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Mar 2021
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A Simple Method for the Isolation and in vitro Expansion of Highly Pure Mouse and Human Satellite Cells
一种分离和体外扩增高纯度小鼠和人类卫星细胞的简单方法   

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Abstract

Satellite cells (SCs) are muscle stem cells capable of regenerating injured muscle. The study of their functional potential depends on the availability of methods for the isolation and expansion of pure SCs, which retain myogenic properties after serial passages in vitro. Here, we describe a protocol for the isolation and in vitro expansion of highly pure mouse and human SCs based on ice-cold treatment (ICT). The ICT is carried out by briefly incubating the dish containing a heterogeneous mix of adherent muscle mononuclear cells on ice for 15-30 min, which leads to the detachment only of the SCs, and gives rise to SC cultures with 95-100% purity. This approach can also be used to passage the cells, allowing SC expansion over extended periods of time without compromising their proliferation or differentiation potential. Overall, the ICT method is cost-effective, accessible, technically simple, reproducible, and highly efficient.


Graphic abstract:


Figure 1. Satellite cell isolation using the ice-cold treatment method.


Keywords: Satellite cell isolation (卫星细胞分离), In vitro expansion (体外扩增), Skeletal muscle (骨骼肌), Pax7 (Pax7), MyoD (MyoD)

Background

The exceptional regenerative ability of skeletal muscle is primarily due to a resident population of stem cells called satellite cells (SCs) (Mauro, 1961; Chang and Rudnicki, 2014; Wang et al., 2014). The study of their functional potential depends on the availability of methods for the isolation and expansion of highly pure SCs with preserved myogenic properties after serial passages in vitro (Danoviz and Yablonka-Reuveni, 2012; Keire et al., 2013; Syverud et al., 2014).


Presently, there are three main methods commonly used for the isolation of SCs: pre-plating, fluorescence activated cell sorting (FACS), and magnetic bead isolation.


The pre-plating method is based on the differing adhesive properties of muscle cells, with SCs being the least adherent (Gharaibeh et al., 2008; Danoviz and Yablonka-Reuveni, 2012; Keire et al., 2013; Syverud et al., 2014). Although cheap and straightforward to perform, this method’s main disadvantages are that it is time consuming and gives rise to cultures of variable purity, often with fibroblast contamination and overgrowth by day 7 of culture (Keire et al., 2013).


The FACS sorting method sorts muscle mononuclear cells pre-labeled with SC specific antibodies (Fukada et al., 2004; Sherwood et al., 2004; Montarras et al., 2005; Pasut et al., 2012; Chapman et al., 2013; Liu et al., 2015). At present, FACS sorting is the gold standard for the isolation and study of SCs. Nevertheless, there are several disadvantages to this method, including high cost and the requirement for a FACS sorter instrument. Moreover, this method is time consuming, requires expertise to perform, and cell purity can be variable. The cell labeling step that precedes the sorting procedure can potentially stress or damage the cells, decrease their viability, or alter their functional properties in vitro (Syverud et al., 2014).


Finally, the third method is based on magnetic cell separation (MACS) and uses magnetic columns and SC specific magnetic bead kits (Blanco-Bose et al., 2001). Because this method assumes that all the other cell types are successfully removed from the muscle cell preparation, it is less precise than the FACS sorting method. This method is expensive to perform, time consuming, and stressful for the cells. As for the other two methods, cell purity is variable, and often the SC cultures become overgrown by fibroblasts by day 7 (Keire et al., 2013; Syverud et al., 2014).


The ideal SC isolation technique would permit isolation of pure SCs with minimal manipulation, producing cells that could be expanded ex vivo without losing their stemness and regenerative capacity. Here, we describe a protocol for ice-cold treatment (ICT); this is a simple, inexpensive, and efficient method for the isolation and long-term expansion of highly pure mouse and human SCs that preserves their myogenic potential (Benedetti et al., 2020). In terms of purity of the isolated cell population, the ICT method outperforms others, such as pre-plating or magnetic bead isolation. Furthermore, it is fast and easy to perform—apart from the time required for enzymatic digestion (1.5 h), it involves minimal manipulation of the cells. Another major advantage of the ICT method is that it doubles up as a very gentle passaging technique, allowing long-term serial expansion of SCs ex vivo without altering their proliferation and differentiation properties. In turn, this drastically reduces the number of mice or muscle biopsies required to obtain a sufficient number of cells (Benedetti et al., 2021). The ICT method permits growing mouse and human SCs to be passaged at least 10 times, expanding their number 150- and 300-fold, respectively (Benedetti et al., 2021). This represents a clear advantage over the most commonly used passaging reagent (trypsin), which typically accelerates the differentiation of passaged SCs after only two passages (Danoviz and Yablonka-Reuveni, 2012; Benedetti et al., 2020 and 2021; Fiore et al., 2020).


Overall, the cost-effectiveness, accessibility, and technical simplicity of this protocol, as well as its remarkable efficiency, represent major improvements over existing protocols. The next step will be to test this protocol for the isolation and expansion of stem cells from tissues other than muscle.

Materials and Reagents

  1. Scalpel and surgical blades (Securelab, size 24)

  2. 70 µm cell strainers (Falcon, catalog number, catalog number: 352350)

  3. 40 µm cell strainers (Falcon, catalog number, catalog number: 352340)

  4. 100 mm tissue culture dishes (Falcon, catalog number: 430167)

  5. 35 mm tissue culture dishes (Falcon, catalog number: 353001)

  6. 60 mm tissue culture dishes (Falcon, catalog number: 353002)

  7. 50 ml polypropylene centrifuge tubes (Falcon, catalog number: 352098)

  8. 10 ml serological pipettes (Falcon, catalog number: 357551)

  9. Dissection boards (Styrofoam board)

  10. Cover slips 24 × 50 (Menzel, catalog number: 15737592)

  11. Pipette tips (Corning)

  12. Parafilm (Sigma, catalog number: P7793)

  13. WT C57BL/6J mice aged 4-6 weeks (The Jackson Laboratory)

  14. Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, catalog number: D5671)

  15. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7030)

  16. Hydrophobic PAP pen for immunostaining (Sigma-Aldrich, catalog number: Z377821)

  17. Human muscle biopsies [i.e., gluteus maximus biopsies obtained from patients undergoing surgery; Benedetti et al. (2021)]

  18. Vectashield mounting medium (Vector Laboratories, catalog number: H-1000-10)

  19. Hoechst 33342 staining dye (Abcam, catalog number: ab228551)

  20. Primary antibodies:

    Pax7 (Developmental Studies Hybridoma Bank)

    MyoD (Santa Cruz Biotechnology, catalog number: sc-760)

    MyoG (F5D) (Developmental Studies Hybridoma Bank)

    MyHC (MF20) (Developmental Studies Hybridoma Bank)

  21. Fluorescently labeled secondary antibodies:

    Goat anti-rabbit Alexa Fluor 488 (1:1,000, Abcam, catalog number: 150077)

    Goat anti-mouse Alexa Fluor 555 (1:1,000, Thermo Fisher Scientific, catalog number: A28180)

  22. 0.1% gelatin (Stem Cell Technologies, catalog number: 07903)

  23. L-glutamine (Sigma-Aldrich, catalog number: 59202C)

  24. Chicken embryo extract (Seralab, catalog number: CE-650-J)

  25. Goat serum (Sigma-Aldrich, catalog number: G9023)

  26. Horse serum (Thermo Fisher Scientific, catalog number: 26050088)

  27. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F2442), heat inactivated at 56°C for 30 min

