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Vascular Smooth Muscle Cell Isolation and Culture from Mouse Aorta
小鼠主动脉血管平滑肌细胞的分离和培养   

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Jia Li
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Abstract

Vascular smooth muscle cells (SMC) in the ascending thoracic aorta arise from neural crest cells, whereas SMCs in the descending aorta are derived from the presomitic mesoderm. SMCs play important roles in cardiovascular development and aortic aneurysm formation. This protocol describes the detailed process for explanting ascending and descending SMCs from mouse aortic tissue. Conditions for maintenance and subculture of isolated SMCs and characterization of the vascular SMC phenotype are also described.

Keywords: Tissue culture(组织培养), Smooth muscle cells(平滑肌细胞), Cell biology(细胞生物学)

Background

Vascular smooth muscle cells (SMCs) make up the muscular medial layer of arteries. Larger elastic arteries, such as the aorta, have multiple concentric lamellae consisting of aligned smooth muscle cells sandwiched between elastin fibers. The elastin and collagen present within the medial layer of elastic arteries allow it to distribute the force generated by the heart throughout the vessel wall (Wagenseil and Mecham, 2009). Smaller muscular arteries, by contrast, have only an internal and external elastic lamina bounding the smooth muscle layer. These arteries are downstream in the arterial tree and thus bear less force from blood flow.

Vascular smooth muscle cells, unlike cardiac and skeletal muscle cells, are capable of modulating their phenotype in response to vascular injury or environmental cues. Under normal physiologic conditions, quiescent, contractile SMCs populate the artery wall and contract to regulate vascular tone and keep blood flow continuous in response to pulsatile pressures. Contractile SMCs are characterized by high expression of smooth muscle-specific contractile genes, including smooth muscle specific α-actin and myosin heavy chain (Owens et al., 2004). However, in response to injury or mitogenic stimuli, SMCs downregulate expression of the contractile genes and take on a synthetic phenotype: they proliferate rapidly, migrate into the site of injury, and remodel the extracellular matrix by synthesizing both matrix-digesting enzymes and new matrix proteins. Many vascular diseases are associated with a synthetic SMC phenotype, including atherosclerosis (Owens, 1995).

SMCs located in different areas of the body actually arise from diverse embryonic lineages (Majesky, 2007). For example, the SMCs populating the ascending thoracic aorta and the cerebrovasculature are derived from neural crest cells. However, SMCs in the descending thoracic aorta come from mesodermal origins. These distinctions affect the ultimate properties of the SMCs, so it is important when designing experiments to use SMCs from the same developmental origin where the phenotype of interest arises.

In this protocol, we give detailed instructions for isolating and culturing SMCs from the ascending and descending thoracic aortas in the mouse. We have previously used this technique to isolate SMCs from genetically modified mice and their wild-type littermates to provide an in vitro system for looking at the effect of genetic changes on SMC phenotype (Cao et al., 2010; Kuang et al., 2012; Kuang et al., 2016; Papke et al., 2013; Kwartler et al., 2014).

