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Method Article

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

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DOI:

10.3791/51354

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April 30th, 2014

 ,  ,  , 
1Department of Biology, University of Alabama at Birmingham, 2Nutrition Métabolisme Aquaculture, INRA UR1067, 3Laboratoire de Physiologie et Genomique des Poissons, INRA UR1037

In This Article

Summary

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.

Abstract

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. teleost fish) and full regeneration following appendage loss (i.e. 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.

Introduction

Considerable understanding of mammalian myogenesis has been obtained through the recapitulation of this process in both primary mouse (Mus musculus) myoblast cultures and the well-described mouse-derived cell line, C2C125. Beginning in the 1950s6, these cultures have led to much advancement in the understanding of the murinemyogenic program and, by extension, myogenesis in other vertebrates. Additionally, single cell myofiber explant techniques have increased out understanding of interactions between satellite cells and surrounding myofibers7-9.Cell cultures are particularly attractive for investigations of myogenesis due to the short time from precursor to differentiated cell10, relative ease of transfection for RNAi11-14, transgenic15,16 and overexpression studies14,17,18 , in vitro expansion followed by in vivo transplantation18-20, and even comparison of myogenic precursor cells and their regulating agents across taxa21,22. While differences due to the artificial environment of the culture system have been described5,23 , these in vitro systems have proven to be indispensible to our dissection of the intricate program governing the formation of multinucleated, terminally differentiated myofibersfrom mononucleated proliferative progenitor cells known as myosatellite cells (MSCs) among the mammals.

Outside of the class Mammalia, however, the conservation and/or divergence of mechanisms controlling myogenesis are poorly understood, largely due to the difficulty in culturing myogenic precursor cells (MPCs) and myoblasts from various taxa. Indeed, primary myoblast cultures have only been described in three birds24-26, one reptile27, a few amphibians28-30, and some fishes1,3,4,31-33. Continuous myogenic cell lines from vertebrates other than rodents34-36are even more rare, with the only non-mammalian myogenic cell line being derived from Japanese quail (Cortunix japonica), QM737. Despite many attempts at immortalization, a teleost myogenic cell line remains elusive and a protocol for efficient transfection of these cells was only published this year15. Thus, clear and well-optimized protocols for culturing primary MPCs and myoblasts from a variety of vertebrates are very much needed to not only further expand our knowledge of the evolution of the myogenic program, but to employ the power of comparative physiology to make breakthroughs in the treatment of human skeletal muscle diseases and disorders.

While the literature contains many reports of MPC/myoblast isolation38-49, it is common for authors to describe the protocols for such isolations in brief, often incomplete, formats. Further, the most instructive protocols reported have been developed for mice50-53, and some of these rely on antibody selection54,55 or fluorescence transgenes56,57, making these protocols unusable or impractical inmost non-rodent species utilized by muscle biologists. With little known about piscine, amphibian, and reptile myogenesis, a detailed and thorough protocol, described with audiovisual guidance and with demonstrated efficiency in distantly related species, would be most helpful to the field.

First described by Powell and colleagues in 198958, the following protocol was initially developed to isolate MPCs and myoblasts from salmonid fishes (namely, rainbow trout, Oncorhynchus mykiss, and Atlantic salmon, Salmo salar) and some larger cyprinids (i.e. goldfish, Carassiusauratusauratus). In 2000, Fauconneau and Paboeuf optimized a primary myoblast culture for rainbow trout59, and minoroptimizations made that protocol utilizable in several smaller minnows of the Danioninae clade (zebrafish, Danio rerio, and giant danio, Devario aequipinnatus)32 due to the many genetic tools available for zebrafish work and thus its close relatives. Teleost fish are attractive organisms for study due to their divergent growth strategy (at least in most species). Large salmonids, like most fishes, grow indeterminately, with growth potential unfettered by an asymptote at maturity, even in old age60-62. Unlike zebrafish, large danionins such as the giant danio63 and moustached danio display growth potentials typical of teleost fish, making their direct juxtaposition an ideal platform for understanding whether MPC cell fate choice plays a role in skeletal muscle hyperplasia versus hypertrophy.

Likewise, we have demonstrated that this protocol can be used with mice and axolotls, with relatively high cell yield and viability indices. Urodele salamanders, such as the Mexican axolotl (Ambystomamexicanum),possess the remarkable ability to regenerate tissues, including entire limbs and tails64-66. This characteristic makes these amphibians interesting models of skeletal muscle wasting and aging. Using the protocol described below, a similar approach can be undertaken as has been done in many fish species, providing an even wider comparative context for such studies. As many truly comparative biologists appreciate, the most meaningful advances in basic biology and translational biomedicine can be made when data are analyzed within the widest spectrum (here, the entire vertebrate lineage).