  28. Dulbecco’s Phosphate-Buffered Saline (PBS) with MgCl2 and CaCl2 (Sigma-Aldrich, catalog number: D8662)

  29. Dulbecco’s Phosphate-Buffered Saline (PBS), modified without MgCl2 and CaCl2 (Sigma-Aldrich, catalog number: D8537)

  30. Penicillin-streptomycin (pen/strep) solution (Sigma-Aldrich, catalog number: P0781)

  31. Gentamycin solution (Sigma-Aldrich, catalog number: G1397)

  32. Formaldehyde solution 4% (Sigma-Aldrich, catalog number: 1004968350)

  33. Methanol (Sigma-Aldrich, catalog number: 34860)

  34. Collagenase type II (Sigma-Aldrich, catalog number: C6885)

  35. Collagenase/dispase (Roche, catalog number: 11097113001)

  36. Ethanol (Sigma-Aldrich, catalog number: 51976)

  37. 70% ethanol (see Recipes)

  38. Collagenase type II digestion solution (see Recipes)

  39. Collagenase/dispase digestion solution (see Recipes)

  40. Growth medium (see Recipes)

  41. Differentiation medium (see Recipes)

  42. Neutralization buffer (see Recipes)

  43. Hoechst solution (see Recipes)

Equipment

  1. Microsurgery scissors (Fine Science Tool, catalog number: 14184-09)

  2. Microsurgery tweezers (Fine Science Tool, catalog number: 11252-00)

  3. Pipettes (Gilson, P10, 20, 200, 1000)

  4. Humidified chamber (prepared by wetting paper towels with distilled water and placing them in a plastic container with a lid)

  5. Centrifuge (Eppendorf, model: 5702)

  6. Vertical Autoclave (Falc, model: ATV80)

  7. Temperature regulated shaking water bath (GLS, catalog number: 1083)

  8. Biosafety cabinet (Gelaire, model: BSB4 A)

  9. Laboratory chemical fume hood (ESCO, Frontier Acela)

  10. CO2 incubator Thermo Forma (Thermo Fisher Scientific, model: 3110)

  11. Zeiss Axioskop 2 Plus microscope (Carl Zeiss)

  12. Phase-contrast microscope (Nikon Eclipse, model: TS100)

  13. Pipet Controllers for serological pipettes (Falcon)

  14. Hemocytometer counting chamber Neubauer improved (BLAUBRAND, model: BR717810)

  15. Ice machine

Software

  1. ZEISS ZEN 2 Blue edition (Carl Zeiss) (downloa from https://www.zeiss.com/microscopy/int/products/microscope-software.html)

  2. ImageJ 1.53a (download from https://imagej.nih.gov/ij/download.html)

Procedure

  1. Isolation of muscle satellite cells

    Notes:

    1. Experimental procedures should be performed only after obtaining ethical approval.

    2. Follow the same procedure for both mouse and human muscle.


    Isolation of mouse satellite cells
    1. Day 1: Preparation prior to muscle dissection

      1. Prepare 70% ethanol (see Recipe).

      2. Autoclave dissection instruments (scissors and tweezers).

      3. Add 3 ml of DMEM supplemented with 1% pen/strep into 60 mm cell culture dishes (one dish/mouse or one dish per gram of human muscle biopsy). These will be used to collect the dissected muscle.

      4. Coat the cell culture dishes with 0.1% gelatin:

        Coat 35 mm and 100 mm cell culture dishes with 1 ml and 5 ml of 0.1% gelatin, respectively, ensuring that the entire surface of the dish is evenly coated, and incubate for 30 min in a cell incubator at 37°C with 5% CO2. Next, remove excess gelatin (without washing the dishes), and leave the dishes open to air dry for 10-15 min under the hood. These dishes will be used for cell culture.

        Note: The 35 mm dishes will be needed one day following isolation.

      5. Prepare collagenase type II and collagenase/dispase digestion solutions (see Recipes).

      6. Prepare enzyme neutralization buffer (see Recipes).

      7. Prepare cell growth medium (see Recipes).

      8. Prepare cell differentiation medium (see Recipes).

      9. Sterilize a scalpel and surgical blades.

      10. Pre-warm a water bath at 37°C.

    2. Day 1: Dissection of mouse hindlimb muscles

      For a schematic representation of the dissection process, see Figure 2.



      Figure 2. Dissection of mouse quadriceps, tibialis anterior, and gastrocnemius muscles.


      1. Euthanize a mouse [i.e., by cervical dislocation; Benedetti et al. (2021)].

      2. Pin the mouse to a dissection board and spray it with 70% ethanol.

      3. Using dissection scissors, make an incision on the skin above the ankle, and peel off the skin over the entire length of the hindlimbs to expose the muscles below.

      4. Collect the tibialis anterior, quadriceps, and gastrocnemius muscles from both legs (Figure 2).

      5. Place the dissected muscles in a 60 mm dish containing DMEM supplemented with 1% pen/strep, and remove any hair to minimize the risk of contamination.

      6. Transfer the dishes under a biosafety cabinet and finely cut up the muscles into 1 mm pieces using a sterile scalpel (Figure 1b).

        Note: Remove and discard any fat from the dissected muscle.

      7. Transfer the minced muscles into a 50 ml tube and centrifuge at 300 × g for 5 min at room temperature (RT).

    3. Day 1: Muscle digestion I (Figure 1c).

      1. Aspirate the supernatant with a 10 ml serological pipette and resuspend the minced muscles in 10 ml of collagenase type II digestion solution per gram of tissue (see Recipes).

        Note: It is important to use a pipette to remove the supernatant, as the pellet is not stable in this step.

      2. Seal the tubes with parafilm and transfer them to a shaking (100 rpm) pre-warmed (37°C) water bath for 45 min.

        Note: Position the tubes horizontally so that they are completely submerged in water.

      3. Spray the tubes with 70% ethanol and transfer them to a biosafety cabinet.

      4. Block the digestion enzyme by adding 10 ml of enzyme neutralization buffer per gram of tissue (see Recipes).

      5. Pipette up and down with a 10 ml serological pipette five times.

      6. Centrifuge at 300 × g for 5 min at RT.

    4. Day 1: Muscle digestion II (Figure 1d).

      1. Aspirate the supernatant with a 10 ml serological pipette and resuspend the pellet in 10 ml of collagenase/dispase digestion solution per gram of tissue (see Recipes).

      2. Seal the tubes with parafilm and transfer them into a shaking (100 rpm) pre-warmed (37°C) water bath (as in Step A3b) for 30 min.

      3. Spray the tubes with 70% ethanol and transfer them to a biosafety cabinet.

      4. Block the digestion by adding 10 ml of enzyme neutralization buffer per gram of tissue (see Recipes).

      5. Pipette up and down with a 10 ml serological pipette five times.

      6. Sieve the digested muscles through a 70 µm cell strainer into a new 50 ml tube.

      7. Sieve the digested muscles through a 40 µm cell strainer into a new 50 ml tube.

        Note: An adequately digested muscle will pass through the strainers easily. If necessary, the muscle digest can be passed again through another strainer to get rid of most of the cell debris and undigested muscle.

      8. Centrifuge the tube with muscle digest at 300 × g for 5 min at RT.

      9. Discard the supernatant and resuspend the pellet in 10 ml of DMEM 10% FBS. Count the number of muscle mononuclear cells using trypan blue.

        Note: At this step, you can combine the pellets if more than 1 mouse was used. On average, the number of cells released after digestion from 3, 4-8-weeks-old mice is around 107 cells.

    5. Day 1: Pre-plating step (Figure 1e-1f).

      1. Plate the cells at 105/ml in a 100 mm dish (uncoated) in 20 ml of DMEM 10% FBS 1% pen/strep and incubate at 37°C for 1 h.

      2. Collect the non-adhered cells in a 50 ml tube and centrifuge at 300 × g for 5 min at RT.

      3. Discard the supernatant and resuspend the cell pellet in DMEM 10% FBS 1% pen/strep.

      4. Count the resuspended cells and plate them at 105/ml into a 100 mm dish (uncoated) in 20 ml of DMEM 10% FBS 1% pen/strep. Incubate the cells at 37°C for 1 h (as in Step A5a).

      5. Collect and transfer the non-adhered cells into a 50 ml tube.

        Note: On average, the pre-plating steps remove around 50% of strongly adherent cells such as fibroblasts.

