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Isolation of Primary Human Skeletal Muscle Cells

原代人骨骼肌细胞的分离

Janelle M. Spinazzola Janelle M. Spinazzola
Emanuela Gussoni Emanuela Gussoni

发布: 2017年11月05日第7卷第21期 DOI: 10.21769/BioProtoc.2591 浏览次数: 11671

评审: Antoine de MorreeHui ZhuYann Simon Gallot

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Abstract

Primary myoblast culture is a valuable tool in research of muscle disease, pathophysiology, and pharmacology. This protocol describes techniques for dissociation of cells from human skeletal muscle biopsies and enrichment for a highly myogenic population by fluorescence-activated cell sorting (FACS). We also describe methods for assessing myogenicity and population expansion for subsequent in vitro study.

Keywords: Skeletal muscle (骨骼肌) search Myoblast isolation (成肌细胞分离) search Tissue dissociation (组织解离) search Fluorescence-activated cell sorting (FACS) (荧光激活细胞分选（FACS）) search

Background

Primary human myoblasts from muscle biopsies are a valuable resource for modeling human muscle disease in vitro. Alterations in myoblast proliferation, differentiation, and fusion are features shared by many neuromuscular disorders, and can be used to assay cell-based and pharmacological therapies. Human skeletal muscle biopsies, especially those affected by disease, often contain extensive populations of non-myogenic cells such as adipocytes and fibroblasts. Thus, it is important to purify a myogenic population for in vitro study of skeletal muscle development and disease. Early studies of muscle disease involved use of tissue explants or unpurified dissociated cells (Geiger and Garvin, 1957; Herrmann et al., 1960; Goyle et al., 1967; Bishop et al., 1971), and later, Blau and Webster introduced a pre-plating technique to remove fibroblasts (Blau and Webster, 1981). Here, we describe an effective technique for dissociation of mononuclear cells from human muscle biopsies, and purification of a highly myogenic population utilizing FACS with the cell surface markers CD56 and CD82 (see Note 1). We recently demonstrated that CD82 is an excellent myogenic marker in both human fetal and adult skeletal muscle that is also retained on activated and differentiating myogenic progenitors (Alexander et al., 2016). This protocol also describes methods to culture these myoblasts and confirm a myogenic population by in vitro fusion assay. Isolation and expansion of these cells from normal individuals and from individuals with muscle disorders will help accelerate the development of therapies for human disorders such as muscular dystrophies.

Materials and Reagents

  1. Dissociation of primary human skeletal muscle tissue (see Note 2)
    1. Protected sterile disposable scalpels with stainless steel blade size #10 (Aspen Surgical, Bard-ParkerTM, catalog number: 372610 )
    2. Sterile 10 cm tissue culture-treated plastic dishes (Corning, Falcon®, catalog number: 353003 )
    3. Assorted sterile 5, 10, and 25 ml pipettes (Olympus Plastics, catalog numbers: 12-102 , 12-104 , 12-106 )
    4. Sterile 0.22 µm PES (low protein binding) filters (250 and 500 ml volumes) (such as Olympus Plastics, catalog number: 25-227 )
    5. Sterile 15 and 50 ml conical centrifuge tubes (Olympus Plastics, catalog numbers: 21-101 and 21-106 )
    6. BD Falcon sterile nylon cell strainers (100 µm and 40 µm pore sizes) ( Corning, catalog numbers: 352360 and 352340 )
    7. Sterile 1.8 ml CryoTubeTM vials (Thermo Fisher Scientific, Thermo Scientific TM, catalog number: 377267 )
    8. 10x Hank’s balanced saline solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14185052 ) or 10x Dulbecco’s phosphate buffer saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14200075 ), free of calcium chloride, magnesium chloride and magnesium sulfate, diluted to 1x with double distilled water and filter sterilized with a 0.22 µm PES filter
      Note: This solution can be stored at 4 °C or room temperature (RT).
    9. Sterile red blood cell lysis solution (QIAGEN, catalog number: 158904 ) (stored at RT)
    10. Sterile HEPES buffered saline solution, without phenol red (Lonza, catalog number: 12-747F )
    11. Dulbecco’s modified Eagle’s medium (DMEM 4.5 g glucose) (Thermo Fisher Scientific, GibcoTM, catalog number: 10564011 )
    12. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10437 )
    13. 100x penicillin-streptomycin-glutamine (PSG) (Thermo Fisher Scientific, GibcoTM, catalog number: 10378016 )
    14. Calcium chloride dihydrate (Sigma-Aldrich, catalog number: C7902-500G )
    15. Dispase II (Roche Diagnostics, catalog number: 04942078001 )
    16. Collagenase D (Roche Diagnostics, catalog number: 11088882001 2.5g )
    17. Complete growth medium (500 ml) (see Recipes)
    18. 1 M calcium chloride solution (CaCl2·2H2O, FW 147) (see Recipes)
    19. Dispase stock solution (see Recipes)
    20. Collagenase D stock solution (see Recipes)
    21. Sterile freezing medium (see Recipes)