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Protocol

Ethics Statement: All experimentation involving vertebrate animals described herein was approved in advance by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham and is consistent with guidelines established by the Office of Laboratory Animal Welfare, National Institutes of Health of the U.S. Department of Health and Human Services.

1. Preparation for Culture

  1. Prepare the base medium as follows: 9 mM NaHCO3 (1.51 g per 2 L), 20 mM HEPES (9.53 g per 2 L) in DMEM (13.48 g/L; 26.96 g per 2 L).
    1. Determine pH and adjust to 7.40 with HCl or NaOH.
    2. Determine osmolality and adjust to ~ 300 mOsm with NaCl (if needed).
    3. Filter sterilize using (2) 1 L vacuum sterilization systems and store at 4 °C protected from light for up to 2-3 months.
      NOTE: See Tables 1 and 2 for complete reagent, tools, consumables, and equipment information.
  2. Prepare poly-L-lysine-treated plates by dissolving 5 mg of γ-irradiated poly-L-lysine (> 300,000 mw) in 50 ml of ultrapure sterile water. Mix gently by inversion for 10 min at room temperature, ensuring full dissolution of lysine polymers.
  3. Pipette the necessary amount of poly-L-lysine solution into each well, according to the plate type (Table 3). Allow plates to stand in laminar flow hood for 5 minutes.
  4. Next, remove poly-L-lysine solution and wash twice with sterile water. Allow plates to air dry in laminar flow hood for 20 minutes.
  5. Obtain 1 mg of laminin (Engelbreth-Holm-Swarm origin) and dissolve in 50 ml of base medium to a final concentration of 20 μg/ml. Apply laminin solution to poly-L-lysine-treated cell culture plates at 2 μg/cm2. (see Table 3 for different plate sizes)
  6. Gently swirl the solution to ensure full well coverage. Seal plates with laboratory tape and place in incubator for 24 hours. The incubator should be set to appropriate temperature for species being used (see Table 7), and no additional gases need to be added to incubator.
    NOTE: Laminin must be allowed to adhere to plates for 24 hours before seeding cells. Failure to do so will result in poor to no cell adherence.
  7. Prepare media as described in Table 4. Complete media is best prepared the day of use.
  8. In preparation for tissue isolation, assemble the following items and autoclave: 300-500 ml beaker(s); glass Petri dishes; scalpel handles; forceps (fine and coarse); dissection scissors; and several sheets of water-repellant autoclave paper.
    NOTE: For each person assisting with the dissection process, (2) scalpel handles, (1) forceps (type depending on personal preference), (1) dissection scissors, and (5-10) sheets of water-repellant autoclave paper are sufficient.
  9. In addition to the autoclaved items above, assemble 70% ethanol, a weight balance, sterile 50 ml conical tubes (number depends on size of culture), and ice.

2. Tissue Dissection

  1. Before beginning, ensure that a sufficient stock of fish is available.
    NOTE: The age of the fish to be used is of critical importance. While fish of two different species may be of similar weights, a younger fish of one species will possess more MPCs than an older fish of another species. As a general rule, younger fish, especially when working with salmonids, are best. For danionins, fish as old as one year can be utilized optimally, while salmonids of age 4-6 months or younger (up to 15 g) are optimal.
  2. For each 5 g of tissue to be dissected, aliquot 25 ml of isolation medium into a sterile 50 ml conical tube.
  3. Weigh, record mass, and number the tube(s). Place tubes on ice.
  4. Euthanize 2-3 fish at a time by immersion in > 300 mg/L sodium bicarbonate-buffered tricaine methanesulfonate. Allow opercular movement to cease for > 5 minutes and then submerse fish in 70% ethanol (contained in the sterile beakers) for 30 seconds.
  5. Remove fish from 70% ethanol and place on water-repellant autoclave paper. Directly behind the opercula, use a scalpel to make a superficial, shallow incision. Remove the scales and skin by grasping the skin at the aforementioned incision and pull toward tail of fish.
  6. Following skin removal, excise the epaxial, fast-glycolytic ‘white’ muscle of the fish from both sides (avoiding the slow-oxidative ‘red’ muscle located near the lateral line) and place in isolation medium. Discard the remainder of the fish as required by institution.
    NOTE: While it is entirely possible to culture MPCs originating from red muscle, nearly every publication using teleost MPCs has utilized cells isolated from the epaxial myotome.
  7. Repeat steps 2.4-2.6 until a sufficient quantity of muscle is obtained. During dissection, ensure that pooled muscle tissue remains on ice to maintain cell viability.