Materials and Reagents

  1. For initial explant
    1. Dissection material: 2 sterile forceps, 2 sterile tweezers, 1 sterile scissor, 2 sterile scalpels
    2. Disposable scalpels (Aspen Surgical, catalog number: 371621 )
    3. Four 60 mm tissue culture dishes per sample (Corning, catalog number: 430589 )
    4. Syringe, 10 ml
    5. Syringe filter , 0.22 µm pore
    6. 500 ml filtration unit, 0.22 µm pore (EMD Millipore, catalog number: SCGPU01RE )
    7. Parafilm
    8. Aluminum foil
    9. 2.5% avertin-2,2,2-tribromoethanol (Sigma-Aldrich, catalog number: T48402-25g ) arrives as powder, make 100% stock solution by dissolved 25 g 2,2,2-tribromoethanol in 25 ml of 2-methyl-2-butanol (Sigma-Aldrich, catalog number: 240486-100ML ), then dilute to 2.5% solution in sterile water. Both the stock solution and the dilution are light sensitive, so should be stored in the dark at 4 °C
    10. 70% ethanol
    11. Dulbecco’s phosphate buffered saline (DPBS) with calcium and magnesium (GE Healthcare, HycloneTM, catalog number: SH30028.02 )
    12. Components of aorta biopsy storage media:
      1. Waymouth’s MB 752/1 medium, 500 ml (Thermo Fisher Scientific, GibcoTM, catalog number: 11220035 )
      2. Antibiotic-antimycotic, 100x (Sigma-Aldrich, catalog number: A5955 )
      3. L-glutamine, 100x (Sigma-Aldrich, catalog number: G7513 )
      4. Sodium bicarbonate (Sigma-Aldrich, catalog number: S8761 )
      5. MEM non-essential amino acids (Sigma-Aldrich, catalog number: M7145 )
      6. HEPES buffer (Sigma-Aldrich, catalog number: H0887 )
    13. Collagenase type 1 (Sigma-Aldrich, catalog number: C1639 )
    14. Elastase, Pancreatic type 1 from porcine pancreas (Sigma-Aldrich, catalog number: E1250 )
    15. Soybean trypsin inhibitor (Thermo Fisher Scientific, GibcoTM, catalog number: 17075-029 )
    16. Heat inactivated fetal bovine serum (FBS) (Atlanta Biologicals)
    17. SmBm bullet kit (Lonza, catalog number: CC3182 )
      1. FGF
      2. EGF
      3. Insulin
      4. Gentamicin and included FBS - Do not use!
  2. For continued culture
    1. 500 ml filtration unit, 0.22 µm pore (EMD Millipore, catalog number: SCGPU01RE )
    2. Freeze vials (2 ml)
    3. Fetal bovine serum (FBS, Atlanta Biologicals)
    4. Antibiotic-antimycotic, 100x (Sigma-Aldrich, catalog number: A5955 )
    5. SmBm bullet kit (Lonza, catalog number: CC3182 )
      1. FGF
      2. EGF
      3. Insulin
      Note: This kit includes a tube of gentamicin and a 25 ml aliquot of FBS that are not needed for any step of this protocol. Please do not use them! For more information, see ‘Preparation of Reagents’ below for the preparation of SmBm complete medium, which does not use the gentamicin or the provided FBS.
    6. TrypLE express (Thermo Fisher Scientific, GibcoTM, catalog number: 12604013 )
    7. DMSO
  3. For immunofluorescence to confirm SMC identity
    1. Coverslips (UV treated, please see Data analysis section below for details)
    2. 6 well plates
    3. Hemocytometer
    4. 3-6 mice, preferably aged 4-6 weeks old
    5. SmBm basal media (Lonza, catalog number: CC3181 )
    6. Fetal bovine serum (FBS) (Atlanta Biologicals)
    7. Recombinant human TGF-β1 (rhTGF- β1, R&D Systems)
    8. 16% formaldehyde (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 28906 ). Dilute 1 to 4 to a final concentration of 4% in DPBS with calcium and magnesium
    9. Blocking buffer (0.3% Triton X, 0.5% BSA in DPBS)
    10. DPBS
    11. Smooth muscle α-actin antibody (Sigma-Aldrich, catalog number: A5228 )
    12. Anti-mouse secondary antibody conjugated with fluorescence
    13. Mounting medium with DAPI (Vectashield, catalog number: H-1200 )
  4. Aorta biopsy storage media (see Recipes)
  5. Complete smooth muscle media (see Recipes)
  6. Smooth muscle cell freeze media (see Recipes)
  7. Digestive enzyme mix (see Recipes)

Equipment

Note: The reproducibility of the experiment is not dependent on the specific brand or model of these equipment. Any microscope, tissue culture hood/incubator, centrifuge, etc., will give similar results.

  1. Magnetic stirrer
  2. Dissecting microscope
  3. Sterile tissue culture hood
  4. Sterile tissue culture incubator
  5. 10 ml pipet
  6. Water bath
  7. Tabletop centrifuge
  8. 200 µl pipette
  9. T25 flasks
  10. T75 flasks
  11. Slow freeze container (either a foam container or an isopropanol based container will work)
  12. Hemocytometer
  13. Inverted microscope
  14. Hot bead sterilizer