      6. Centrifuge the tube with non-adhered cells at 300 × g for 5 min at RT.

      7. Discard the supernatant and resuspend the cell pellet in growth medium (see Recipes).

      8. Plate the cells into 0.1% gelatin coated 100 mm dishes at 106 cells/dish. Grow them overnight (Figure 1g).

    6. Day 2: Ice-cold treatment (ICT) (Figure 1h).

      1. Wash the dishes containing the heterogeneous mix of adhered muscle cells three times with 10 ml of PBS (with MgCl2 and CaCl2) at RT.

        Note: Gently pipette PBS all around the dish.

      2. Add 10 ml of ice-cold PBS (without MgCl2 and CaCl2) to each 100 mm dish.

        Note: Both PBS with or without CaCl2 can be used. However, using PBS without CaCl2 will facilitate detachment.

      3. Fill a polystyrene box with ice (0°C) and place the dishes on top of the ice for 15-30 min with occasional gentle manual shaking of the dish (creating a swirling motion).

        Notes:

        1. If purity is of utmost importance (e.g., 99-100% purity), the dishes should not be kept on ice for longer than 30 min. Nevertheless, the dishes can be kept on ice for up to 1 h to increase cell yield, with purity still high at 90%.

        2. The original dishes (from day 1 of the isolation procedure) containing heterogeneous muscle cells can be kept up to day 3 of culture and used for further rounds of ICTs .

      4. Collect the detached cells into a 50 ml tube.

      5. Wash the original dish once more with 10 ml of PBS to collect the remaining detached cells.

        Note: Gently pipette PBS all around the dish.

      6. Centrifuge the cells at 300 × g for 5 min at RT.

      7. Discard the supernatant and resuspend the cells in growth medium.

      8. Count the cells.

        Note: On average, 1.5 × 104-2 × 104 SCs can be collected after 30 min on ice at 24 h of culture of the original dish. A further 4 × 104 SCs can be collected after the second or third ICT at 48 h or 72 h of culture).

      9. Plate the cells into 35 mm dishes precoated with 0.1% gelatin, at a density of 103 cells/dish and place them into a cell incubator.

        Note: At this density, the SC proliferate for up to three days. Afterward, they begin to differentiate. Increasing the plating density will accelerate the time it takes for the myoblasts to differentiate and fuse into myotubes.

      10. Cell culture

        1. Culture the cells in growth medium, changing the medium every two days.

        2. To induce myoblast differentiation, remove growth medium and replace it with differentiation medium (see Recipes) once the cells reach 80% confluency.

      11. Cell passaging

        1. Place the dishes containing proliferating SCs on ice for up to 30 min.

        2. Follow the steps described in A6.


    Isolation of human muscle satellite cells

    Notes:

    1. Experimental procedures using human muscle biopsies should be performed only after obtaining ethical approval and informed consent.

    2. The number of SCs obtained from human muscle biopsies may vary depending on the age of the donor, with a smaller number of cells typically obtained from older donors.


    1. Preparation prior to muscle dissection

      Proceed as in Step A1.

    2. Muscle cutting

      1. Place the human muscle biopsy in a 60 mm dish containing DMEM supplemented with 1% pen/strep. Work in a biosafety cabinet.

      2. Remove any fat and connective tissue using scissors.

      3. Finely cut up the muscle into 1 mm pieces using a sterile scalpel.

      4. Transfer the minced muscles to a 50 ml tube and centrifuge at 300 × g for 5 min.

    3. Muscle digestion I:

      Proceed as described in A3.

    4. Muscle digestion II:

      Proceed as described in A4.

      Note: On average, 2 × 106 muscle mononuclear cells can be obtained from digestion of 1 g of human muscle biopsy.

    5. Pre-plating:

      Proceed as in A5.

    6. ICT:

      Proceed as in A6.

      To further enrich in SCs the heterogeneous muscle cell culture on day 1 after isolation, it may be necessary to grow it for an additional 3-4 days before proceeding to ICT (when the culture reaches a confluency of about 80%).

      Notes:

      1. On average, 1 × 104 SCs can be collected after the first ICT.

      2. A further 2 × 104 SCs can be collected after the second or third ICT at 48 h or 72 h of culture.

    7. Cell culture

      Proceed as in Step A7.

      Note: Human SCs proliferate slower than mouse SCs, with a doubling time of 46 h.


  2. Analysis of mouse and human satellite cell specific marker expression by immunofluorescence staining

    1. Grow the cells in a 35 mm dish.

    2. Aspirate the medium and wash the dishes once with 1 ml of PBS with MgCl2 and CaCl2.

    3. Fix the cells by adding 4% PFA (1 ml/35 mm dish) and incubate for 10 min at room temperature.

      Note: PFA is toxic and must be used under a chemical fume hood.

    4. Discard the PFA into a toxic waste container and wash three times with 1 ml of PBS for 5 min each.

    5. Permeabilize the cells by adding cold methanol (1 ml/35 mm dish) and incubate at -20°C for 6 min.

    6. Discard the methanol into a toxic waste container and wash three times with 1 ml of PBS for 5 min each.

    7. Remove the PBS and mark the border of the dish with a hydrophobic pen.

    8. Block by adding PBS containing 5% goat serum (100 μl/35 mm dish) for 30 min at RT.

    9. Remove the blocking solution and incubate the cells with the primary antibodies (100 μl/35 mm dish) at the appropriate dilution [Pax7 (1:10), MyoD (1:50), Myogenin (1:20), and Myosin Heavy Chain (1:20) diluted in sterile filtered 4% BSA/PBS] overnight in a humidified chamber at 4°C.

    10. Remove the primary antibody solution and wash three times with 1 ml of PBS for 5 min each.

    11. Incubate the cells with the secondary antibodies (100 μl/35 mm dish) [goat anti-rabbit Alexa Fluor 488 (1:1,000) and goat anti-mouse Alexa Fluor 555 (1:1,000) diluted in sterile filtered 1%BSA/PBS] for 1h at room temperature in a humified chamber in the dark.

    12. Remove the secondary antibody solution and wash three times with 1 ml of PBS for 5 min each.

    13. Counterstain the nuclei with Hoechst solution (see Recipes) (100 μl/35 mm dish) for 5 min at room temperature in the dark.

    14. Remove the Hoechst solution and wash three times with 1 ml of PBS for 5 min each.

    15. Mount the coverslips with Vectashield mounting medium (30 μl/35 mm dish).

    16. Analyze under a fluorescence microscope.

Data analysis

Cell morphology was analyzed under a bright field microscope (Figures 3A and 4A). To analyze the expression of myogenic markers such as Pax7, MyoD, Myogenin, and Myosin Heavy Chain (MHC), the cells were stained as described in the procedure section (Figure 3B and 3D). Mouse SC purity was above 99% after ICT isolation. SC purity was assessed by immunofluorescence staining for Pax7 and MyoD, by counting the percentage of SCs positive for Pax7 and/or MyoD at day 3 of culture in growth medium after ICT (Figure 3B and 3C) (Benedetti et al., 2021).

    The purity of human SCs isolated using the ICT approach is typically above 97% after ICT. Because human SCs in culture rapidly downregulate the expression of Pax7, SC purity was assessed by immunofluorescence staining for myogenin, by counting the percentage of SCs positive for myogenin at day 5 of culture in growth medium (Figure 4B and 4C) (Benedetti et al., 2021). The differentiation potential of human SCs was analyzed by performing immunofluorescence staining with an anti-Myosin Heavy Chain (MHC) antibody (Figure 4D).



Figure 3. Isolation and characterization of mouse muscle SCs using the ICT method.

