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Isolation of Primary Human Skeletal Muscle Cells
原代人骨骼肌细胞的分离   

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Cell Stem Cell
Dec 2016

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), Myoblast isolation (成肌细胞分离), Tissue dissociation (组织解离), Fluorescence-activated cell sorting (FACS) (荧光激活细胞分选(FACS))

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

  1. Dissociation of primary human skeletal muscle tissue
    Note: All steps in this protocol should be performed in a sterile laminar flow biosafety cabinet using sterile tissue culture technique. Human skeletal muscle can only be obtained following approval from the Institutional IRB. We obtain de-identified, discarded skeletal muscle tissue under a protocol approved by Boston Children’s Hospital IRB.
    1. Pre-weigh one 10 cm tissue culture plate with lid, and place the tissue sample to be dissociated in a second (non pre-weighed) 10 cm tissue culture plate.
    2. Using sterile scalpels, remove any connective tissue from the muscle biopsy. Tissue should be kept moist. Add a few drops of sterile 1x HBSS as necessary to prevent it from drying out. Place muscle tissue in the pre-weighed 10 cm tissue culture plate, replace lid, and weigh the plate again. Subtract from this number the tare of the empty plate to calculate the amount of muscle tissue to be dissociated.
    3. Thaw frozen aliquots of dispase II and collagenase D in a 37 °C water bath. Thawed collagenase D stock solution (see Recipes) and dispase II stock (see Recipes) will be added at a volume of 3.5 ml each per gram of muscle tissue to be dissociated. Thaw only the amounts of collagenase D and dispase II necessary for dissociation. If an excess of enzymes is thawed, it can be refrozen once and re-used.
    4. Using sterile scalpels, mince muscle tissue until it resembles a fine paste. During mincing, add a few drops of sterile 1x HBSS to prevent exposed tissue from drying out. Tissue should always appear moist, but with no excess of liquid.
    5. After tissue is finely minced, add equal amounts of the thawed dispase II and collagenase D solutions. The final concentration will be 5 mg/ml for collagenase D and 1.2 U/ml for dispase II in this solution. Pipette minced tissue and enzyme solution up and down through a sterile wide-bore 25 ml pipette a few times.
    6. Incubate plate in a humidified 5% CO2 incubator set to 37 °C for 15 min.
    7. Pipette the digestion solution up and down through a sterile 25 ml pipette a few times and incubate again for 15 min. Repeat this step additional 1-2 times, until the slurry easily passes through a sterile 5 ml pipette and all tissue chunks are dissolved. The total digestion time will range between 45 min and 1 h 15 min.
    8. Add 2 volumes of complete growth medium (see Recipes) (based on the total volume of dispase II and collagenase D) to the digested slurry and filter the digestion solution through a 100 µm cell strainer over a 50 ml conical tube. Change cell strainer if it appears clogged.
    9. Pellet cells for 10 min at 1,100 x g, RT.
    10. Resuspend the pellet in 1 volume of complete growth medium (i.e., 3 ml) and add 7 volumes (i.e., 21 ml) of red blood cell lysis solution. Invert the tube a few times and then filter the solution through a 40 µm cell strainer over a 50 ml conical tube.
    11. Count cells using a hemocytometer, then pellet the cells for 10 min at 1,100 x g, RT. Expect approximately 107 cells/gram tissue from postnatal skeletal muscle and 108 cells/gram tissue from fetal skeletal muscle. Cell numbers vary among individuals.
    12. Freeze cells at a concentration of 1 x 107 cells/ml in ice-cold freezing medium (see Recipes). Store cryovials at -80 °C overnight, then transfer them to -150 °C where they can be permanently stored until necessary. Cell freezing is not required if all reagents and FACS equipment are immediately available.

  2. Purification of myoblasts from dissociated human skeletal muscle mononuclear cells
    Note: All steps in this protocol should be performed in a sterile laminar flow biosafety cabinet using sterile tissue culture technique. Cell sorting should be performed in as clean an environment as possible, including sorting human cells under a BSL-2 biosafety cabinet, if possible.
    1. Thawing of cryopreserved sample prior to FACS
      Cryopreserved cells should be carefully thawed and plated 1 day prior to cell sorting. This allows the cells to recover from the freezing process before undergoing FACS.
      1. Pre-warm complete growth medium in a 37 °C water bath. Then, pipette 10 ml pre-warmed medium into a sterile 50 ml conical tube.
      2. Coat sterile tissue culture-treated plates (10 cm plate) with 10 ml 0.1% gelatin (see Recipes) for 1 h at 37 °C, then remove the gelatin solution by aspiration. Let plates dry briefly in the biosafety cabinets and replace lid.
      3. Carefully and quickly thaw a vial of cryopreserved, dissociated cells in a 37 °C water bath and transfer the cells into the 50 ml conical tube with 15 ml pre-warmed proliferation medium using a 1 ml pipette. Rinse the inside of the cryovial with fresh complete growth medium to remove as many cells as possible. This step should be performed very quickly as the DMSO used during the cryopreservation process is toxic to the cells at RT.
      4. Plate the cells in the pre-warmed medium onto sterile, tissue-culture treated plates at approximately 0.5-1 x 107 cells/10 cm plate or 1.5-3 x 107 cells/15 cm plate. If using a 15 cm plate, add 15 ml pre-warmed medium to bring the total medium volume to 25 ml.
        Note: No plate coating is required for this step.
      5. Incubate the cells in a humidified 5% CO2 incubator set to 37 °C overnight. If plating cells several days in advance, change the growth medium every other day and do not allow confluence to exceed 80%.
    2. Preparation of sample for FACS
      1. Pre-warm complete growth medium and 1x DPBS in a 37 °C water bath. Place the 5% FBS/HBSS (see Recipes) on ice.
      2. Check the cells under a phase contrast microscope with 10x magnification. Ensure that there is no contamination and that the cells look healthy.
        Note: There will be many floating, live cells in your culture, which is normal for dissociated human skeletal muscle. It is also likely that there will be small clumps of cells in the culture, and the number of clumps will vary. These clumps will be filtered out prior to cell sorting. Additionally, the dissociation process results in a large amount of debris in addition to cells. This will make the culture appear ‘dirty’ (i.e., little black specks, etc.), but again, this is normal and should not be considered contamination. This debris will be removed during the FACS sample preparation process.
      3. Wash the cells with 5 ml (10 cm plate) or 10 ml (15 cm plate) of 1x DPBS at least twice or ideally three times.
      4. Pipette 2 ml (10 cm plate) or 5 ml (15 cm plate) of TrypLE ExpressTM dissociation enzyme onto the plate and incubate in a humidified 5% CO2 incubator set to 37 °C for 2 min.
      5. Check under the microscope if cells have lifted and are now floating freely in the medium. If cells still adhere to the plate, gently tap the bottom of the plate to loosen the cells and return the plate to the incubator for another minute.
      6. After incubation, remove the cells by gently swirling the medium, pipetting the cells a few times and pooling the cells to one side of the plate by tilting it at an angle (~45°), then carefully pipette the medium into the 50 ml conical tube.
        Note: When the plate is tilted at an angle, the cells can be seen on the surface of the plate as a light opaque coating. Repeatedly rinse gently the cells off the plate using the trypsin solution (TrypLE ExpressTM Dissociation Enzyme with Phenol Red) until this coating is no longer visible.
      7. Repeat step B2f with growth medium (5 ml for 10 cm plate and 10 ml for 15 cm plate) to collect any remaining cells and quench the TrypLETM.
        Note: Prolonged exposure of the cells to undiluted TrypLE ExpressTM may negatively affect the health of the cells. Addition of growth medium to the trypsinized cells will quench this effect.
      8. Check the plate under a phase contrast microscope at 10x magnification for the presence of cells. There should be very few cells on the surface of the plate after this process.
      9. Centrifuge the 50 ml conical tubes containing the cells and washes at 1,100 x g at 4 °C for 10 min to pellet the cells.
      10. Remove the supernatant, and resuspend the cells in 10 ml 5% FBS/HBSS.
      11. Determine the cell concentration using a hemocytometer or other cell counting device.
      12. For FACS controls, use 5 ml round-bottom test tubes and set aside 2.5 x 105 cells in 500 µl 5% FBS/HBSS for each of the following controls:
        1. ‘Unstained’ control
        2. Calcein blue single color control
        3. CD56 single color control
        4. CD82 single color control
      13. Pipette the ‘unstained’ control sample through the strainer cap of a 5 ml round-bottom test tube. Keep on ice.
        Note: Some FACS machines may require tubes that are different in diameter/size from the tube specified in this protocol. Check in advance that the tubes fit in the FACS machine.
      14. Centrifuge the remaining cells (to be labeled with both CD56 and CD82 antibodies or single color controls) for 10 min at 1,100 x g at 4 °C.
      15. Resuspend cells at a concentration of 1 x 107/ml in 5% FBS/HBSS.
      16. Primary antibody incubation: add CD56 and CD82 antibodies to the appropriate cell solutions at a concentration of 5 µl per 1 x 106 cells.
      17. To gate for live cells, add calcein blue at a concentration of 0.5 µl per 1 x 106 cells to the appropriate cell solutions. Gently mix and place on ice protected from the light for 30 min.
        Note: Calcein blue is a cell viability dye and is used in this protocol to discriminate live from dead cells during the FACS.
      18. After this incubation, wash the cells 1 x in 2ml of 5% FBS/HBSS.
      19. Centrifuge the cells for 10 min at 1,100 x g at 4 °C.
      20. Resuspend the CD56 and CD82 single color controls in 500 µl of 5% FBS/HBSS, and pipette through the strainer cap of a 5 ml round-bottom test tube. Store on ice in the dark.
      21. Resuspend the CD56/CD82/calcein blue stained cells in 1 ml of 5% FBS/HBSS, and pipette through the strainer cap of a 5 ml round-bottom test tube. Store on ice in the dark.
      22. Prepare collection tube for CD56+CD82+ sorted cells by pipetting 500 µl of growth medium into a new tube. Store on ice.