Procedure

  1. Preparation of reagents
    1. To make aorta biopsy storage media, add the following to the bottle of 500 ml. Waymouth’s MB 752/1 medium.
      1. 5 ml of antibiotic/antimycotic
      2. 6.25 ml of L-glutamine
      3. 15 ml of sodium bicarbonate
      4. 5 ml of MEM non-essential amino acids
      5. 5 ml of HEPES buffer
      Note: Please note that we do not remove any of the basal media to compensate for the addition of the additives. The final volume of aorta biopsy storage media will therefore be 536.25 ml. The media can be stored at 4 °C for up to 6 months or until the expiration date from the manufacturer on the bottle, whichever is sooner.
    2. Preparation of avertin solution
      1. Add 15.5 ml tert-amyl alcohol to 25 g avertin (2-2-2-tribromoethanol) and stir the solution on a magnetic stirrer until avertin is completely dissolved. This will probably take overnight. The stock should be stored in a dark bottle at room temperature and capped tightly.
      2. Combine 0.5 ml avertin stock and 39.5 ml normal saline or PBS in a 50 ml graduated cylinder.
      3. Drop in magnetic stir bar, seal graduate cylinder with Parafilm and completely wrap cylinder with aluminum foil to exclude all light.
      4. Stir overnight to dissolve.
      5. Filter the working solution through 0.2 micron filter into a dark bottle. Keep working solution at 4 °C.
      6. Stock and working solution containers will be properly labeled (name, date made, concentration and initials of lab member).
      7. Stock solution should be discarded after four months, and working solution should be discarded after 2 weeks.
      8. All solutions should not be used if a precipitate or discoloration is detected.
    3. To make complete smooth muscle media (SmBm complete), filter the following through a 0.22 µm vacuum filter and add to a bottle of 500 ml SmBm basal media from Lonza. This media can be stored at 4 °C for up to 2 months or until the expiration date from the manufacturer on the bottle, whichever is sooner. It is important to note that the aliquot of gentamicin contained in the SmBm bullet kit should not be added.
      1. 100 ml FBS
      2. Aliquot of FGF contained in the bullet kit (1 ml).
      3. Aliquot of EGF contained in the bullet kit (0.5 ml).
      4. Aliquot of insulin contained in the bullet kit (0.5 ml).
      5. 5 ml of antibiotic/antimycotic
      Note: We do not remove any of the basal media to compensate for the addition of the additives. The final volume of SmBm complete will therefore be 607 ml.
    4. Preparation of digestive enzymes and other reagents
      1. Thaw and filter one 500 ml bottle of FBS and store at 4 °C in 50 ml aliquots.
      2. To make smooth muscle cell freeze media, combine 45 ml of SmBm complete with 5 ml filtered FBS and 5 ml sterile DMSO. 
      3. Collagenase type 1 is received as a powder. The final concentration needed for a 3-4 h digestion is 1.00 mg/ml. To prepare the stock solution, add 2.5 ml aorta biopsy storage media to re-suspend the 50 mg collagenase. Mix well. Make 500 µl aliquots and store at -20 °C.
      4. Soybean trypsin inhibitor is received as a powder. The final concentration needed for a 3-4 h digestion is 0.25 mg/ml. To prepare the stock solution, take 2.5 mg and dissolve in 10 ml aorta biopsy storage media, then make 10 aliquots, 1 ml/aliquots
      5. Elastase type 1 is received as a liquid. The bottle can be stored at 4 °C. Do not aliquot as the reagent is not stable when aliquoted and stored at -20 °C. The final concentration needed for a 3-4 h digestion is 0.1875 mg/ml.

  2. Dissection of the mouse to remove aortic tissue
    1. Anesthetize the experimental mice (3-6 mice/explant) by intraperitoneal (IP) injection with 2.5% avertin. Use 0.5 ml of solution for each mouse. To determine when anesthesia is effective, pinch the toe of the mouse hard between your nails. If the mouse kicks or reacts at all, the anesthesia is incomplete. Wait a couple of additional minutes and test again until there is no response to the toe pinch. If anesthesia is still incomplete after 5-7 min you can dose the mouse again with an additional 0.3 ml of avertin.
    2. Place the mouse under a light source for dissection.
    3. Cut the skin of the mouse from the abdomen to the top of the thorax.
    4. Lift the sternum with tweezers and cut the diaphragm. Then cut away the lower part of the ribcage to expose the heart, remove the lungs and thymus, and cut away the esophagus, expose the aorta running along the spine. See Figure 1 and Video 1.