A. Representative bright field images of the mouse muscle cell culture. SCs were isolated by ICT from heterogeneous mononuclear cells. The ICT-isolated SCs at day 2 (D2) and day 4 (D4) in growth medium (GM) and at day 3 (D3) after adding differentiation medium (DM). B. Representative immunofluorescence images of ICT-isolated SCs stained for Pax7 (red), MyoD (green), and nuclei (blue). C. Graph showing the percentage of cells positive for Pax7 and/or MyoD at D3 of culture in growth medium after ICT (n = 3 independent experiments). D. Representative immunofluorescence images of ICT-isolated SCs stained for myosin heavy chain (MHC) (red) and nuclei (blue) after four days in growth medium followed by three days in DM. Scale bar = 100 μm. Error bars represent mean ± SEM (Benedetti et al., 2021).



Figure 4. Isolation and characterization of human muscle SCs (hSCs) using the ICT method.

A. Representative bright field images of the heterogeneous human muscle mononuclear cell culture and isolated SCs at day 3 (D3) and day 10 (D10) in growth medium (GM), and at D10 after adding differentiation medium (DM). B. Representative immunofluorescence images of human SCs stained for Myogenin (red) and nuclei (blue). C. Graph showing the percentage of myogenin+ cells after ICT at day 5 of culture in GM (n = 3 independent experiments). D. Representative immunofluorescence images of human SCs stained for myosin heavy chain (MHC) (red) and nuclei (blue) after 10 days in GM followed by 10 days in DM. Scale bar = 100 µm. Error bars represent mean ± SEM (Benedetti et al., 2021).

Recipes

  1. 70% ethanol

    Add 70 ml of 100% ethanol to 30 ml of ddH2O

    Store at room temperature

  2. Collagenase type II digestion solution

    Dissolve collagenase type II powder in PBS with MgCl2 and CaCl2 at a concentration of 0.4 mg/ml, for a total of 10 ml per gram of tissue.

  3. Collagenase/dispase digestion solution

    Dissolve collagenase/dispase powder in PBS MgCl2 and CaCl2 free at a concentration of 1 mg/ml, for a total of 10 ml for gram of tissue.

  4. Growth medium

    Supplement DMEM medium with 20% horse serum, 3% chicken embryo extract, 1% penicillin/streptomycin, and 1% L-Glutamine. Store at 4°C.

  5. Differentiation medium

    Supplement DMEM medium with 5% horse serum, 1% chicken embryo extract, 1% penicillin/streptomycin, and 1% L-Glutamine. Store at 4°C.

  6. Neutralisation buffer

    Supplement DMEM medium with 10% FBS and 1% penicillin/ streptomicin.

  7. Hoechst solution

    Dilute Hoechst stain 1:1,000 in PBS

Acknowledgments

This work was supported by a grant from The Dutch Duchenne Parent Project NL (DPP NL) to BLO; research grants from Parent Project Italy (PP, Italy) to MB and from the University of Rome (RP11715C7D238352, RM118164275C7EBE and RM11916B7E20311C to MB, and AR11715C7F9E158E, AR11816436905518 and AR11916B7E2A7B64 to AB). This protocol describes the work published in Benedetti et al. (2021).

Competing interests

The authors declare no competing financial or non-financial interests.

Ethics

All procedures involving mice were approved by the Italian Ministry for Health and were conducted according to the EU regulations and the Italian Law on Animal Research. Ethical Approval ID: 82945.19. valid until 18/01/2023.

All patients gave informed consent to undergo intra-operative muscle biopsy and to publish the clinical and laboratory data obtained.

References

  1. Benedetti, A., Fiore, P. F., Madaro, L., Lozanoska-Ochser, B. and Bouche, M. (2020). Targeting PKCtheta Promotes Satellite Cell Self-Renewal. Int J Mol Sci 21(7): 2419.
  2. Benedetti, A., Cera, G., De Meo, D., Villani, C., Bouche, M. and Lozanoska-Ochser, B. (2021). A novel approach for the isolation and long-term expansion of pure satellite cells based on ice-cold treatment. Skelet Muscle 11(1): 7.
  3. Blanco-Bose, W. E., Yao, C. C., Kramer, R. H. and Blau, H. M. (2001). Purification of mouse primary myoblasts based on α7 integrin expression. Exp Cell Res 265(2): 212-220.
  4. Chang, N. C. and Rudnicki, M. A. (2014). Satellite cells: the architects of skeletal muscle. Curr Top Dev Biol 107: 161-181.
  5. Chapman, M. R., Balakrishnan, K. R., Li, J., Conboy, M. J., Huang, H., Mohanty, S. K., Jabart, E., Hack, J., Conboy, I. M. and Sohn, L. L. (2013). Sorting single satellite cells from individual myofibers reveals heterogeneity in cell-surface markers and myogenic capacity. Integr Biol (Camb) 5(4): 692-702.
  6. Danoviz, M. E. and Yablonka-Reuveni, Z. (2012). Skeletal muscle satellite cells: background and methods for isolation and analysis in a primary culture system. Methods Mol Biol 798: 21-52.
  7. Fiore, P. F., Benedetti, A., Sandona, M., Madaro, L., De Bardi, M., Saccone, V., Puri, P. L., Gargioli, C., Lozanoska-Ochser, B. and Bouche, M. (2020). Lack of PKCtheta Promotes Regenerative Ability of Muscle Stem Cells in Chronic Muscle Injury. Int J Mol Sci 21(3) : 932.
  8. Fukada, S., Higuchi, S., Segawa, M., Koda, K., Yamamoto, Y., Tsujikawa, K., Kohama, Y., Uezumi, A., Imamura, M., Miyagoe-Suzuki, Y. et al. (2004). Purification and cell-surface marker characterization of quiescent satellite cells from murine skeletal muscle by a novel monoclonal antibody. Exp Cell Res 296(2): 245-255.
  9. Gharaibeh, B., Lu, A., Tebbets, J., Zheng, B., Feduska, J., Crisan, M., Peault, B., Cummins, J. and Huard, J. (2008). Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique. Nat Protoc 3(9): 1501-1509.
  10. Keire, P., Shearer, A., Shefer, G. and Yablonka-Reuveni, Z. (2013). Isolation and culture of skeletal muscle myofibers as a means to analyze satellite cells. Methods Mol Biol 946: 431-468.
  11. Liu, L., Cheung, T. H., Charville, G. W. and Rando, T. A. (2015). Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting. Nat Protoc 10(10): 1612-1624.
  12. Mauro, A. (1961). Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 9: 493-495.
  13. Montarras, D., Morgan, J., Collins, C., Relaix, F., Zaffran, S., Cumano, A., Partridge, T. and Buckingham, M. (2005). Direct isolation of satellite cells for skeletal muscle regeneration. Science 309(5743): 2064-2067.
  14. Pasut, A., Oleynik, P. and Rudnicki, M. A. (2012). Isolation of muscle stem cells by fluorescence activated cell sorting cytometry. Methods Mol Biol 798: 53-64.
  15. Sherwood, R. I., Christensen, J. L., Conboy, I. M., Conboy, M. J., Rando, T. A., Weissman, I. L. and Wagers, A. J. (2004). Isolation of adult mouse myogenic progenitors: functional heterogeneity of cells within and engrafting skeletal muscle. Cell 119(4): 543-554.
  16. Syverud, B. C., Lee, J. D., VanDusen, K. W. and Larkin, L. M. (2014). Isolation and Purification of Satellite Cells for Skeletal Muscle Tissue Engineering. J Regen Med 3(2).
  17. Wang, Y. X., Dumont, N. A. and Rudnicki, M. A. (2014). Muscle stem cells at a glance. J Cell Sci 127(Pt 21): 4543-4548.