  3. Fluorescence-activated cell sorting
    It is beyond the scope of this chapter to review FACS or flow cytometry in detail. Gating specifications are briefly indicated (Figure 1).
    1. Determine optimal excitation voltages and compensation values using the ‘no stain’ and single color controls.
    2. Determine the live cell population gating for calcein blue positive cells.
    3. Determine the double positive (DP) CD56+/CD82+ and double negative (DN) populations. Gate and sort for the DP cell population.


      Figure 1. Example of FACS gating technique for human myogenic cells immunostained with anti-CD56 and anti-CD82 antibodies using calcein AM blue as vital dye marker. A. Unstained control sample, gate on single cells; B. Calcein blue signal (calcein AM blue) to gate on live cells; C. Gated live cells stained for CD82-PE and CD56-APC. DN = double negative cells; DP = double positive cells.

  4. In vitro culture and analysis of human fetal skeletal myoblasts
    1. In vitro cell culture
      Note: All steps in this protocol should be performed in a sterile laminar flow biosafety cabinet using sterile tissue culture technique.
      1. Coat sterile tissue culture-treated plates (10 cm plate) with 10 ml 0.1% gelatin (see Recipes) for 1 h in a humidified 5% CO2 incubator set to 37 °C, then remove the gelatin solution by aspiration. Let plates dry briefly in the biosafety cabinets and replace lid.
      2. Pre-warm complete growth medium in a water bath set to 37 °C.
      3. Resuspend sorted CD56/CD82 double positive cells at 0.5-1 x 106 cells/10 ml complete growth medium and plate on coated plates. Gently rock plate(s) to evenly distribute cells, and then incubate in a CO2 incubator. Sorted cells will be small and have a bright, rounded appearance and should attach within 1 day post-sorting (Figure 2).
      4. Propagate the cells to 65-75% confluency. This should take approximately 2-3 days; however, if necessary, replace the medium with fresh growth medium every 2 days until the plate is at 65-75% confluency.


        Figure 2. In vitro culture of FACS sorted CD56 and CD82 double negative (DN) and double positive (DP) human cells. (A and B) On day 1 after sorting, both DN and DP cells appear bright and rounded, while some cells have flattened and firmly adhered to the plate. (C and D) On day 7 after sorting (4 days in growth medium and 3 days in differentiation medium [see Recipes]), DN cells remain mononuclear, whereas DP cells have fused and formed multinucleated myotubes (arrows). (E and F) Examples of immunostaining for myosin heavy chain (MHC) and desmin on double-positive cells are shown.

