluor 488-conjugated donkey antimouse, 1:200 dilution in PBS) to the cultures and incubate for 2 h at 37 °C.
Rinse with PBS.
Mount the coverslips with Citiflour-mounting solution onto slides.
Visualize the coverslips, using a Zeiss LSM 510 confocal microscope; or a similar instrument could be used.
Timing: 2 d
Immunostaining myotubes with the embryonic myosin heavy chain antibody
Rinse coverslips with PBS.
Fix the cells in −20 °C methanol for 5–7 min.
After 5 min, gently wash the coverslips in PBS in order to remove the excess fixing agent and then further incubate in PBS supplemented with 1% BSA and 0.05% saponin (permeabilization solution) for 5 min.
Block the cells for 30 min with 10% goat serum and 1% BSA.
Incubate the cells overnight with the primary antibody against embryonic MHC (F1.652, IgG,) diluted (1:5) in the permeabilization solution.
Wash the cells with PBS and incubate with the secondary antibody (Cy3-conjugated antimouse, 1:200 dilution in PBS) for 2 h.
After incubating in the secondary antibody for 2 h, the coverslips are finally rinsed with PBS and then the coverslips with Citiflour-mounting solution onto slides are mounted. Visualize the coverslips using a confocal microscope or similar instrument. Cultures used for immunostaining are simultaneously counterstained with DAPI, a classic fluorescent nuclear and chromosome counterstaining marker, and used to identify the nuclei and indicate the chromosome-banding patterns. DAPI selectively binds to double-stranded DNA (dsDNA) and, thus, shows little to no background staining in the cytoplasm. Immunostained coverslips can be stored for more than 1 year. The counting is done at the time of fusion index analysis of the success of the culture system. Generally, we perform this analysis soon after preparing the coverslips. Count nuclei in 20 randomly chosen microscope fields from a minimum of six separate muscle culture experiments.
Timing: 2 d
Electrophysiological characterization of the myotubes using patch-clamp electrophysiology
Place the glass coverslips with the cultured skeletal muscle myotubes in a chamber on the microscope stage.
Continuously perfuse the chamber (2 ml min−1) with the extracellular solution through the inline heater (35 °C) by gravity.
Pull a glass pipette (three-step pull, heat = ramp value, pull = 0, velocity = 30, time = 200, pressure = 300) with the electrode puller and fill with the intracellular solution. The electrode resistance should be 6–8 MΩ.
Place the electrode into the headstage; apply positive pressure (approximately 2 cm3 from a 10 cm3 syringe) to the pipette before touching the extracellular solution.
Bring the electrode close to the target cell; compensate for the pipette offset; touch a cell with the tip of the electrodes (seal position) under visual control.
Apply a −5 mV seal test; compensate pipette capacitance.
Apply a −70 mV holding (voltage clamp mode).
Form a gigaseal.
Rupture the cell membrane by the application of short suction pulses; measure resting membrane potential in I = 0 mode; compensate for whole cell capacitance and resistance.
Set the amplifier gain to 2 and apply a 2 kHz low-pass filter. Digitize signals at 20 kHz.
Correct all membrane potential data by subtracting a 15 mV tip potential. This is calculated using the Axon's pClamp 8 program.
Measure membrane resistance and capacitance using 50 ms voltage steps, from −85 to −95 mV, without any whole-cell or series resistance compensation followed by single-exponential fitting (τ = RmCm).
Measure the sodium and potassium currents in voltage clamp mode using 10 mV voltage steps from a −85 mV holding potential. Measure the sodium channel current amplitude at −25 mV, where no potassium channel activation is observed. Measure the potassium channel current at +15 mV at the end of the voltage step, when sodium channels are inactivated.
Evoke action potentials with 1 s depolarizing current injections from a −85 mV holding potential.
Timing: 1 d
Troubleshooting advice can be found in Table 1.
From microcantilever fabrication through immunocytochemical and electrophysiological characterization:
Steps 1–8, microcantilever fabrication (days 1–14/21): 2–3 weeks
Steps 10–22, clean and coat the microcantilevers and glass coverslips with DETA (days 14–21): 2–3 d
Steps 23–40, cell culture including dissection, tissue isolation and plating (days 16–23): 4–6 h
Steps 40A–C (days 26–33) electrophysiological characterization: 1 d; immunocytochemical characterization: 2 d; immunostaining myotubes for α-actin: 2 d
Glass coverslips and cantilevers coated with DETA are analyzed by contact angle and XPS. XPS has been shown to provide a good quantitative indicator of monolayer formation. The contact angle, along with XPS data, indicates that the surfaces are covered by a complete monolayer of DETA.
In the cultures, myoblast fusion begins after 24 h in the defined culture system and after 36–48 h maximal fusion is reached. Spontaneous contractions of the multinucleated myotubes are observed by the end of the second day (Fig. 5).
Immunocytochemistry combined with confocal microscopy and electrophysiological methods are then used to characterize the myotubes. Myotubes are observed to be labeled by the skeletal muscle markers for α-actin and the embryonic myosin heavy chain to facilitate unambiguous identification (Fig. 6).
In half of the cantilevers from each experiment, one will observe that dissociated muscle cells are aligned along the long axis of the cantilever to form contracting myotubes (Fig. 7).
Electrophysiological recordings from the myotubes indicate the presence of voltage-dependent inward and outward currents in voltage clamp mode. In current clamp mode, the myotubes fire single action potentials (Fig. 8).
We would like to acknowledge Matthew P. Daniels at NHLBL-NIH, DARPA ITO grant number N65236-01-1-7400 and National Institutes of Health (NIH) grant number 1 RO5 N5050452-03.
About this article
Developing a novel serum-free cell culture model of skeletal muscle differentiation by systematically studying the role of different growth factors in myotube formation
In Vitro Cellular & Developmental Biology - Animal (2009)