简介

[摘要]卫星细胞(SCs)是一种能够再生受伤肌肉的肌肉干细胞。对其功能潜力的研究取决于分离和扩展纯 SCs 的方法的可用性,这些方法在体外连续传代后仍保留生肌特性。在这里,我们描述了一种基于冰冷处理 (ICT) 的高纯度小鼠和人类 SCs的分离和体外扩增协议。ICT 是通过将含有粘附肌肉单核细胞异质混合物的培养皿在冰上短暂孵育 15-30 分钟来进行的,这会导致仅 SCs 脱离,并产生纯度为 95-100% 的 SC 培养物。这种方法也可用于细胞传代,允许 SC 扩展延长时间,而不会影响其增殖或分化潜力。总体而言,ICT 方法具有成本效益、可访问性、技术简单、可重复和高效的特点。

[背景]骨骼肌非凡的再生能力主要是由于称为卫星细胞 (SC) 的干细胞常驻种群(Mauro,1961;Chang 和 Rudnicki,2014;Wang等,2014)。对其功能潜力的研究取决于在体外连续传代后分离和扩增具有保留肌原特性的高纯度 SCs 的方法的可用性(Danoviz 和 Yablonka-Reuveni,2012 年;Keire等人,2013 年;Syverud等人。 , 2014)。

目前,常用的分离 SCs 的方法主要有三种:预镀、荧光激活细胞分选 (FACS) 和磁珠分离。

预镀方法基于肌肉细胞不同的粘附特性,SCs 的粘附性最低(Gharaibeh等,2008;Danoviz 和 Yablonka-Reuveni,2012;Keire等,2013;Syverud等, 2014) 。尽管成本低廉且易于执行,但该方法的主要缺点是耗时且培养物纯度不一,通常在培养第 7 天时会出现成纤维细胞污染和过度生长(Keire等人,2013 年)。

分选方法各种肌肉的单核细胞的FACS预标记与SC特异性抗体(深田等人,2004;舍伍德等人,2004; Montarras等人,2005; Pasut等人,2012;查普曼等人,2013。 ; Liu等人,2015 年)。目前,FACS 分选是分离和研究 SCs 的金标准。然而,这种方法有几个缺点,包括成本高和需要 FACS 分拣仪。此外,这种方法很耗时,需要专业知识才能执行,而且细胞纯度可能会发生变化。分选程序之前的细胞标记步骤可能会对细胞造成压力或损坏,降低其活力,或改变其体外功能特性(Syverud等,2014)。

最后,第三种方法基于磁细胞分离 (MACS),并使用磁柱和 SC 特定磁珠套件(Blanco-Bose等,2001)。由于此方法假定所有其他细胞类型都已从肌肉细胞制剂中成功去除,因此它不如 FACS 分选方法精确。这种方法执行起来昂贵、耗时且对细胞有压力。至于其他两种方法,细胞纯度是可变的,SC 培养物通常在第 7 天被成纤维细胞过度生长(Keire等人,2013 年;Syverud等人,2014 年)。

理想的 SC 分离技术将允许以最少的操作分离纯 SCs,产生可以在体外扩增而不会失去其干性和再生能力的细胞。在这里,我们描述了冰冷处理 (ICT) 的协议;这是一种简单、廉价且有效的方法,用于分离和长期扩增高纯度小鼠和人类 SCs,并保留其生肌潜力(Benedetti等人,2020 年)。在分离细胞群的纯度方面,ICT 方法优于其他方法,例如预镀或磁珠分离。此外,它快速且易于执行——除了酶消化所需的时间(1.5 小时)外,它还涉及最少的细胞操作。ICT 方法的另一个主要优点是它兼作一种非常温和的传代技术,允许体外长期连续扩增 SCs,而不会改变其增殖和分化特性。反过来,这大大减少了获得足够数量细胞所需的小鼠或肌肉活检数量(Benedetti等,2021)。ICT 方法允许正在生长的小鼠和人类 SCs 至少传代 10 次,将它们的数量分别扩大 150 倍和 300 倍(Benedetti等人,2021 年)。这代表了与最常用的传代试剂(胰蛋白酶)相比的明显优势,后者通常仅在两次传代后加速传代 SCs 的分化(Danoviz 和 Yablonka-Reuveni,2012 年;Benedetti等人,2020 年和 2021 年;Fiore等人。 , 2020)。

总体而言,该协议的成本效益、可访问性和技术简单性,以及其卓越的效率,代表了对现有协议的重大改进。下一步将是测试该协议是否从肌肉以外的组织中分离和扩增干细胞。

关键字:卫星细胞分离, 体外扩增, 骨骼肌, Pax7, MyoD

材料和试剂

 

  1. 手术刀和手术刀片(Securelab,24 号)
  2. 70 µm 细胞过滤器(Falcon,目录号,目录号:352350)
  3. 40 µm 细胞过滤器(Falcon,目录号,目录号:352340)
  4. 100 mm 组织培养皿(Falcon,目录号:430167)
  5. 35 mm 组织培养皿(Falcon,目录号:353001)
  6. 60 mm 组织培养皿(Falcon,目录号:353002)
  7. 50 ml 聚丙烯离心管(Falcon,目录号:352098)
  8. 10 ml血清移液管(Falcon,目录号:357551)
  9. 解剖板(泡沫聚苯乙烯板)
  10. 盖玻片 24 × 50(Menzel,目录号:15737592) 
  11. 移液器吸头 (Corning)
  12. 封口膜(Sigma,目录号:P7793 
  13. 4-6 周龄的 WT C57BL/6J 小鼠(杰克逊实验室)
  14. Dulbecco 改良 Eagle 培养基(DMEM)(Sigma-Aldrich,目录号:D5671)
  15. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7030)
  16. 用于免疫染色的疏水性 PAP 笔(Sigma-Aldrich,目录号:Z377821 
  17. 人肌肉活组织检查[,从接受手术的患者获得的臀大肌活组织检查;贝内代蒂等人(2021)]
  18. Vectashield 安装介质(Vector Laboratories,目录号:H-1000-10)
  19. Hoechst 33342染色染料(Abcam,目录号:ab228551 
  20. 一抗:

Pax7(发展研究杂交瘤银行)

MyoD(Santa Cruz Biotechnology,目录号:sc-760)

MyoG (F5D)(发育研究杂交瘤银行)

MyHC (MF20)(发育研究杂交瘤银行)

  1. 荧光标记的二抗:

山羊抗兔 Alexa Fluor 488(1:1,000,Abcam,目录号:150077)

山羊抗小鼠 Alexa Fluor 555(1:1,000,Thermo Fisher Scientific,目录号:A28180)

  1. 0.1% 明胶(Stem Cell Technologies,目录号:07903)
  2. L-谷氨酰胺(Sigma-Aldrich,目录号:59202C)
  3. 鸡胚提取物(Seralab,目录号:CE-650-J)
  4. 山羊血清(Sigma-Aldrich,目录号:G9023)
  5. 马血清(Thermo Fisher Scientific,目录号:26050088)
  6. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F2442),在 56°C 下热灭活 30 分钟
  7. 含有MgCl 2和 CaCl 2 的Dulbecco 磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:D8662)
  8. Dulbecco 磷酸盐缓冲盐水(PBS),不含MgCl 2和 CaCl (Sigma-Aldrich,目录号:D8537)
  9. 青霉素-链霉素(pen/strep)溶液(Sigma-Aldrich,目录号:P0781)
  10. 庆大霉素溶液(Sigma-Aldrich,目录号:G1397)
  11. 4%甲醛溶液(Sigma-Aldrich,目录号:1004968350)
  12. 甲醇(Sigma-Aldrich,目录号:34860)
  13. II型胶原酶(Sigma-Aldrich,目录号:C6885 
  14. 胶原酶/分散酶(Roche,目录号:11097113001 
  15. 乙醇(Sigma-Aldrich,目录号:51976)
  16. 70% 乙醇(见配方)
  17. II型胶原酶消化溶液(见食谱)
  18. 胶原酶/分散酶消化溶液(见食谱)
  19. 生长培养基(见食谱)
  20. 分化培养基(见食谱)
  21. 中和缓冲液(见配方)
  22. Hoechst 解决方案(见食谱)

 

设备

 