    2. To passage cells
      1. Coat sterile tissue culture-treated plates with 0.1% gelatin as above (as in step D1a).
      2. Pre-warm the following in a water bath set to 37 °C: 1x DPBS, TrypLETM Express dissociation enzyme, and complete growth medium.
      3. Remove medium from plate by aspiration and wash the cells twice with 10 ml (10 cm plate) 1x DPBS. Remove DPBS by aspiration.
      4. Pipette 2 ml TrypLETM Express onto the plate and incubate in a humidified 5% CO2 incubator set to 37 °C for 2-3 min. Gently remove the cells from the plate by pipetting up and down a few times before transferring cells into a sterile conical tube. Wash any remaining cells from the surface of the plate with additional complete growth medium.
      5. Centrifuge the cells at 1,100 x g at RT for 10 min.
      6. Resuspend the cells in 10 ml fresh complete growth medium.
      7. Determine the cell concentration using a hemocytometer and plate the cells at 0.5-1 x 106 cells in 10 ml complete growth medium/10 cm plate.
      8. Cells should be passaged every 2-3 days and should not be grown past 70% confluency.
        Note: Cells should never reach 100% confluency when proliferating, as they will begin to differentiate and fuse on contact. The high serum growth medium will lower in serum concentration over time and will not be able to prevent fusion.
    3. To freeze cells
      1. Trypsinize and centrifuge cells as in steps D2c-D2e.
      2. Resuspend cells in ice-cold freezing medium (10% DMSO in complete growth medium) at desired cell concentration (106-107/ml).
      3. Store cryovials at -80 °C overnight then transfer to -150 °C where they can be permanently stored until necessary.
    4. To perform an in vitro fusion assay
      1. Coat 4-well chamber slides with 0.1% gelatin.
      2. Trypsinize the cells (with TrypLE ExpressTM Dissociation Enzyme with Phenol Red) and determine the cell concentration as described above, then plate 20,000 cells in 500 µl complete growth medium/well.
      3. Incubate the cells in a humidified 5% CO2 incubator set to 37 °C until cells are ~80% confluent.
      4. When cells are ~80% confluent, remove the growth medium from each well and replace with 500 µl pre-warmed differentiation medium (see Recipes). Incubate the cells in a humidified 5% CO2 incubator set to 37 °C overnight.
        Note: Low serum medium induces differentiation and fusion of myoblasts in culture (Yaffe and Saxel, 1977).
      5. Replace the differentiation medium in each well daily during the course of the fusion assay.
      6. Monitor the differentiation of the cells using a phase contrast microscope at 10x or 20x magnification. Fusion should occur within 1 week of exposure to differentiation medium (Figure 2). When mature myotubes of > 10 nuclei/myotube are present, perform the following immunocytochemistry protocol to visualize the cells by fluorescence microscopy.
    5. Immunofluorescence for in vitro fusion assay
      Note: This immunocytochemical protocol may also be utilized for the detection of other myogenic markers of proliferating or differentiating cells.
      1. Thaw 4% PFA (see Recipes) at RT.
      2. Carefully wash the cells with 200 µl 1x PBS/well. Remove PBS using a 1,000 µl pipette.
      3. Fix the cells with 200 µl 4% PFA for 20 min at RT; remove fixation liquid using a pipette, then permeabilize the cells with 200 µl permeabilization solution (see Recipes) for 3 min at RT.
      4. Remove permeabilization solution using a pipette, and then block the cells for 30 min at RT with 200 µl blocking solution (see Recipes).
      5. Prepare the primary antibody solution by diluting the anti-human desmin antibody 1:100 in fresh blocking solution. Incubate the cells with primary antibody solution overnight at 4 °C.
      6. Wash the cells 3 times with 1x PBS for 5 min at RT. The plate may be gently agitated on a rotating shaker.
      7. Prepare the secondary antibody solution by diluting the anti-mouse antibody 1:1,000 in blocking solution. Incubate the cells in the dark with secondary antibody solution for 1 h at RT.
      8. Wash the cells 3 times with 1x PBS for 5 min at RT in the dark. The plate may be gently agitated on a rotating shaker.
      9. Mount the cells with Vectashield with DAPI and store at 4 °C protected from light with aluminum foil.
      10. Visualize the cells by fluorescent microscopy using ultraviolet/DAPI and FITC/GFP filter sets for DAPI and desmin, respectively.

Data analysis

In vitro fusion assay–fusion index calculation

  1. After immunofluorescent staining of myotubes, count the following in each of five random fields per well (x 4 wells/sample):
    1. # of total nuclei
    2. # of nuclei within myotubes
  2. Calculate fusion index (%) as ((# nuclei within myotubes/# total nuclei) x 100).
  3. Average the fusion index of the five fields.
  4. Compare the fusion index of sorted myoblasts versus unsorted.

Notes

  1. We would like to note that FACS with either CD56 or MCAM in conjunction with CD82 as enriching markers is a highly effective method for isolating human fetal myogenic progenitors. We refer the readers to the following protocol describing use of MCAM as a positive selection marker in cells sorted from human fetal tissue (Lapan and Gussoni, 2012). It is not appropriate to use CD82 and MCAM for adult human skeletal muscle, since endothelial cells express MCAM in adult muscle.
  2. Institutional review and protocol approval are required prior to collection and processing of human tissue. All personnel handling human tissue must receive appropriate safety and human subject education training.

Recipes

  1. Growth medium (500 ml)
    1. 395 ml of high glucose (4.5 g) Dulbecco’s modified Eagle’s medium (DMEM)
    2. 100 ml of fetal bovine serum (FBS)
    3. 5 ml of 100x penicillin-streptomycin-glutamine (PSG)
    4. Sterilize by filtering the solution through a 500 ml 0.22 µm PES filter unit
    5. Store at 4 °C and use within 1 month
    Note: FBS varies considerably between companies and between lots. Therefore, we recommend testing several different FBS samples with the in vitro fusion assay described here.
  2. Dispase II stock solution
    1. Dissolve 1 g powder dispase II in 100 ml HEPES buffered saline solution
    2. Add 316 ml of high glucose (4.5 g) DMEM to generate a stock solution of 2.4 U/ml
    3. Filter-sterilize the solution through a PES 500 ml filter; aliquot into 15 ml conical tubes (10 ml/tube)
    4. Store aliquots at -20 °C
  3. 1 M calcium chloride solution (CaCl2·2H2O, FW 147)
    1. Dissolve 1.47 g powder in 10 ml of double distilled water
    2. Store at 4 °C
  4. Collagenase D stock solution
    1. Dissolve 2.5 g powder collagenase D in 250 ml solution of 1x HBSS supplemented with 1.25 ml of 1 M calcium chloride solution
    2. Sterilize by filtering through a 250 ml PES filter unit
    3. Aliquot in 15 ml conical tubes (10 ml/tube) and stored at -20 °C
  5. Sterile freezing medium
    1. Combine 90% FBS and 10% dimethyl sulfoxide (DMSO)
    2. Prepare freezing medium and immediately store on ice
    3. Unused sterile freezing medium can be stored at 4 °C for up to 4 weeks
  6. Differentiation medium (50 ml)
    1. 48.5 ml of low glucose (1 g) Dulbecco’s modified Eagle’s medium (DMEM)
    2. 1 ml of horse serum (HS)
    3. 0.5 ml of 100x penicillin-streptomycin-glutamine (PSG)
    4. Sterilize by filtering the solution through a 150 ml 0.22 µm PES filter unit
    5. Store at 4 °C and use within 1 month
  7. Sterile 5% FBS/HBSS solution (50 ml)
    1. Add 2.5 ml of FBS to 1x HBSS
    2. Sterilize by filtering the solution through a 50 ml 0.22 µm PES filter unit
    3. Store at 4 °C
  8. 0.1% gelatin
    1. Add 0.5 g gelatin to 500 ml of double distilled water. DO NOT SHAKE
    2. Sterilize the solution by autoclaving for 20 min and store at 4 °C
  9. 4% paraformaldehyde (4% PFA)
    1. Dilute 16% paraformaldehyde with 1x PBS
    2. USE CAUTION as paraformaldehyde is extremely toxic; it is recommended that paraformaldehyde be used in a fume hood for safety. Aliquot and store at -20 °C
    3. Aliquots should not be repeatedly frozen and thawed; discard unused PFA after initial use
  10. Permeabilization solution
    Mix 50 µl of Triton X-100 with 10 ml of 1x PBS
  11. Blocking solution
    Mix together 1 ml of fetal bovine serum (FBS), 10 µl of Triton X-100, and 9 ml of 1x PBS

Acknowledgments

This work is supported by a grant from the Muscular Dystrophy Association #479606 (EG) and by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number 1R01AR069582-01 (EG). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
This protocol was modified from previous work, specifically from the listed references 1 and 7.