3. Mechanical Dissociation

  1. Pour one tube of 25 ml/5 g of muscle tissue into a glass Petri dish. Using two scalpel handles with attached #10 scalpel blades, mince tissue to a slurry or purée consistency. A “back-and-forth” motion of pulling the scalpel blades past each other works best.
    NOTE: While tissue homogenizers may seem appropriate, the number of viable cells will be dramatically reduced, if not abolished.
  2. Using a 25 ml serological pipette and serological pipettor, remove the slurried tissue and replace in the original conical tube.
  3. Repeat steps 3.1-3.2 until all tissue in all tubes has been mechanically dissociated.
  4. Centrifuge tissue for 5 minutes at 300 x g and 10 °C.
  5. Following centrifugation, discard the 25 ml of media supernatant with a 25 ml serological pipette, vacuum aspirator, or by careful decanting.
  6. Add 25 ml of wash medium to each tube, resuspend tissue, and recentrifuge as above.
  7. Wash twice with wash medium, removing the supernatant each time.

4. Enzymatic Dissociation

  1. During step 3.4, prepare collagenase solution by combining 0.22 g of Type IV collagenase and 44 ml of base medium (or sufficient quantity at 0.11 g/0.22 ml).
  2. Stir in a beaker for 10-15 minutes at 4 °C. Filter sterilize with a 50 ml syringe and a 0.45 μm syringe filter.
    NOTE: More than one syringe filter may be needed, depending on the collagenase preparation. Collagenase obtained from vendors other than Worthington Biochemical poses more difficulty during filtration. Collagenase is used to dissociate peptide bonds in collagen that constitute the endomysium. This digestion will remove that protective barrier of the myofibers.
  3. For each g of muscle tissue, resuspend tissue in a 0.2% collagenase solution. For 5 g of muscle tissue, add 10 ml of collagenase solution and 15 ml of base medium.
  4. Add 10 μl of PSF per ml of collagenase solution/base medium (e.g. 250 μl to 25 ml). Ensure that tissue is well suspended, as this will affect the efficiency of the enzymatic digestion.
  5. Incubate muscle tissue in collagenase for 60 minutes at 18 °C with gentle rocking. Depending on the degree of mechanical dissociation and species, collagenase digestion may be extended to 90 minutes, although this may affect cell viability.
  6. Following collagenase digestion, centrifuge conical tubes at 300 x g for 5 minutes at 12 °C. Discard the supernatant and resuspend tissue in 25 ml of wash medium.
  7. Recentrifuge at 300 x g for 5 minutes at 12 °C. Repeat with washing medium a second time.
  8. After washing tissue twice, resuspend muscle tissue in 25 ml of wash medium and triturate with a 10 ml serological pipette until the tissue/medium homogenate passes in and out of the pipette with relative ease. 5-10 triturations is usually sufficient.
    1. Repeat with a 5 ml serological pipette and then a 16 gauge metal cannula attached to a syringe.
  9. Centrifuge conical tubes at 300 x g for 5 minutes at 12 °C. Decant supernatant.
  10. During the above 5 minute centrifugation, prepare the trypsin digestion solution by combining 0.25 g of trypsin with 5 ml of base medium.
  11. Stir at 4 °C for 10-15 minutes and filter sterilize with a 20 ml syringe and 0.45 μm syringe filter.
  12. Retrieve conical tubes and add 100 μl of trypsin solution and 4.9 ml of dissociation medium for every 1 g of muscle tissue to every tube. For example, add 500 μl of trypsin solution and 24.5 ml of dissociation medium to 5 g of muscle tissue.
    NOTE: Trypsin is a serine protease that will separate myogenic precursor cells from the basal lamina.
  13. Resuspend tissue in the trypsin/dissociation medium well. Incubate for 20 minutes at 18 °C.
  14. Following the first trypsin incubation, centrifuge conical tubes at 300 x g for 1 min at 12 °C.
  15. Neutralize trypsin by combining the supernatants with isolation medium at a concentration of 1 volume of supernatant to 4 volumes of isolation medium. Store at 4 °C during the second trypsin digestion.
  16. To the remaining pellet of tissue, repeat steps 4.12-4.15, combining the supernatant following step 4.15 with the supernatant/isolation medium from the first trypsin digestion.
  17. ALTERNATIVELY: The collagenase and trypsin digestions may be combined to maximize cell yields.
    1. To do this, triturate the collagenase digest using a metal cannula (6 in length and 16 gauge works best) attached to a 20 ml syringe until the homogenate easily passes through the cannula.
    2. To the homogenate, add 500 μl of trypsin solution (prepared in step 4.10) and incubate at 18 °C for 20 minutes.
    3. Following the incubation, centrifuge conical tubes at 300 x g for 1 minute at 12 °C.
    4. Neutralize trypsin by combining the supernatants with isolation medium at a concentration of 1 volume of supernatant to 4 volumes of isolation medium.
    5. Store at 4 °C during the second trypsin digestion.
    6. Proceed with step 4.18 as with the standard protocol.
  18. Dispense the final supernatant/isolation medium mixture into 50 ml conical tubes and centrifuge at 300 x g for 10 minutes at 12 °C.
  19. Remove the supernatants taking care not to disturb the cell pellets. Pipette 2 ml of complete medium into each tube, dissolving each cell pellet.
  20. Combine cells suspended in complete medium in one 50 ml conical tube.
  21. Rinse each tube with 1 ml of complete medium, combining with the pool of cells resuspended previously.
  22. Triturate with a metal cannula and syringe 5-10 times.
  23. Filter cell suspension through a 100 μm cell strainer, rinsing with complete medium.
  24. Filter the cell suspension through a 40 μm cell strainer.
  25. Add sufficient complete medium to 50 ml and centrifuge at 300 x g for 10 minutes at 15 °C.