  1. 显微手术剪刀(Fine Science Tool,目录号:14184-09)
  2. 显微手术镊子(Fine Science Tool,目录号:11252-00)
  3. 移液器 (Gilson, P10, 20, 200, 1000)
  4. 加湿室(通过用蒸馏水润湿纸巾并将其放入带盖的塑料容器中制备)
  5. 离心机(Eppendorf,型号:5702)
  6. 立式高压釜(Falc 型号:ATV80)
  7. 调温振荡水浴(GLS,目录号:1083)
  8. 生物安全柜(Gelaire,型号:BSB4 A)
  9. 实验室化学通风柜(ESCO,Frontier Acela)
  10. CO 2培养箱 Thermo Forma(Thermo Fisher Scientific,型号:3110)
  11. 蔡司 Axioskop 2 Plus 显微镜(卡尔蔡司)
  12. 相差显微镜(Nikon Eclipse,型号:TS100)
  13. 用于血清移液器的移液器控制器 (Falcon)
  14. 血球计数室 Neubauer 改进型(BLAUBRAND,型号:BR717810 
  15. 制冰机

 

软件

 

  1. ZEISS ZEN 2 Blue edition (Carl Zeiss)(从https://www.zeiss.com/microscopy/int/products/microscope-software.html 下载
  2. ImageJ 1.53a(从https://imagej.nih.gov/ij/download.html下载

 

程序

 

  1. 肌肉卫星细胞的分离

笔记:

  1. 只有在获得伦理批准后才能进行实验程序。
  2. 对小鼠和人体肌肉执行相同的程序。

 

小鼠卫星细胞的分离

  1. 第 1 天:肌肉解剖前的准备工作
  1. 准备 70% 乙醇(见配方)。
  2. 高压灭菌器解剖仪器(剪刀和镊子)。
  3. 将 3 ml 补充有 1% pen/strep 的 DMEM 添加到 60 mm 细胞培养皿中(每克人类肌肉活检一盘/小鼠或一盘)。这些将用于收集解剖肌肉。
  4. 涂上 0.1% 明胶的细胞培养皿:

分别用 1 ml 和 5 ml 0.1% 明胶涂覆 35 mm 和 100 mm 细胞培养皿,确保培养皿的整个表面均匀涂覆,并在 37°C 5% 的细胞培养箱中孵育 30 分钟一氧化碳接下来,去除多余的明胶(不洗盘子),将盘子打开,在引擎盖下风干 10-15 分钟。这些培养皿将用于细胞培养。

注意:隔离后一天将需要 35 毫米培养皿。

  1. 制备 II 型胶原酶和胶原酶/分散酶消化溶液(参见食谱)。
  2. 准备酶中和缓冲液(参见食谱)。
  3. 准备细胞生长培养基(见食谱)。
  4. 准备细胞分化培养基(见食谱)。
  5. 消毒手术刀和手术刀片。
  6. 在 37°C 下预热水浴。
    1. 第 1 天:解剖小鼠后肢肌肉

有关解剖过程的示意图,请参见图 2。

 

 

图 2. 解剖小鼠股四头肌、胫前肌和腓肠肌。

 

  1. 安乐死一只老鼠 [,通过颈椎脱位;贝内代蒂等人(2021)]。
  2. 将鼠标固定在解剖板上,并用 70% 乙醇喷洒。
  3. 使用解剖剪刀,在脚踝上方的皮肤上做一个切口,然后在后肢的整个长度上剥离皮肤,露出下面的肌肉。
  4. 从双腿收集胫骨前肌、股四头肌和腓肠肌 (图 2)。
  5. 将解剖的肌肉放在含有 DMEM 的 60 毫米盘子中,并辅以 1% 笔/链球菌,并去除任何头发以最大程度地减少污染风险。
  6. 将菜肴转移到生物安全柜下,并使用无菌手术刀将肌肉切成 1 毫米的碎片(图 1b)。

注意:从解剖肌肉中取出并丢弃任何脂肪。

  1. 将切碎的肌肉转移到 50 ml 管中,并在室温 (RT) 下以 300 × g离心5 分钟。
  1. 第 1 天:肌肉消化 I(图 1c)。
  1. 用 10 ml 血清移液管吸出上清液,并将切碎的肌肉重新悬浮在每克组织10 ml的 II 型胶原酶消化溶液中(参见食谱)。

注意:使用移液器去除上清液很重要,因为在这一步中沉淀不稳定。

  1. 用封口膜密封管子并将它们转移到摇晃 (100 rpm) 预热 (37°C) 水浴中 45 分钟。

注意:水平放置管子,使它们完全浸没在水中。

  1. 用 70% 乙醇喷洒管并将它们转移到生物安全柜
  2. 通过每克组织添加 10 ml 酶中和缓冲液来阻断消化酶(参见食谱)。
  3. 用 10 ml 血清移液管上下吸管五次。
  4. 在室温下以 300 × g离心5 分钟。
  1. 第 1 天:肌肉消化 II(图 1d)。
  1. 用 10 ml 血清移液管吸出上清液,并将沉淀重悬在每克组织10 ml胶原酶/分散酶消化溶液中(参见食谱)。
  2. 用封口膜密封管子,并将它们转移到摇晃 (100 rpm) 预热 (37°C) 水浴(如步骤 A3b)中 30 分钟。
  3. 用 70% 乙醇喷洒管并将它们转移到生物安全柜
  4. 通过每克组织添加 10 ml 酶中和缓冲液来阻止消化(参见食谱)。
  5. 用 10 ml 血清移液管上下吸管五次。
  6. 通过 70 µm 细胞过滤器将消化的肌肉筛入新的 50 ml 管中。
  7. 通过 40 µm 细胞过滤器将消化的肌肉筛入新的 50 ml 管中。

注意:充分消化的肌肉将很容易通过过滤器。如有必要,肌肉消化物可以再次通过另一个过滤器,以去除大部分细胞碎片和未消化的肌肉。

  1. 在室温下以 300 × g 的速度将肌肉消化管离心5 分钟。
  2. 弃去上清液,将沉淀重悬在 10 ml DMEM 10% FBS 中。使用台盼蓝计算肌肉单核细胞的数量。

注意:在此步骤中,如果使用了 1 只以上的鼠标,您可以合并颗粒。平均而言,3、4-8 周龄小鼠消化后释放的细胞数量约为 10 7 个细胞。

  1. 第 1 天:预镀步骤(图 1e-1f)。
  1. 将细胞以 10 /ml接种在100 毫米培养皿(未包被)中,置于 20 毫升 DMEM 10% FBS 1% 笔/链球菌中,并在 37°C 下孵育 1 小时。
  2. 将未粘附的细胞收集在 50 ml 管中,并在室温下以 300 × g离心5 分钟。
  3. 丢弃上清液并在 DMEM 10% FBS 1% pen/strep 中重新悬浮细胞颗粒。
  4. 对重悬的细胞进行计数,并以 10 /ml 的浓度将它们接种到 100 毫米的培养皿(未包被)中,放入20 毫升的 DMEM 10% FBS 1% pen/strep 溶液中。将细胞在 37°C 下孵育 1 小时(如步骤 A5a)。 
  5. 收集并转移未粘附的细胞到 50 ml 管中。

注意:平均而言,预镀步骤会去除约 50% 的强贴壁细胞,例如成纤维细胞。

  1. 在 RT 下以 300 g将带有非粘附细胞的管子离心5 分钟。
  2. 丢弃上清液并将细胞沉淀重悬在生长培养基中(参见食谱)。
  3. 将细胞以 10 6 个细胞/盘放入0.1%明胶涂层的100毫米盘中。一夜之间种植它们(图 1g)。
  1. 第 2 天:冰冷处理 (ICT)(图 1h)。
  1. 在室温下用 10 ml PBS(含MgCl 2和 CaCl 清洗含有粘附肌肉细胞异质混合物的菜肴三次

注意:轻轻地在盘子周围吸取 PBS。

  1. 向每个 100 毫米的培养皿中加入 10 毫升冰冷的 PBS(不含MgCl 2和 CaCl 

注意:可以使用含或不含 CaCl 2 的PBS 然而,使用不含 CaCl 2 的PBS会促进分离。

  1. 用冰块 (0°C) 填充聚苯乙烯盒,将盘子放在冰上 15-30 分钟,偶尔轻轻手动摇动盘子(产生旋转运动)。

笔记:

  1. 如果纯度是最重要的(例如,99-100% 纯度),则盘子在冰上的放置时间不应超过 30 分钟。尽管如此,培养皿可以在冰上放置长达 1 小时以提高细胞产量,纯度仍高达 90%。
  2. 含有异质肌肉细胞的原始菜肴(从隔离程序的第 1 天开始)可以保留至培养的第 3 天,并用于进一步的 ICT 轮次。
  1. 将分离的细胞收集到 50 ml 管中。
  2. 用 10 ml PBS 再次清洗原始培养皿以收集剩余的分离细胞。

注意:轻轻地在盘子周围吸取 PBS。

  1. 在室温下以 300 g离心细胞5 分钟。
  2. 丢弃上清液并将细胞重新悬浮在生长培养基中。
  3. 计数细胞。

注意:在原始培养皿培养 24 小时后,在冰上放置 30 分钟后,平均可以收集1.5 × 10 -2 × 10 SCs。在培养 48 小时或 72 小时的第二次或第三次 ICT 后,可以进一步收集4 × 10 SCs。

  1. 将细胞板放入预涂有 0 的 35 毫米盘子中1% 明胶,密度为 10 3细胞/培养皿,并将它们放入细胞培养箱中。

注意:在此密度下,SC 最多可增殖三天。之后,他们开始分化。增加电镀密度将加快成肌细胞分化和融合成肌管所需的时间。

  1. 细胞培养
  1. 在生长培养基中培养细胞,每两天更换一次培养基。
  2. 为了诱导成肌细胞分化,一旦细胞达到 80% 汇合,去除生长培养基并用分化培养基替换它(参见食谱)。
  1. 细胞传代
  1. 将含有增殖 SCs 的菜肴放在冰上长达 30 分钟。
  2. 按照 A6 中描述的步骤进行操作。

 

人肌肉卫星细胞的分离

笔记:

  1. 只有在获得伦理批准和知情同意后才能进行使用人体肌肉活检的实验程序。
  2. 从人类肌肉活检中获得的 SCs 数量可能因供体的年龄而异,通常从老年供体那里获得的细胞数量较少。

 

  1. 肌肉解剖前的准备

按照步骤 A1 进行操作。

  1. 肌肉切割
  1. 将人体肌肉活检组织放入含有 DMEM 的 60 毫米盘中,并辅以 1% 笔/链球菌。生物安全柜中工作。
  2. 使用剪刀去除任何脂肪和结缔组织。
  3. 使用无菌手术刀将肌肉切成 1 毫米的碎片。
  4. 将切碎的肌肉转移到 50 ml 管中,并以 300 × g离心5 分钟。
  1. 肌肉消化I:

按照 A3 中的说明进行操作。

  1. 肌肉消化二:

按照 A4 中的说明进行操作。

注:平均而言,消化 1 g 人体肌肉活检组织可获得2 × 10 6肌肉单核细胞。

  1. 预镀:

按照 A5 中的步骤进行。

  1. 信息通信技术:

按照 A6 中的步骤进行。

为了在分离后的第 1 天进一步丰富 SCs 异质肌肉细胞培养物,可能需要在进行 ICT 之前将其再生长 3-4 天(当培养物达到约 80% 的汇合度时)。

笔记:

  1. 第一次 ICT 之后,平均可以收集1 × 10 4 个SC。
  2. 在培养 48 小时或 72 小时的第二次或第三次 ICT 后,可以再收集2 × 10 SCs。
  1. 细胞培养

按照步骤 A7 进行操作。

注意:人类 SCs 的增殖速度比小鼠 SCs 慢,倍增时间为 46 小时。

 

  1. 免疫荧光染色分析小鼠和人卫星细胞特异性标志物的表达
  1. 在 35 毫米培养皿中培养细胞。
  2. 吸出培养基并用 1 ml PBS 和MgCl 2和 CaCl 2洗盘子一次
  3. 通过添加 4% PFA(1 ml/35 mm 培养皿)固定细胞并在室温下孵育 10 分钟。

注意:PFA 是有毒的,必须在化学通风柜下使用。

  1. 将 PFA 丢弃到有毒废物容器中,用 1 ml PBS 洗涤 3 次,每次 5 分钟。
  2. 加入冷甲醇(1 ml/35 mm 培养皿)使细胞通透,并在 -20°C 下孵育6 分钟。 
  3. 将甲醇倒入有毒废物容器中,用 1 ml PBS 洗涤 3 次,每次 5 分钟。 
  4. 取出 PBS 并用疏水笔标记盘子的边界。
  5. 在 RT 中加入含有 5% 山羊血清(100 μl/35 mm 培养皿)的 PBS 封闭 30 分钟。
  6. 去除封闭液,用适当稀释度的一抗(100 μl/35 mm 培养皿)孵育细胞 [Pax7 (1:10)、MyoD (1:50)、Myogenin (1:20) 和 Myosin Heavy Chain (1:20) 稀释在无菌过滤的 4% BSA/PBS] 中,在 4°C 的加湿室中过夜。
  7. 去除一抗溶液,用 1 ml PBS 洗涤 3 次,每次 5 分钟。
  8. 用二抗(100 μl/35 mm 培养皿)[山羊抗兔 Alexa Fluor 488 (1:1,000) 和山羊抗小鼠 Alexa Fluor 555 (1:1,000) 用无菌过滤的 1% BSA/PBS 稀释细胞孵育细胞] 在室温下,在黑暗中的腐殖室中放置 1 小时。
  9. 去除二抗溶液,用 1 ml PBS 洗涤 3 次,每次 5 分钟。
  10. 用 Hoechst 溶液(见食谱)(100 μl/35 mm 培养皿)在室温下在黑暗中复染细胞核 5 分钟。
  11. 取出 Hoechst 溶液,用 1 ml PBS 洗涤 3 次,每次 5 分钟。
  12. 使用 Vectashield 安装介质(30 μl/35 mm 培养皿)安装盖玻片。
  13. 在荧光显微镜下分析。

 

数据分析

 

在明场显微镜下分析细胞形态(图 3A 和 4A)。为了分析 Pax7、MyoD、Myogenin 和 Myosin Heavy Chain (MHC) 等生肌标志物的表达,按照程序部分的描述对细胞进行染色(图 3B 和 3D)。ICT 隔离后,小鼠 SC 纯度高于 99%。SC 纯度通过 Pax7 和 MyoD 的免疫荧光染色评估,通过计算 ICT 后在生长培养基中培养第 3 天 Pax7 和/或 MyoD 呈阳性的 SCs 的百分比(图 3B 和 3C)(Benedetti,2021)。

  使用 ICT 方法分离的人类 SCs 的纯度在 ICT 后通常高于 97%。因为培养中的人类 SCs 迅速下调 Pax7 的表达,SC 纯度通过肌细胞生成素的免疫荧光染色评估,通过计算生长培养基中培养第 5 天的肌细胞生成素阳性 SCs 的百分比(图 4B 和 4C)(Benedetti等人。 , 2021)。通过使用抗肌球蛋白重链 (MHC) 抗体进行免疫荧光染色来分析人类 SCs 的分化潜能(图 4D)。

 

 

图 3. 使用 ICT 方法对小鼠肌肉 SCs 进行分离和表征。

A.小鼠肌肉细胞培养的代表性明场图像。SCs 通过 ICT 从异质单核细胞中分离出来。ICT 隔离的 SCs 在第 2 天 (D2) 和第 4 天 (D4) 在生长培养基 (GM) 中以及在添加分化培养基 (DM) 后的第 3 天 (D3)。B.针对 Pax7(红色)、MyoD(绿色)和细胞核(蓝色)染色的 ICT 隔离 SCs 的代表性免疫荧光图像。C.图表显示了在 ICT 后在生长培养基中培养的第 3 天 Pax7 和/或 MyoD 阳性细胞的百分比(n = 3 个独立实验)。D.生长培养基中四天后在 DM 中染色三天后,ICT 分离的 SCs 的代表性免疫荧光图像被染色为肌球蛋白重链 (MHC)(红色)和细胞核(蓝色)比例尺 = 100 μ米。误差棒代表平均值 ± SEM (Benedetti et al ., 2021)。