References

  1. Alexander, M. S., Rozkalne, A., Colletta, A., Spinazzola, J. M., Johnson, S., Rahimov, F., Meng, H., Lawlor, M. W., Estrella, E., Kunkel, L. M. and Gussoni, E. (2016). CD82 is a marker for prospective isolation of human muscle satellite cells and is linked to muscular dystrophies. Cell Stem Cell 19(6): 800-807.
  2. Bishop, A., Gallup, B., Skeate, Y. and Dubowitz, V. (1971). Morphological studies on normal and diseased human muscle in culture. J Neurol Sci 13(3): 333-350.
  3. Blau, H. M. and Webster, C. (1981). Isolation and characterization of human muscle cells. Proc Natl Acad Sci U S A 78(9): 5623-5627.
  4. Geiger, R. S. and Garvin, J. S. (1957). Pattern of regeneration of muscle from progressive muscular dystrophy patients cultivated in vitro as compared to normal human skeletal muscle. J Neuropathol Exp Neurol 16(4): 523-543.
  5. Goyle, S., Kalra, S. L. and Singh, B. (1967). The growth of normal & dystrophic human skeletal muscle in tissue culture. Neurol India 15(4): 149-151.
  6. Herrmann, H., Konigsberg, U. R. and Robinson, G. (1960). Observations on culture in vitro of normal and dystrophic muscle tissue. Proc Soc Exp Biol Med 105: 217-221.
  7. Lapan, A. D. and Gussoni, E. (2012). Isolation and characterization of human fetal myoblasts. Methods Mol Biol 798: 3-19.
  8. Yaffe, D. and Saxel, O. (1977). A myogenic cell line with altered serum requirements for differentiation. Differentiation 7(3): 159-166.

简介

原代成肌细胞培养是研究肌肉疾病,病理生理学和药理学的有用工具。 该协议描述了通过荧光激活细胞分选(FACS)从人类骨骼肌活检中分离细胞并富集高度肌原细胞的技术。 我们还描述了用于评估随后的体外研究中肌原性和群体扩张的方法。
【背景】来自肌肉活组织检查的原代人成肌细胞是模拟体外人体肌肉疾病的有价值的资源。成肌细胞增殖,分化和融合的改变是许多神经肌肉疾病所共有的特征,并且可以用于测定基于细胞的和药理学的治疗。人类骨骼肌活组织检查,尤其是那些受疾病影响的人,通常含有大量的非肌原细胞,如脂肪细胞和成纤维细胞。因此,纯化肌原细胞进行骨骼肌发育和疾病的体外研究是非常重要的。肌肉疾病的早期研究涉及使用组织外植体或未纯化的分离细胞(Geiger和Garvin,1957; Herrmann等人,1960; Goyle等人,1967; Bishop 1971年),后来,Blau和Webster引入了一种预先电镀技术去除成纤维细胞(Blau and Webster,1981)。在这里,我们描述了一个有效的技术解离人类肌肉活检单核细胞,并利用FACS与细胞表面标记CD56和CD82(见注1)纯化高度肌原体人口。我们最近证明,CD82是人类胎儿和成人骨骼肌中优良的生肌标志物,其也保留在活化的和分化的肌原性祖细胞上(Alexander等人,2016)。该方案还描述了培养这些成肌细胞的方法,并通过体外融合测定确认肌原细胞群。这些细胞从正常个体和肌肉障碍个体中分离和扩增将有助于加速人类疾病如肌营养不良症的治疗方法的开发。

关键字:骨骼肌, 成肌细胞分离, 组织解离, 荧光激活细胞分选(FACS)

材料和试剂

  1. 原代人骨骼肌组织的解离(参见注释2)
    1. 受保护的无菌一次性手术刀,带有#10号不锈钢刀片(Aspen Surgical,Bard-Parker TM,目录号:372610)
    2. 无菌的10厘米组织培养处理的塑料盘(Corning,Falcon ,产品目录号:353003)
    3. 各种无菌5,10和25毫升移液器(奥林巴斯塑料,产品目录号:12-102,12-104,12-106)
    4. 无菌0.22微米PES(低蛋白质结合)过滤器(250和500毫升体积)(如奥林巴斯塑料,目录号:25-227)
    5. 无菌15和50毫升锥形离心管(奥林巴斯塑料,目录号:21-101和21-106)
    6. BD Falcon无菌尼龙细胞过滤器(100μm和40μm孔径)(Corning,目录号:352360和352340)
    7. 无菌的1.8ml CryoTube TM小瓶(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:377267)
    8. 10x Hank平衡盐溶液(HBSS)(Thermo Fisher Scientific,Gibco TM,目录号:14185052)或10x Dulbecco's磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM TM目录号:14200075),不含氯化钙,氯化镁和硫酸镁,用双蒸水稀释至1倍,用0.22微米PES过滤器过滤灭菌。 注意:此解决方案可以存储在4°C或室温(RT)下。
    9. 无菌红细胞裂解液(QIAGEN,目录号:158904)(保存在RT)
    10. 无酚红HEPES缓冲盐水溶液(Lonza,目录号:12-747F)
    11. Dulbecco改良的Eagle培养基(DMEM 4.5g葡萄糖)(Thermo Fisher Scientific,Gibco TM,产品目录号:10564011)
    12. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10437)
    13. 100x青霉素 - 链霉素 - 谷氨酰胺(PSG)(Thermo Fisher Scientific,Gibco TM,产品目录号:10378016)
    14. 氯化钙二水合物(Sigma-Aldrich,目录号:C7902-500G)
    15. Dispase II(Roche Diagnostics,目录号:04942078001)
    16. 胶原酶D(Roche Diagnostics,目录号:11088882001 2.5g)
    17. 完整的生长培养基(500毫升)(见食谱)
    18. 1M氯化钙溶液(CaCl 2•2H 2 O,FW 147)(见食谱)
    19. Dispase储备溶液(见食谱)
    20. 胶原酶D原液(见食谱)
    21. 无菌冷冻介质(见食谱)