      Figure 1. Mouse thoracic cavity. Picture shows the mouse after chest has been opened. Blue indicates the lung tissue and green the thymus tissue which must both be removed to give better access to the heart and aorta.

      Video 1. Opening of mouse thoracic cavity and removal of tissues. This video shows the first part of the dissection from the opening of the mouse thoracic cavity to the removal of the lungs and trachea.

    5. Place the mouse under a dissecting microscope.
    6. Remove the remaining tissues and use microdissection scissors or forceps to remove all of the fat around the top of the heart, the ascending aorta, and its branches. See Figure 2.


      Figure 2. Exposed heart and descending aorta in a mouse. Picture shows the mouse after chest has been opened and lungs, thymus, and other tissues have been removed. The red arrow indicates the heart, which should be pulled up slightly to expose the ascending aorta and carotid branches. The red arrowhead indicated the descending aorta which should be clipped below the diaphragm.

    7. Gently pull up on the heart with forceps, without breaking the ascending aorta. Use forceps or scissors to gently separate the descending aorta from the spine. Clip the descending aorta just below the diaphragm. Clip the carotid arteries to release the ascending aorta. See Video 2. Put the heart, ascending, and descending aortas which are all connected in a 60 mm dish with sterile DPBS.

      Video 2. Removal of mouse heart and aorta. This video shows the second part of the dissection: removing the heart and aorta.

    8. Place the dish under the dissecting microscope and remove any fatty or connective tissues from the ascending and descending aorta. See Figure 3. Separate the ascending and descending aorta by making a cut just distal to the origin of the left common carotid artery. Put ascending and descending aorta separately in new dishes with DPBS labeled according to the region of the aorta and the genotype of the mouse.
    9. Working quickly but carefully, repeat for each experimental mouse until all the tissue has been separated. Pool ascending aortas and descending aortas for each explant.
    10. Move all the dishes with aortic tissue to the tissue culture hood.


      Figure 3. Appearance of the dissected aorta. Whole mount image showing the aortic tissue after it has been removed from the mouse and completely cleaned. Ascending aorta is marked by a red arrow.

  3. Explanting of the tissue
    Note: Please note that all steps for the rest of the protocol should be carried out under a tissue culture hood using standard sterile tissue culture techniques.
    1. Prepare the enzyme mix
      1. Thaw one aliquot each of collagenase and trypsin inhibitor at 4 °C before beginning the dissection of the mice.
      2. For each explant, use 5 ml of aorta biopsy storage media. If you are doing a 3-4 h digestion during the day, add 250 µl collagenase, 100 µl trypsin inhibitor, and 324 µl* elastase for each 5 ml of aorta biopsy storage media. For an overnight (15-18 h) digestion, use 1:10 dilution so 25 µl collagenase, 10 µl trypsin inhibitor, and 32.4 µl* elastase.
        *Note: The amount of elastase to add was calculated based on the mass and volume of the bottle. If you order elastase from a different source, you will need to recalculate the amount of this enzyme to add.
    2. Pour 70% ethanol, DPBS, and aorta biopsy storage media into three separate 60 mm tissue culture dishes.
    3. Using forceps, take each isolated aorta from its storage tube and wash the piece of tissue in ethanol, then DPBS, then biopsy media. Do not leave the tissue in ethanol for more than about 10-15 sec, though leaving it in DPBS or media for longer is OK.
    4. Place each piece of tissue into the lid of a 60 mm tissue culture dish. Using a scalpel and forceps, cut tissue into small pieces-make sure to cut the tissue both circumferentially and longitudinally to increase the surface area exposed to the digestion mix. The pieces will be variable in size, but the average size should be 1-2 mm in diameter.
    5. Place the chopped up tissue pieces into a new 60 mm tissue culture dish containing the 5 ml of media plus digestive enzyme mix made up in step C1. Label the dish.
    6. Place dish into incubator (37 °C). If doing a 3-4 h digestion, check at 2 h and every 30 min thereafter. If doing an overnight digestion, leave overnight, check at 15 h and every hour thereafter.
    7. When the tissue looks mostly digested with several floating single cells, but still some large clumps of tissue, pipet up and down tissue and media 5-10 times using a 10 ml pipet to help release cells from tissue. Place back into incubator for 30 more minutes. Also, remove filtered FBS from fridge to warm up to room temperature and place complete SmBM media into water bath to warm up to 37 °C.
    8. At the end of incubation time, look at tissue under microscope to make sure that the cells are free from the tissue. See Figure 4. If the tissue has been well digested (there should still be some intact pieces of tissue), add 2.5 ml of Serum and pipet up and down ~5 times. Next add 2.5 ml of complete SmBM media and pipet up and down ~5 times.