 

 

图 4. 使用 ICT 方法分离和表征人类肌肉 SCs (hSCs)。

A.在第 3 天 (D3) 和第 10 天 (D10) 在生长培养基 (GM) 中以及在添加分化培养基 (DM) 后的第 10 天,异质人肌肉单核细胞培养物和分离的 SCs 的代表性明场图像。B.针对肌细胞生成素(红色)和细胞核(蓝色)染色的人类 SCs 的代表性免疫荧光图像。C.图显示了在 GM 中培养第 5 天 ICT 后肌细胞生成素 + 细胞的百分比(n = 3 个独立实验)。D.GM 中10 天后,在 DM 中 10 天后,针对肌球蛋白重链 (MHC)(红色)和细胞核(蓝色)染色的人类 SC 的代表性免疫荧光图像比例尺 = 100 µm。误差棒代表平均值 ± SEM (Benedetti et al ., 2021)。

 

食谱

 

  1. 70%乙醇

将 70 ml 100% 乙醇加入 30 ml ddH O

在室温下储存

  1. II型胶原酶消化液

将 II 型胶原酶粉末溶解在含有 MgCl 2和 CaCl 2 的PBS 中,浓度为 0.4 毫克/毫升,每克组织总共溶解10 毫升。

  1. 胶原酶/分散酶消化液

将胶原酶/分散酶粉末溶解在 PBS MgCl 2和 CaCl 2 中,浓度为 1 毫克/毫升,每克组织总共需要 10 毫升。

  1. 生长培养基

用 20% 马血清、3%鸡胚提取物、1% 青霉素/链霉素和 1% L-谷氨酰胺补充 DMEM 培养基储存在 4°C。

  1. 分化培养基

用 5% 马血清、1%鸡胚提取物、1% 青霉素/链霉素和 1% L-谷氨酰胺补充 DMEM 培养基储存在 4°C。

  1. 中和缓冲液

用 10% FBS 和 1% 青霉素/链霉素补充 DMEM 培养基。

  1. 赫斯特解决方案

在 PBS 中以 1:1,000稀释 Hoechst 染色剂

 

致谢

 

这项工作得到了荷兰杜兴父项目 NL (DPP NL) 对 BLO 的资助;意大利父项目(PP,意大利)对 MB 和罗马大学的研究资助(RP11715C7D238352、RM118164275C7EBE 和 RM11916B7E20311C 到 MB,以及 AR11715C7F9E158E、AR1181516186E 到 AB )。该协议描述了 Benedetti等人发表的工作(2021)。

 

利益争夺

 

作者声明没有相互竞争的财务或非财务利益。

 

伦理

 

所有涉及小鼠的程序均经意大利卫生部批准,并根据欧盟法规和意大利动物研究法进行。伦理批准 ID:82945.19。有效期至 18/01/2023。

所有患者均知情同意接受术中肌肉活检并公布获得的临床和实验室数据。

 

参考

 

  1. Benedetti, A., Fiore, PF, Madaro, L., Lozanoska-Ochser, B. 和 Bouche, M. (2020)。靶向 PKCtheta 促进卫星细胞自我更新。 Int J Mol Sci 21(7): 2419。
  2. Benedetti, A.、Cera, G.、De Meo, D.、Villani, C.、Bouche, M. 和 Lozanoska-Ochser, B.(2021 年)。基于冰冷处理的纯卫星细胞分离和长期扩增的新方法。骨骼肌11(1):7。
  3. Blanco-Bose, WE, Yao, CC, Kramer, RH 和 Blau, HM (2001)。基于 α7 整合素表达的小鼠原代成肌细胞的纯化。Exp Cell Res 265(2): 212-220。
  4. Chang, NC 和 Rudnicki, MA (2014)。卫星细胞:骨骼肌的建筑师。 Curr Top Dev Biol 107:161-181。
  5. Chapman, MR, Balakrishnan, KR, Li, J., Conboy, MJ, Huang, H., Mohanty, SK, Jabart, E., Hack, J., Conboy, IM 和 Sohn, LL (2013)。从单个肌纤维中分选单个卫星细胞揭示了细胞表面标志物和肌生成能力的异质性。 Integr Biol (Camb) 5(4): 692-702。
  6. Danoviz, ME 和 Yablonka-Reuveni, Z. (2012)。骨骼肌卫星细胞:在原代培养系统中分离和分析的背景和方法。分子生物学方法798:21-52。
  7. Fiore, PF, Benedetti, A., Sandona, M., Madaro, L., De Bardi, M., Saccone, V., Puri, PL, Gargioli, C., Lozanoska-Ochser, B. 和 Bouche, M. (2020)。缺乏 PKCtheta 促进肌肉干细胞在慢性肌肉损伤中的再生能力。Int J Mol Sci 21(3):932。              
  8. Fukada, S., Higuchi, S., Segawa, M., Koda, K., Yamamoto, Y., Tsujikawa, K., Kohama, Y., Uezumi, A., Imamura, M., Miyagoe-Suzuki, Y 。等人(2004)。新型单克隆抗体对小鼠骨骼肌静止卫星细胞的纯化和细胞表面标志物表征。 Exp Cell Res 296(2):245-255。
  9. Gharaibeh, B.、Lu, A.、Tebbets, J.、Zheng, B.、Feduska, J.、Crisan, M.、Peault, B.、Cummins, J. 和 Huard, J. (2008)。通过预板技术从小鼠骨骼肌中分离出含有干细胞的缓慢粘附的细胞部分。国家协议3(9):1501-1509。 
  10. Keire, P.、Shearer, A.、Shefer, G. 和 Yablonka-Reuveni, Z.(2013 年)。骨骼肌肌纤维的分离和培养作为分析卫星细胞的一种手段。 方法 Mol Biol 946:431-468。
  11. Liu, L.、Cheung, TH、Charville, GW 和 Rando, TA (2015)。通过荧光激活细胞分选分离骨骼肌干细胞。 Nat Protoc 10(10): 1612-1624。
  12. Mauro, A. (1961)。骨骼肌纤维的卫星细胞J Biophys Biochem Cytol 9:493-495。
  13. Montarras, D.、Morgan, J.、Collins, C.、Relaix, F.、Zaffran, S.、Cumano, A.、Partridge, T. 和 Buckingham, M. (2005)。用于骨骼肌再生的卫星细胞的直接分离。科学309(5743):2064-2067。
  14. Pasut, A.、Oleynik, P. 和 Rudnicki, MA (2012) 通过荧光激活细胞分选细胞术分离肌肉干细胞。分子生物学方法798:53-64。
  15. Sherwood, RI, Christensen, JL, Conboy, IM, Conboy, MJ, Rando, TA, Weissman, IL 和 Wagers, AJ (2004)。成年小鼠成肌祖细胞的分离:骨骼肌内和移植细胞的功能异质性。 单元格119(4):543-554。
  16. Syverud, BC, Lee, JD, VanDusen, KW 和 Larkin, LM (2014)。用于骨骼肌组织工程的卫星细胞的分离和纯化。J Regen Med 3(2)。
  17. Wang, YX, Dumont, NA 和 Rudnicki, MA (2014)。肌肉干细胞一目了然J Cell Sci 127(第 21 篇):4543-4548。
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引用:Benedetti, A., Cera, G., De Meo, D., Villani, C., Bouche, M. and Lozanoska-Ochser, B. (2021). A Simple Method for the Isolation and in vitro Expansion of Highly Pure Mouse and Human Satellite Cells. Bio-protocol 11(23): e4238. DOI: 10.21769/BioProtoc.4238.
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