  2. 从解离的人骨骼肌单核细胞纯化成肌细胞
    1. 冷冻保存的样本在FACS之前解冻
      1. 无菌0.22μmPES过滤器(500 ml容量)(Thermo Fisher Scientific,目录号:569-0020)
      2. 无菌50毫升锥形离心管(奥林巴斯塑料,目录号:21-106)
      3. 无菌组织培养处理的塑料盘子(10或15厘米大小)(Corning,Falcon,产品目录号:353003和353025)
      4. 含有解离的未纯化的原代细胞的CryoTube TM小瓶(Simport,目录号:T309-2A)
      5. Dulbecco改良的Eagle培养基(DMEM)(DMEM 4.5g葡萄糖)(Thermo Fisher Scientific,Gibco TM,产品目录号:10564011)
      6. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10437)
      7. 100x青霉素 - 链霉素 - 谷氨酰胺(PSG)(Thermo Fisher Scientific,Gibco TM,产品目录号:10378016)
      8. 完整的生长介质(见食谱)
    2. 制备FACS样品
      1. 无菌0.22μmPES过滤器(50 ml容量)Cole Palmer Steriflip-GP过滤器,0.22μmPES产品编号UX-29969-20(EMD Millipore,产品目录号:SCGP00525)
      2. 无菌15和50毫升锥形离心管(奥林巴斯塑料,目录号:21-101和21-106)
      3. 无菌5毫升带细胞滤网帽的圆底试管(Corning,Falcon ,产品目录号:352054)
      4. 分离未纯化的原代细胞,在FACS前一天解冻
      5. 无菌1×HBSS(从10×储备液中稀释)(Thermo Fisher Scientific,Gibco TM,产品目录号:14185052)
      6. 无菌1×DPBS(从10×储备液稀释)(Thermo Fisher Scientific,Gibco TM,目录号:14200075)
      7. TrypLE Express TM解离酶与酚红(Thermo Fisher Scientific,Gibco TM,产品目录号:12605010)
      8. 抗体
        1. APC抗CD56抗体,克隆HCD56(BioLegend,目录号:318310)
        2. PE抗CD82抗体,克隆ASL-24(BioLegend,目录号:342103)
      9. 钙黄绿蓝(1毫克小瓶)(Thermo Fisher Scientific,目录号:C1430)
        注:重悬于200μl二甲基亚砜(DMSO,AmericanBio,目录号:AB03091-00050)中,取25μl等分样品,-20°C保存。使用0.5微升的钙黄绿素/ 10 6 细胞。
      10. 无菌5%FBS溶液(50毫升)(见食谱)
    3. 荧光激活细胞分选(FACS)
      FACS或带有细胞过滤器盖的5ml圆底管(Corning,Falcon,产品目录号:352235)

  3. 人类骨骼肌成肌细胞的体外培养和分析
    1. 无菌50毫升锥形离心管(奥林巴斯塑料,目录号:21-106)
    2. 无菌的10厘米组织培养处理的塑料盘(Corning,Falcon ,产品目录号:353003)
    3. 无菌0.22μmPES过滤器(50 ml容量)Cole Palmer Steriflip-GP过滤器,0.22μmPES产品编号UX-29969-20(EMD Millipore,产品目录号:SCGP00525)
    4. 无菌1×HBSS(从10×储备液中稀释)(Thermo Fisher Scientific,Gibco TM,产品目录号:14185052)
    5. 使用苯酚红TrypLE TM Express Express解离酶(Thermo Fisher Scientific,Gibco TM,产品目录号:12605010)
    6. 将Dulbecco改良的Eagle's培养基(DMEM)4.5g葡萄糖用于增殖培养基(Thermo Fisher Scientific,Gibco TM,目录号:10564011)和DMEM 1g葡萄糖用于分化培养基(Thermo Fisher Scientific,Gibco) TM ,目录号:10567014)
    7. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10437)
    8. 100x青霉素 - 链霉素 - 谷氨酰胺(PSG)(Thermo Fisher Scientific,Gibco TM,产品目录号:10378016)
    9. 来自猪皮的明胶A型(Sigma-Aldrich,目录号:G1890)
    10. 马血清(Thermo Fisher Scientific,Gibco TM,产品目录号:16050122)
    11. 完整的生长介质(见食谱)
    12. 分化培养基(50毫升)(见食谱)
    13. 0.1%明胶(见食谱)

  4. 免疫荧光用于体外融合测定
    1. 铝箔
    2. 4孔室载玻片,Nunc Lab-Tek II Permanox(Thermo Fisher Scientific,目录号:177437)
    3. 10×Dulbecco磷酸盐缓冲盐水(PBS)(从10×储备液中稀释)(Thermo Fisher Scientific,Gibco TM,目录号:14200075)
      注意:用双蒸水稀释至1倍。存放在RT。
    4. 抗体(储存在4℃)
      1. 抗肌球蛋白重链(Developmental Studies Hybridoma Bank,MF-20)或抗人结蛋白(克隆D33)(Abcam,目录号:ab8470)
      2. AffiniPure F(Ab')2 Alexa Fluor 594驴抗小鼠IgG(H + L)(Jackson ImmunoResearch,目录号:715-586-150)(避光)
    5. 带有DAPI(Vector Laboratories,目录号:H-1500)的Vectashield HardSet固定介质
    6. Triton X-100(Sigma-Aldrich,目录号:T8787-250ml)
    7. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10437)
    8. 16%多聚甲醛(PFA)(Electron Microscopy Sciences,目录号:15710-S),稀释至4%
    9. 4%多聚甲醛(PFA)(见食谱)
    10. 透化溶液(见食谱)
    11. 阻止解决方案(见食谱)

设备

  1. 原代人骨骼肌组织的解离
    1. 台式离心机(Beckman Coulter,型号:Allegra 6R,目录号:366816)
    2. Bright-Line TM血细胞计数器(0.1mm)(Hausser Scientific,目录号:1492)
    3. 无菌层流生物安全柜(SterilGard II类A / B3)(Baker公司,型号:SG400)
    4. -150°C冰箱,带有液氮储存备用水箱(VIP
    5. 加湿的5%CO 2培养箱设定为37℃(Thermo Fisher Scientific,Thermo Scientific TM,型号:Forma TM Series II 3110,目录号码:3110)
    6. 流式细胞仪:Becton Dickinson Aria II配有4个激光器和一个生物安全柜

  2. 从解离的人骨骼肌单核细胞纯化成肌细胞
    1. 无菌层流生物安全柜(SterilGard II类A / B3)(Baker公司,型号:SG400)
    2. 设置为37°C的水浴(Sheldon Manufacturing,SHEL LAB ®,型号:SWB15)
    3. 台式离心机(Beckman Coulter,型号:Allegra 6R,目录号:366816)
    4. 倒置显微镜(尼康,型号:TMS-F,目录号:210775)
    5. Bright-Line TM血细胞计数器(0.1mm)(Hausser Scientific,目录号:1492)
    6. 细胞分选仪,如Becton Dickinson Aria II配备4个激光器和一个生物安全柜

  3. 人类骨骼肌成肌细胞的体外培养和分析
    1. 1,000μl移液器
    2. 旋转摇床
    3. 无菌层流生物安全柜(SterilGard II类A / B3)(Baker公司,型号:SG400)
    4. 设置为37°C的水浴(Sheldon Manufacturing,SHEL LAB ®,型号:SWB15)
    5. 加湿的5%CO 2培养箱设定为37℃(Thermo Fisher Scientific,Thermo Scientific TM,型号:Forma TM Series II 3110,目录号码:3110)
    6. 台式离心机(Beckman Coulter,型号:Allegra 6R,目录号:366816)
    7. 倒置显微镜(尼康,型号:TMS-F,目录号:210775)
    8. Bright-Line TM血细胞计数器(0.1mm)(Hausser Scientific,目录号:1492)
    9. 具有落射荧光功能的倒置显微镜,包括紫外/ DAPI和FITC / GFP滤光片组(如尼康,型号:Eclipse E1000)

