Myofiber Isolation: A Technique to Obtain Single Myofibers from Harvested Mouse Extensor Digitorum Longus (EDL) Muscle

Abstract

Source: Hüettner, S.S. et al. Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo. J. Vis. Exp. (2021)

This video demonstrates the isolation of single myofibres from harvested murine Extensor Digitorum Longus (EDL) muscle. The isolated myofibres can be cultured to study myofiber-associated muscle stem cell activity.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. EDL muscle isolation and collagenase digestion

1. Spray all equipment with 70% ethanol to avoid contamination.
2. Sacrifice the mouse in accordance with the national regulations for animal experimentation.
3. Spray the hindlimbs of the mouse with 70% ethanol. Use hardened fine curved scissors (24 mm cutting edge) and fine forceps (Dumont 7, curved or straight) to remove the skin and to expose the underlying muscles. Avoid any contact of the underlying muscles with fur (increases the risk of contamination).
4. Remove the surrounding fascia with fine curved forceps without damaging the underlying muscles (Figure 1A). Close the forceps to avoid bending.
5. Use curved forceps to expose the distal tendons of the TA (tibialis anterior) and EDL muscle. To remove the TA, grab the distal TA tendon with forceps and cut with fine Vannas spring scissors (5 mm cutting edge, 0.35 mm tip diameter). While holding the TA at the tendon, pull it towards the knee and cut the muscle close to the knee (Figure 1B), the EDL muscle is now exposed.
   NOTE: Make sure that only the TA tendon is grabbed in this step, otherwise the underlying EDL might get damaged. When cutting off the TA muscle make sure that the tendons at the knee can be seen easily afterwards.
6. Lift the distal EDL tendon with fine curved forceps and cut with fine Vannas spring scissors (Figure 1C). Expose the proximal EDL tendon by carefully pulling the EDL towards the knee. Cut the proximal tendon with fine Vannas spring scissors. Transfer the EDL muscle to the 37°C preheated 2.5 mL of collagenase digestion solution, in the 15 mL reaction tube. The collagenase solution is prepared as according to this procedure:
   1. Prepare collagenase digestion solution by dissolving 0.2% (w/v) collagenase type 1 (from Clostridium histolyticum) in DMEM (Dulbecco's modified Eagle's medium with 4.5 g/L glucose and sodium pyruvate), filter through 0.22 µm filter. (Figure 1D).
      NOTE: Make a small incision at the outer knee connective tissue to fully expose the proximal EDL tendon. Make sure to only grab the tendons and not to stretch the EDL too much.
7. Repeat steps 1.3. to 1.6 with the second EDL. Add both EDL muscles to the same 15 mL reaction tube filled with 2.5 mL collagenase digestion solution.
8. Incubate the EDL muscles in the reaction tube at 37°C in a circulating water bath.
   NOTE: Incubation time depends on several factors, such as collagenase activity, age of the mouse and amount of fibrotic tissue. The typical incubation time for EDL muscles of adult mice (2-6 months of age) is 1-1.5 h and for aged mice (18 months of age) 1.5-2 h.
9. To avoid excessive digestion of the muscles, check the muscles during the digestion time. Stop the digestion when muscles loosen up and single myofibers are visible (Figure 1E). Transfer muscles carefully with the large bore pipette to the first well of the prewarmed 12-well plate containing myofiber isolation medium (Figure 1G), which is prepared as follows:
   1. Supplement DMEM (Dulbecco's modified Eagle's medium with 4.5 g/L glucose and sodium pyruvate) with 20% FBS (fetal bovine serum), filter through 0.22 µm filter. Add medium to the pre-coated isolation plates (12-well plate, 2-4 mL isolation medium per well) approximately 30 min before the isolation and equilibrate in a humidified 37°C incubator with 5% CO₂.

2. Myofiber dissociation

1. For the following steps use a stereo binocular microscope, preferentially equipped with a heating plate (37 °C). Use the large bore pipette to flush the muscles with warm isolation medium until single myofibers are released. Dissociate the muscles with the large bore pipette until the desired number of myofibers are floating freely in the solution.
   NOTE: Avoid excessive trituration to reduce the risk of damaged myofibers. Using a heating plate for myofiber dissociation is strongly advised since temperature drops during the isolation process, which will result in myofiber death.

Results

Figure 1. Mouse EDL dissection and single myofiber isolation. (A) The skin of the mouse hindlimb is removed and the muscle surrounding fascia is pulled away to expose the TA (tibialis anterior) muscle. (B) The TA muscle is removed to expose the EDL (extensor digitorum longus) muscle, marked by the white arrow. (C) The EDL muscle is dissected by cutting its tendons. (D) Two EDL muscles are collagenase digested. (E) Appearance of collagenase digested muscle with visible single myofibers loosening up from the tissue core. (F) Large and small-bore Pasteur pipette with scale. (G) The EDL muscles (marked by black arrows) are physically triturated using a large bore Pasteur pipette. (H) Single intact myofibers are thin and shiny and can be individually collected for culture and analysis. (I) Hypercontracted and dead myofibers are unsuitable for culture and analysis. The microscopic images of 2H and 2I were captured with a microscope using a N-Achroplan 5x objective. Scale bars are 200 µm.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Collagenase type 1	Sigma	C0130
DMEM (Dulbecco’s modified Eagle’s medium with 4.5 g/l glucose and sodium pyruvate)	GibCo	41966029	Cell culture medium
Fetal bovine serum	Gibco	10270-106	Fetal bovine serum