5. Counting, Dilution, and Seeding of Cells

  1. Following the final centrifugation in step 4.25, remove supernatant by careful decanting or with a serological pipette. Resuspend the cell pellet in 5 ml of complete medium.
  2. With cells fully resuspended, remove 20 μl of the cell suspension and place in a 1.5 ml microcentrifuge tube.
  3. Add 5 μl of Trypan blue dye and wait 5 minutes. Using a hemocytometer, determine the number of viable cells.
  4. Upon determination, dilute cells to the needed concentration. Teleost MPCs are best seeded at 150,000-200,000 cells per cm2. For a six-well plating strategy, diluting cells to 1.5 x 106 - 2.0 x 106 per ml supports sufficient proliferation and differentiation.
  5. Retrieve poly-L-lysine-treated, laminin-coated plates and seed cells. (See Table 5 for plate-specific dilutions and plating volumes.) NOTE: Table 6 depicts average number of cells isolated per gram of muscle tissue isolated from various teleost species. Rainbow trout (O. mykiss) exhibit fewer MPCs per gram of muscle tissue than do zebrafish (D. rerio) and will thereby require more fish to isolate from for proper seeding.
  6. Seal plates with laboratory tape and place in chilling incubator at the appropriate temperature. MPCs from all species listed here (save for mice) may be incubated under normal atmospheric conditions without carbon dioxide (CO2) supplementation. (See Table 7.)
  7. ALTERNATIVELY: Some laboratories have adopted a protocol that calls for allowing MPCs to adhere to the culture substrata for 40 minutes.
    1. Then rinse plates with washing media to remove any loosely attached and non-adherent cells. Warning: This step is very important to avoid “non-specific” adhesion of other cells (e.g. fibroblasts) to the culture substrata.
    2. Add complete media and place cells in the incubator and care for as detailed below.

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Results

Twenty-four hours post-seeding, myogenic precursor cells (MPCs) should be visible attached to the laminin substratum (see Figures 1a and 1d). Following seeding, cells (MPCs) adopt a spindle-like shape, indicative of this cell type (Figure 1) and are MyoD1+ (Figure 2). In Danio species, MPCs appear to be more compact with smaller bipolar processes than do MPCs from Oncorhynchus and Salmo species. However, over four days of cultu...

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Discussion

The myogenic program, in whichever species examined, can be most easily studied through an in vitro system. Indeed, upon isolation, myogenic precursor cells (MPCs) in fish or myosatellite cells (MSCs) in mammals readily enter this highly regulated process involving the proliferation, cell cycle withdrawal, and terminal differentiation of myoblasts and the fusion of those myoblasts into nascent myotubes. The general lack of transgenic gene reporter strains of piscine species (with the possible exception of the ze...

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Disclosures

There is nothing to disclose.