软件

  1. 细胞分选分析软件(FlowJo: https://www.flowjo.com/solutions/flowjo

程序

  1. 原始人类骨骼肌肉组织的解离
    注意:本议定书中的所有步骤应使用无菌组织培养技术在无菌层流生物安全柜中进行。人体骨骼肌肉只有经机构IRB批准才能获得。根据波士顿儿童医院IRB批准的方案,我们获得去除身份的废弃骨骼肌肉组织。
    1. 预先称量一个10厘米组织培养板与盖子,并将组织样本分离在第二个(未预先称重)10厘米组织培养板。
    2. 使用无菌手术刀,从肌肉活检组织中删除任何结缔组织。组织应保持湿润。根据需要添加几滴无菌1x HBSS以防止干燥。将肌肉组织放入预先称重的10厘米组织培养板中,更换盖子,并重新称重板。
      。从这个数字减去空盘子的皮重,以计算肌肉组织的数量
    3. 在37℃水浴中解冻分散酶II和胶原酶D的冷冻等分试样。将解冻的每克肌肉组织中加入每3.5ml体积的解冻胶原酶D原液(参见食谱)和分散酶II原液(参见食谱)。只解冻解离所需的胶原酶D和分散酶II的量。如果过量的酶被解冻,它可以重新冻结一次,并重新使用。
    4. 使用无菌手术刀,剁碎肌肉组织,直到它类似于良好的糊状物。在切碎过程中,加入几滴无菌1x HBSS以防止暴露的组织变干。
      组织应该总是显得潮湿,但是没有多余的液体
    5. 将组织细碎后,加入等量的融化的分散酶II和胶原酶D溶液。在该溶液中胶原酶D的最终浓度将是5mg / ml,而对于分散酶II是1.2U / ml。
      用无菌的大口径25毫升移液管吸取切碎的组织和酶溶液上下
    6. 将培养板置于加湿的5%CO 2培养箱中37℃15分钟。
    7. 上下消化溶液通过无菌25毫升吸管几次,再次孵育15分钟。重复此步骤1-2次,直到浆液容易地通过无菌的5ml移液管并且所有组织块被溶解。
      总消化时间在45分钟到1小时15分钟之间
    8. 将2体积的完全生长培养基(参见配方)(基于分散酶II和胶原酶D的总体积)加入消化的浆液中,并通过100μm细胞过滤器在50ml锥形管上过滤消化溶液。如果出现堵塞,请更换滤网。
    9. 在1100×g,RT下沉淀细胞10min。
    10. 在1体积的完全生长培养基(即,3ml)中重悬沉淀并加入7体积(即,21ml)红细胞裂解溶液。翻转管几次,然后通过一个40毫米的细胞过滤器,通过一个50毫升的锥形管过滤溶液。
    11. 用血细胞计数器对细胞进行计数,然后将细胞在1,100×g,RT下沉淀10分钟。期望来自出生后骨骼肌的约10 7个细胞/克组织和来自胎儿骨骼肌的10 8个细胞/克组织。细胞数量因人而异。
    12. 在冰冷的冷冻介质中以1×10 7细胞/ ml的浓度冷冻细胞(参见食谱)。将冷冻保存在-80°C过夜,然后将它们转移到-150°C,在那里它们可以永久保存直到需要。
      如果所有试剂和FACS设备都立即可用,则不需要细胞冷冻
  2. 从分离的人骨骼肌单核细胞中纯化成肌细胞
    注意:本议定书中的所有步骤应使用无菌组织培养技术在无菌层流生物安全柜中进行。如果可能的话,细胞分选应尽可能在干净的环境中进行,包括在BSL-2生物安全柜内分选人类细胞。
    1. 在FACS之前解冻冷冻保存的样品 冷冻保存的细胞应仔细解冻,并在细胞分选前1天进行铺板。这允许细胞在进行FACS之前从冷冻过程中恢复。
      1. 在37℃水浴中预热完全生长培养基。然后,吸取10毫升预热介质到无菌50毫升锥形管。
      2. 用10ml 0.1%明胶(参见食谱)在37℃涂覆无菌组织培养处理的平板(10cm平板)1小时,然后通过抽吸除去明胶溶液。让盘子在生物安全柜中短暂干燥并更换盖子。
      3. 在37℃水浴中小心快速解冻一瓶冷冻保存,解离的细胞,并使用1ml移液管将细胞转移到具有15ml预热增殖培养基的50ml锥形管中。用新鲜的完全生长培养基冲洗冷冻管内部以尽可能多地除去细胞。这个步骤应该很快进行,因为冷冻保存过程中使用的DMSO在室温下对细胞有毒性。
      4. 将预热的培养基中的细胞在约0.5-1×10 7个细胞/ 10cm平板或1.5-3×10 7个/平方厘米的板上铺在无菌的组织培养处理的平板上>细胞/ 15厘米板。如果使用15厘米板,加入15毫升预热介质,使总体积达到25毫升。
        注意:此步骤不需要镀膜。
      5. 将细胞置于设定为37℃的潮湿的5%CO 2培养箱中孵育过夜。如果提前几天接种细胞,每隔一天更换生长培养基,不允许合并超过80%。
    2. 制备FACS样品
      1. 在37℃水浴中预热完全生长培养基和1x DPBS。将5%FBS / HBSS(见食谱)放在冰上。
      2. 用10倍放大倍数的相差显微镜检查细胞。确保没有污染,细胞看起来健康。
        注意:你的文化中会有许多漂浮的活细胞,这是正常的解离人骨骼肌。培养物中也可能有细小的细胞团,团块的数量也会有所不同。在细胞分选之前,这些团块将被过滤掉。另外,解离过程除细胞外还产生大量的碎片。这将使文化显得“脏”(即黑色的小斑点等),但这又是正常的,不应该被视为污染。在FACS样品制备过程中,这些碎片将被移除。
      3. 用5ml(10cm平板)或10ml(15cm平板)的1xDPBS洗涤细胞至少两次或理想三次。
      4. 移取2ml(10cm平板)或5ml(15cm平板)的TrypLE Express TM解离酶到平板上并在潮湿的5%CO 2培养箱组到37℃2分钟。
      5. 在显微镜下检查细胞是否已经升高,现在在培养基中自由漂浮。如果细胞仍粘附在平板上,轻轻敲打平板的底部松开细胞,并将平板放回培养箱一分钟。
      6. 孵育后,轻轻旋转培养基,吸取细胞数次,通过倾斜角度(〜45°)将细胞集中到培养皿的一侧,然后小心地将培养基吸入50 ml圆锥管。
        注意:当板倾斜一定角度时,可以在板的表面上看到细胞,作为不透光的涂层。使用胰蛋白酶溶液(TrypLE Express TM与Phenol Red的解离酶)轻轻反复冲洗平板上的细胞,直到该涂层不再可见。
      7. 用生长培养基重复步骤B2f(10厘米板5毫升,15厘米板10毫升)收集剩余的细胞并猝灭TrypLE TM TM。
        注意:将细胞长时间暴露于未稀释的TrypLE Express TM可能会对细胞的健康产生负面影响。向胰蛋白酶消化的细胞中加入生长培养基可以抑制这种效应。
      8. 在相差显微镜下放大10倍,观察细胞的存在。
        在这个过程之后,板的表面上应该有很少的细胞
      9. 离心含有细胞的50ml锥形管,并在4℃下以1,100gxg洗涤10分钟以沉淀细胞。
      10. 去除上清液,并重悬细胞在10毫升5%FBS / HBSS。