  2. Purification of myoblasts from dissociated human skeletal muscle mononuclear cells
    1. Thawing of cryopreserved sample prior to FACS
      1. Sterile 0.22 µm PES filter (500 ml volume) (Thermo Fisher Scientific, catalog number: 569-0020 )
      2. Sterile 50 ml conical centrifuge tubes (Olympus Plastics, catalog number: 21-106 )
      3. Sterile tissue culture-treated plastic dishes (10 or 15 cm size) (Corning, Falcon®, catalog numbers: 353003 and 353025 )
      4. CryoTubeTM vial containing dissociated unpurified primary cells (Simport, catalog number: T309-2A )
      5. Dulbecco’s modified Eagle’s medium (DMEM) (DMEM 4.5 g glucose) (Thermo Fisher Scientific, GibcoTM, catalog number: 10564011 )
      6. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10437 )
      7. 100x penicillin-streptomycin-glutamine (PSG) (Thermo Fisher Scientific, GibcoTM, catalog number: 10378016 )
      8. Complete growth medium (see Recipes)
    2. Preparation of sample for FACS
      1. Sterile 0.22 µm PES filter (50 ml volume) Cole Palmer Steriflip-GP Filter, 0.22 µm PES Item # UX-29969-20 (EMD Millipore, catalog number: SCGP00525 )
      2. Sterile 15 and 50 ml conical centrifuge tubes (Olympus Plastics, catalog numbers: 21-101 and 21-106 )
      3. Sterile 5 ml round bottom test tubes with cell strainer caps (Corning, Falcon®, catalog number: 352054 )
      4. Dissociated unpurified primary cells, thawed one day prior to FACS
      5. Sterile 1x HBSS (diluted from 10x stock) (Thermo Fisher Scientific, GibcoTM, catalog number: 14185052 )
      6. Sterile 1x DPBS (diluted from 10x stock) (Thermo Fisher Scientific, GibcoTM, catalog number: 14200075 )
      7. TrypLE ExpressTM dissociation enzyme with phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 12605010 )
      8. Antibodies
        1. APC anti-CD56 antibody, Clone HCD56 (BioLegend, catalog number: 318310 )
        2. PE anti-CD82 antibody, Clone ASL-24 (BioLegend, catalog number: 342103 )
      9. Calcein blue (1 mg vial) (Thermo Fisher Scientific, catalog number: C1430 )
        Note: Resuspend in 200 µl dimethyl sulfoxide (DMSO, AmericanBio, catalog number: AB03091-00050 ) aliquot in 25 µl aliquots and store at -20 °C (stock). Use 0.5 µl stock calcein/106 cells.
      10. Sterile 5% FBS solution (50 ml) (see Recipes) 
    3. Fluorescence-activated cell sorting (FACS)
      FACS or 5 ml round-bottom tubes with cell strainer caps (Corning, Falcon®, catalog number: 352235 )

  3. In vitro culture and analysis of human skeletal myoblasts
    1. Sterile 50 ml conical centrifuge tubes (Olympus Plastics, catalog number: 21-106 )
    2. Sterile 10 cm tissue culture-treated plastic dishes (Corning, Falcon®, catalog number: 353003 )
    3. Sterile 0.22 µm PES filter (50 ml volume) Cole Palmer Steriflip-GP Filter, 0.22 µm PES Item # UX-29969-20 (EMD Millipore, catalog number: SCGP00525 )
    4. Sterile 1x HBSS (diluted from 10x stock) (Thermo Fisher Scientific, GibcoTM, catalog number: 14185052 )
    5. TrypLETM Express Dissociation Enzyme with Phenol Red (Thermo Fisher Scientific, GibcoTM, catalog number: 12605010 )
    6. Dulbecco’s modified Eagle’s medium (DMEM) 4.5 g glucose for proliferation medium (Thermo Fisher Scientific, GibcoTM, catalog number: 10564011 ) and DMEM 1 g glucose for differentiation medium (Thermo Fisher Scientific, GibcoTM, catalog number: 10567014 )
    7. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10437 )
    8. 100x penicillin-streptomycin-glutamine (PSG) (Thermo Fisher Scientific, GibcoTM, catalog number: 10378016 )
    9. Gelatin Type A from porcine skin (Sigma-Aldrich, catalog number: G1890 )
    10. Horse serum (Thermo Fisher Scientific, GibcoTM, catalog number: 16050122 )
    11. Complete growth medium (see Recipes)
    12. Differentiation medium (50 ml) (see Recipes)
    13. 0.1% gelatin (see Recipes)