Acknowledgements

The authors would like to extend many thanks to Drs. Josep Planas and Juan Castillo for their professional expertise in the development and application of this culture protocol to small fishes and amphibians. Thanks are also due to the countless individuals who have tirelessly assisted with the dissection and dissociation of muscle tissue from many fish (both in species and number), including Matthew Charging, Delci Christensen, Zachary Fowler, Brooke Franzen, Nathan Froehlich, Kira Marshall, Ben Meyer, Ethan Remily, and Sinibaldo Romero. This work was supported by University of Alabama at Birmingham Department of Biology start-up funds, Center for Protease Research NIH Grant # 2P20 RR015566, NIH NIAMS Grant # R03AR055350, and NDSU Advance FORWARD NSF Grant #HRD-0811239 to PRB. Support was also provided by the UAB Nutrition Obesity Research Center award # P30DK056336, NIH NIDDK. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Table 1. Detailed Reagent Information
ReagentCompany (Preferred v. Alternate)Catalog Number (Preferred v. Alternate)Quantity per Culture
γ-irradiated poly-L-lysineSigma-Aldrich (MP Biomedicals)P5899 (ICN19454405)5 mg
DMEM (high glucose)Sigma-Aldrich (cellgro)MT-50-003-PB (D7777)2 L
LamininBD Biosciences (Sigma-Aldrich)CB-40232 (L2020)1 mg
Sodium Bicarbonate (NaHCO3)Fisher Scientific (Sigma-Aldrich)BP328-500 (S5761)1.51 g
HEPES (C8H18N2O4S)Fisher Scientific (Sigma-Aldrich)BP310-1 (H6147)9.53 g
Antibiotic/AntimycoticThermo Scientific (Sigma-Aldrich)SV3007901 (A5955)17-20 mL
Gentamicin SulfateLonza (Sigma-Aldrich)BW17-519Z (G1397)2-3 mL
Donor Equine SeraThermo Scientific (Sigma-Aldrich)SH3007403 (H1270)75 mL
Fetal Bovine SeraThermo Scientific (Sigma-Aldrich)SH3007103 (F2442)25 mL
Collagenase (Type IV)Worthington (Sigma-Aldrich)LS004189 (C9891)0.44 g
Trypsin (from Pancreas)MP Biomedicals (Sigma-Aldrich)ICN15357125 (T5266)1 g
Table 2. Consumables, Tools and Equipment
ConsumableToolsEquipment
Cell Culture PlatesForceps (Coarse)Serological Pipettor
Sterile 50 mL Conical TubesForceps (Fine)pH Meter
Laboratory TapeScalpel HandlesChilling Incubator (Echotherm)
0.2 μm Vacuum Sterilization SystemsScalpel Blades (#10, #11)Laminar Flow Hood
Water-repellant Autoclave PaperSurgical ScissorsVacuum Manifold
Serological PipettesGlass Petri DishesMicroosmolality Meter
12-16 G Cannulas with Luer Locks
Table 3. Optimized Volumes for Coating Cell Culture Plates
Plate Sizecm^2 per WellPoly-L-lysine*Laminin**
6 well9.51.6 mL1
24 well1.90.320.2
48 well0.950.160.1
96 well0.320.060.03
*0.1 mg/mL concentration** 0.020 mg/mL concentration
Table 4. Media for Isolation, Dissociation, and Culture
ReagentIsolationWashDissociation
Base Medium419.25 mL395.40 mL297.00 mL
Gentamicin Sulfate**0.75 mL0.60 mL-
Donor Equine Serum75.00 mL--
Fetal Bovine Serum***---
* PSF: penicillin/streptomycin/fungizone cocktail (100x); ** 50 mg/mL concentration; *** Characterized
Table 5. Recommended Dilutions and Plating Volumes
Plate Sizecm^2 per WellDilutionPlating Volume
6 well9.51.5-2.0x10^6 cells/mL1 mL
24 well1.91.5-2.0x10^6 cells/mL250 μL
48 well0.951.5-2.0x10^6 cells/mL150 μL
96 well0.321.5-2.0x10^6 cells/mL50-100 μL
Table 6. Average number of cells per g tissue
SpeciesAverage # cells/g tissue
Danio rerio6,400,000
Danio dangila1,783,000
Devario aequipinnatus1,797,000
Oncorhynchus mykiss66,800
Table 7. Recommended Incubation Temperatures
SpeciesTemperature
Danio/ Devario spp.26 - 28 °C
Oncorhynchus/Salmo spp.10* - 18 °C
Ambystoma mexicanum18°C
* Lower temperatures support lower proliferation rates.

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Myogenic Precursor CellsPrimary Cell CultureTissue DissectionMechanical DissociationEnzymatic DissociationCollagenase TreatmentLaminin SubstrateCell PlatingMyogenesis AssayZebrafish Model
Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages
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