      11. 使用血细胞计数器或其他细胞计数设备确定细胞浓度
      12. 对于FACS对照,使用5ml圆底试管,并且将下列对照中的每一个放置在500μl5%FBS / HBSS中的2.5×10 5个细胞:
        1. “未染”控制
        2. 钙黄绿色单色控制
        3. CD56单色控制
        4. CD82单色控制
      13. 吸取未染色的对照样品通过5ml圆底试管的过滤器盖。继续冰。
        注:某些FACS机器可能需要与本协议规定的管径不同的管。提前检查管子是否适合FACS机器。
      14. 在4℃下,将剩余的细胞(用CD56和CD82抗体或单一颜色对照标记)离心10分钟,以1,100μg×g 进行。
      15. 在5%FBS / HBSS中重悬浓度为1×10 7 / ml的细胞。
      16. 一抗孵育:将CD56和CD82抗体以5μl/ 1×10 6个细胞的浓度加入到适当的细胞溶液中。
      17. 为了关闭活细胞,将浓度为0.5μl/ 1x10 6细胞的钙黄绿加入适当的细胞溶液中。
        轻轻混合并置于冰上保护30分钟 注意:钙黄绿素是一种细胞活力染料,在此流程中用于在FACS期间区分活细胞和死细胞。
      18. 孵育后,将细胞在2ml 5%FBS / HBSS中洗涤1次。

      19. 在1100℃下将细胞离心10分钟,4℃
      20. 在500μl的5%FBS / HBSS中重悬CD56和CD82单色对照,并通过5ml圆底试管的过滤器帽进行移液。
        在冰上储存
      21. 在1ml 5%FBS / HBSS中重悬CD56 / CD82 /钙黄绿素染色的细胞,并通过5ml圆底试管的过滤器帽进行移液。
        在冰上储存
      22. 通过将500μl生长培养基移液到新管中来制备用于CD56 + CD82 +分选细胞的收集管。存放在冰上。

  3. 荧光激活细胞分选
    详细回顾FACS或流式细胞术是超出本章的范围。浇注规格简要说明(图1)。

    1. 使用“无污点”和单色控制确定最佳激励电压和补偿值
    2. 确定钙黄绿素阳性细胞的活细胞群门控。
    3. 确定双阳性(DP)CD56 + / CD82 + +和双阴性(DN)人群。门和分类的DP细胞人口。


      图1.使用钙黄绿素AM蓝作为活性染料标记的抗CD56和抗CD82抗体免疫染色的人肌原细胞的FACS门控技术实例A.未染色的对照样品,单细胞门; B. Calcein蓝色信号(钙黄绿素AM蓝色)门禁活细胞; C.对CD82-PE和CD56-APC染色的门控活细胞。 DN =双重阴性细胞; DP =双重阳性细胞。

  4. 人胎儿骨骼肌成肌细胞的体外培养和分析
    1. 体外细胞培养
      注意:本议定书中的所有步骤都应使用无菌组织培养技术在无菌层流生物安全柜中进行。
      1. 在设定为37℃的湿润的5%CO 2培养箱中用10ml 0.1%明胶(见配方)将无菌组织培养处理的平板(10cm平板)包衣1小时,然后除去明胶解决方案的愿望。让盘子在生物安全柜中短暂干燥并更换盖子。
      2. 预热完全生长培养基,水浴温度设定为37°C。
      3. 以0.5-1×10 6个细胞/ 10ml完全生长培养基重悬分选的CD56 / CD82双阳性细胞,并在包被的平板上平板。轻轻摇动平板以均匀分布细胞,然后在CO 2培养箱中孵育。分选后的细胞很小,有明亮的圆形外观,应在分选后1天内附着(图2)。
      4. 将细胞繁殖至65-75%汇合。这应该需要大约2-3天;然而,如果有必要的话,每2天更换新鲜生长培养基的培养基,直到培养皿达到65-75%汇合。


        图2. FACS分选的CD56和CD82双重阴性(DN)和双重阳性(DP)人类细胞的体外培养物(A和B)在分选后的第1天,DN细胞和DP细胞均呈现明亮圆润,有些细胞变平,牢固粘附于平板。 (C和D)在分选后的第7天(在生长培养基中4天,在分化培养基中3天[见食谱]),DN细胞保持单核,而DP细胞融合并形成多核肌管(箭头)。 (E和F)显示双阳性细胞上肌球蛋白重链(MHC)和结蛋白的免疫染色实例。

    2. 传代细胞

      1. 用0.1%明胶(如步骤D1a)涂覆无菌组织培养处理的平板
      2. 在37℃的水浴中预热以下物质:1×DPBS,TrypLE TM快速解离酶和完全生长培养基。
      3. 通过抽吸从平板上除去培养基,用10毫升(10厘米平板)1×DPBS洗涤细胞两次。删除DPBS的愿望。
      4. 移取2ml TrypLE TM Express到平板上,并在设定为37℃的潮湿5%CO 2培养箱中温育2-3分钟。将细胞转移到无菌的锥形管中之前,通过上下移动几次,轻轻地从平板上除去细胞。用另外的完全生长培养基清洗平板表面剩余的细胞。

      5. 在室温下将细胞在1,100×g 离心10分钟
      6. 将细胞重悬于10毫升新鲜的完全生长培养基中。
      7. 使用血细胞计数器确定细胞浓度,并将细胞在10ml完全生长培养基/ 10cm培养板中以0.5-1×10 6细胞进行平板培养。
      8. 细胞应每2-3天传代一次,不应长满70%以上。
        注意:细胞在增殖时不应达到100%融合,因为它们将开始分化并融合在接触上。高血清生长培养基随着时间的推移会降低血清浓度,并且不能防止融合。
    3. 冻结细胞
      1. 按步骤D2c-D2e胰蛋白酶消化和离心细胞。
      2. 重悬细胞在冰冷的冷冻介质(完全生长培养基中的10%DMSO)在所需的细胞浓度(10 6 -10 7 / ml)。

      3. 在-80°C下冷冻保存过夜,然后转移到-150°C,永久保存,直到需要。
    4. 进行体外试验
      1. 外套4孔的幻灯片与0.1%明胶。
      2. 用胰蛋白酶消化细胞(用苯酚红TrypLE Express TM解离酶)并如上所述确定细胞浓度,然后在500μl完全生长培养基/孔中铺平20000个细胞。
      3. 孵育细胞在5%的湿度设置为37°C的孵化器,直到细胞约80%汇合。
      4. 当细胞达到约80%汇合时,从每个孔中取出生长培养基,用500μl预热的分化培养基代替(见食谱)。将细胞在5%CO 2湿润培养箱中37℃培养过夜。
        注:低血清培养基诱导培养的成肌细胞分化和融合(Yaffe and Saxel,1977)。