  4. Immunofluorescence for in vitro fusion assay
    1. Aluminum foil
    2. 4-well chamber slides, Nunc Lab-Tek II Permanox (Thermo Fisher Scientific, catalog number: 177437 )
    3. 10x Dulbecco’s phosphate buffered saline (PBS) (diluted from 10x stock) (Thermo Fisher Scientific, GibcoTM, catalog number: 14200075 )
      Note: Diluted to 1x with double distilled water. Store at RT.
    4. Antibodies (stored at 4 °C)
      1. Anti-Myosin Heavy Chain (Developmental Studies Hybridoma Bank, MF-20) or anti human desmin (clone D33) (Abcam, catalog number: ab8470 )
      2. AffiniPure F(Ab’)2 Alexa Fluor 594 Donkey anti-Mouse IgG (H+L) (Jackson ImmunoResearch, catalog number: 715-586-150 ) (Protect from light)
    5. Vectashield HardSet mounting medium with DAPI (Vector Laboratories, catalog number: H-1500 )
    6. Triton X-100 (Sigma-Aldrich, catalog number: T8787-250ml )
    7. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10437 )
    8. 16% paraformaldehyde (PFA) (Electron Microscopy Sciences, catalog number: 15710-S ), diluted to 4%
    9. 4% paraformaldehyde (PFA) (see Recipes)
    10. Permeabilization solution (see Recipes)
    11. Blocking solution (see Recipes)

Equipment

  1. Dissociation of primary human skeletal muscle tissue
    1. Bench top centrifuge (Beckman Coulter, model: Allegra® 6R , catalog number: 366816)
    2. Bright-LineTM hemocytometer (0.1 mm) (Hausser Scientific, catalog number: 1492 )
    3. Sterile laminar flow biosafety cabinet (SterilGard® Class II Type A/B3) (The Baker Company, model: SG400 )
    4. -150 °C freezer, with liquid nitrogen storage backup tank (VIP® PLUS) (Panasonic, model: MDF-C2156VANC )
    5. Humidified 5% CO2 incubator set to 37 °C (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM Series II 3110 , catalog number: 3110)
    6. FACS: Becton Dickinson Aria II equipped with 4 lasers and a biosafety cabinet

  2. Purification of myoblasts from dissociated human skeletal muscle mononuclear cells
    1. Sterile laminar flow biosafety cabinet (SterilGard® Class II Type A/B3) (The Baker Company, model: SG400 )
    2. Water bath set to 37 °C (Sheldon Manufacturing, SHEL LAB®, model: SWB15 )
    3. Bench top centrifuge (Beckman Coulter, model: Allegra® 6R , catalog number: 366816)
    4. Inverted microscope (Nikon, model: TMS-F , catalog number: 210775)
    5. Bright-LineTM hemocytometer (0.1 mm) (Hausser Scientific, catalog number: 1492 )
    6. Cell sorter, such as Becton Dickinson Aria II equipped with 4 lasers and a biosafety cabinet

  3. In vitro culture and analysis of human skeletal myoblasts
    1. 1,000 µl pipette
    2. Rotating shaker
    3. Sterile laminar flow biosafety cabinet (SterilGard® Class II Type A/B3) (The Baker Company, model: SG400 )
    4. Water bath set to 37 °C (Sheldon Manufacturing, SHEL LAB®, model: SWB15 )
    5. Humidified 5% CO2 incubator set to 37 °C (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM Series II 3110 , catalog number: 3110)
    6. Bench top centrifuge (Beckman Coulter, model: Allegra® 6R , catalog number: 366816)
    7. Inverted microscope (Nikon, model: TMS-F , catalog number: 210775)
    8. Bright-LineTM hemocytometer (0.1 mm) (Hausser Scientific, catalog number: 1492 )
    9. Inverted microscope with epi-fluorescence capabilities including ultraviolet/DAPI and FITC/GFP filter sets (such as Nikon, model: Eclipse E1000 )

Software

  1. Cell sorter analysis software (FlowJo: https://www.flowjo.com/solutions/flowjo)

Procedure

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文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Spinazzola, J. M. and Gussoni, E. (2017). Isolation of Primary Human Skeletal Muscle Cells. Bio-protocol 7(21): e2591. DOI: 10.21769/BioProtoc.2591.

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分类

干细胞 > 成体干细胞 > 肌肉干细胞

细胞生物学 > 细胞分离和培养 > 细胞分离 > 流式细胞术

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