      5. 在融合检测过程中,每天更换每个孔中的分化培养基
      6. 使用相差显微镜在10倍或20倍放大率下监测细胞的分化。融合应在暴露于分化培养基的1周内发生(图2)。当成熟肌管> 10个核/肌管存在,执行以下免疫细胞化学方案,通过荧光显微镜观察细胞。
    5. 免疫荧光用于体外融合检测
      注:这种免疫细胞化学方案也可用于检测增殖或分化细胞的其他肌源性标记物。

      1. 解冻4%PFA(见食谱)
      2. 用200μl1x PBS /孔小心清洗细胞。使用1000μl移液器移除PBS。
      3. 在室温下用200μl4%PFA固定细胞20分钟;使用移液管移除固定液,然后在室温下用200μl透化溶液(参见食谱)透化细胞3分钟。
      4. 使用移液管去除透化溶液,然后用200μl封闭溶液在室温封闭细胞30分钟(见食谱)。
      5. 通过在新鲜的封闭溶液中稀释抗人结蛋白抗体1:100来制备初级抗体溶液。
        在4°C过夜孵育细胞与初级抗体溶液
      6. 用1x PBS洗涤细胞3次5分钟。
        可以在旋转摇床上轻轻摇动平板
      7. 通过在封闭溶液中稀释抗小鼠抗体1:1,000来制备二抗溶液。
        在室温孵育细胞在黑暗中的二抗抗体溶液1小时
      8. 在室温下用1×PBS清洗细胞3次5分钟。
        可以在旋转摇床上轻轻摇动平板
      9. 用DAPI将Vectashield的电池安装好,并用铝箔保存在4°C的光下。
      10. 通过荧光显微镜分别使用DAPI和desmin的紫外线/ DAPI和FITC / GFP滤光片组来显现细胞。

数据分析

融合分析 - 融合指数计算

  1. 对肌管进行免疫荧光染色后,每孔5个随机区(x 4个孔/样品)的每一个中计数如下:

    1. 总核数#
    2. 肌管内的细胞核数量
  2. 计算融合指数(%)为((#肌管内肌管/#总核)×100)。
  3. 平均五个领域的融合指数。

  4. 比较分选的成肌细胞与未分选的融合指数

笔记

  1. 我们希望注意到CD56或MCAM与CD82结合的FACS作为富集标志物是分离人胎儿肌源性祖细胞的高度有效的方法。我们引用读者以下协议描述使用MCAM作为从人胎儿组织分选的细胞中的正选择标记(Lapan和Gussoni,2012)。因为内皮细胞在成人肌肉中表达MCAM,因此使用CD82和MCAM用于成人骨骼肌是不合适的。
  2. 在收集和处理人体组织之前,需要进行机构审查和方案批准。所有处理人体组织的人员都必须接受适当的安全和人文科目的教育培训。

食谱

  1. 生长培养基(500毫升)
    1. 395ml高葡萄糖(4.5g)达尔伯克改良伊格尔培养基(DMEM)
    2. 100毫升的胎牛血清(FBS)
    3. 5毫升100x青霉素 - 链霉素 - 谷氨酰胺(PSG)

    4. 通过500毫升0.22微米PES过滤器单元过滤溶液进行消毒
    5. 在4°C储存并在1个月内使用
    注:FBS公司之间和地段之间差异很大。因此,我们建议用这里描述的体外融合试验来测试几种不同的FBS样品。
  2. Dispase II储备液
    1. 将1克粉末分散酶II溶于100毫升HEPES缓冲盐水溶液中
    2. 加入316ml高葡萄糖(4.5g)DMEM以产生2.4U / ml的储备溶液
    3. 通过PES 500毫升过滤器过滤消毒溶液;等分成15毫升锥形管(10毫升/管)
    4. 将等分试样储存在-20°C
  3. 1M氯化钙溶液(CaCl 2•2H 2 O,FW 147)
    1. 将1.47克粉末溶解在10毫升双蒸水中
    2. 在4°C储存
  4. 胶原酶D储备溶液
    1. 将2.5克粉状胶原酶D溶于250毫升1×HBSS溶液中,并补充1.25毫升1M氯化钙溶液。
    2. 通过250毫升PES过滤装置进行过滤消毒
    3. 分装在15毫升锥形管(10毫升/管)中,并储存在-20°C
  5. 无菌冷冻培养基
    1. 结合90%FBS和10%二甲基亚砜(DMSO)
    2. 准备冰冻介质,立即储存在冰上
    3. 未使用的无菌冷冻培养基可以在4°C储存长达4周
  6. 分化培养基(50毫升)
    1. 48.5ml低葡萄糖(1g)Dulbecco改良的Eagle培养基(DMEM)
    2. 1毫升马血清(HS)
    3. 0.5毫升100x青霉素 - 链霉素 - 谷氨酰胺(PSG)

    4. 通过150毫升0.22微米PES过滤器过滤溶液进行消毒
    5. 在4°C储存并在1个月内使用
  7. 无菌5%FBS / HBSS溶液(50ml)
    1. 添加2.5毫升FBS到1x HBSS

    2. 通过50毫升0.22微米PES过滤器单元过滤溶液进行消毒
    3. 在4°C储存
  8. 0.1%明胶
    1. 将0.5克明胶加入到500毫升的双蒸水中。 不要摇晃

    2. 高压灭菌20分钟,然后储存于4°C,灭菌
  9. 4%多聚甲醛(4%PFA)

    1. 用1x PBS稀释16%多聚甲醛
    2. 多聚甲醛的毒性极大,请谨慎使用。为保证安全,建议在通风橱中使用多聚甲醛。分装和存储在-20°C
    3. 等分试样不应反复冻结和解冻;首次使用后丢弃未使用的PFA
  10. 透化解决方案
    将50μl的Triton X-100与10 ml的1×PBS混合
  11. 阻止解决方案
    将1ml胎牛血清(FBS),10μlTriton X-100和9ml 1x PBS混合在一起。

致谢

这项工作是由肌肉萎缩症协会#479606(EG)和美国国立卫生研究院的关节炎和肌肉骨骼和皮肤疾病的奖项1R01AR069582-01(EG)授予的。这些内容完全是作者的责任,不一定代表国立卫生研究院的官方观点。
该协议是从以前的工作,特别是从列出的参考文献1和7中修改。

参考

  1. Alexander,MS,Rozkalne,A.,Colletta,A.,Spinazzola,JM,Johnson,S.,Rahimov,F.,Meng,H.,Lawlor,MW,Estrella,E.,Kunkel,LM和Gussoni,E. (2016)。 CD82是一种预测性分离人类肌肉卫星细胞的标志物,与肌营养不良症有关。细胞干细胞19(6):800-807。
  2. Bishop,A.,Gallup,B.,Skeate,Y.和Dubowitz,V.(1971)。 正常和患病的人体肌肉的形态学研究
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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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