      Figure 4. Appearance of the digested tissue. Photo of the dish after digestion, with some ‘net-like’ tissue floating in it (black arrow) but a majority of tissue pieces digested.

    9. Transfer the entire contents of the tissue culture dish (should be 10 ml total) into one 15 ml tube.
    10. Spin down at 180 x g (about 700 rpm in a Sorvall Legend T+ tabletop centrifuge) at room temperature for 5 min.
    11. Carefully check to see if there is any floating tissue piece in the tube. If so, use a 200 µl pipette to remove the tissue piece and place it directly into a labeled T25 flask with complete SmBm. Once there are no tissue pieces floating, remove supernatant and resuspend the pellet in 5 ml of complete SmBM media.
      Note: If the supernatant was cloudy, repeat steps C10 and C11 until the supernatant is clear. If the supernatant was clear, proceed to step C12.
    12. Transfer the 5 ml complete SmBm with resuspended cells to a labeled T25 flask. Place flask in incubator.
    13. Allow cells to settle and adhere to the flask. Don’t change media for at least 48 h. If cells still haven’t settled down at this point, don’t remove any media and add 2 ml of media to the flask. Let the cells settle for another 1 to 2 days.

  4. Maintenance and subculture of mouse vascular smooth muscle cells.
    1. Once SMCs grow up, feed the cells with fresh media every 3-4 days.
      1. Prewarm the SmBm complete media bottle in a 37 °C water bath for at least 15 min prior to feeding cells.
      2. Aspirate media from each flask and discard.
      3. Add 5 ml of media for each T25 flask without disturbing the cell layer.
      4. Return flasks to the incubator.
    2. When the cells reach about 70% confluent, or when some areas start to get very tightly packed and almost overgrown, the cells are ready to split. See Figure 5. Passage the SMCs from one T25 flask to one T75 flask.
      1. Prewarm the SmBm complete media bottle in a 37 °C water bath and allow the TrypLE Express reagent to equilibrate at room temperature for at least 15 min prior to splitting cells.
      2. Aspirate media from each flask and discard.
      3. Wash each flask once with 4 ml of DPBS, allow it to cover the cell layer, then aspirate and discard the DPBS.
      4. Add 1 ml TrypLE Express reagent to each flask. Make sure the trypsin is evenly covering the bottom of the flask/cell layer.
      5. Place the flask in the 37 °C incubator for 3 min.
      6. Confirm digestion under microscope. If most cells are round, bang the flask twice on both sides. If most cells are still attached, incubate at room temperature for 1 min and then confirm digestion and bang the flasks twice on both sides.
      7. Add 4 ml of SmBm complete to each flask to neutralize the trypsin enzyme. Pipet up and down to detach all the cells and completely neutralize the enzyme.
      8. Transfer all 5 ml of liquid plus cells to a 15 ml conical tube.
      9. Spin down 5 min at 180 x g in a tabletop centrifuge at room temperature.
      10. Aspirate supernatant, resuspend cell pellet in 10 ml of SmBm complete, and transfer all 10 ml to a labeled T75 flask. Mark the date of split and the passage number (P1 for this first passaging).


        Figure 5. Appearance of confluent SMCs. The left picture shows a 100% confluent field of smooth muscle cells. These cells need to be passaged within 24 h. The right panel shows smooth muscle cells around 70% confluent. Note the spindle-shaped morphology which is characteristic of SMC cultures.

    3. When the SMCs have expanded, the cell aliquots can be frozen. Follow steps D2a-D2i in the above protocol for passaging SMCs. One T75 flask can be split into 2 frozen cell aliquots. A confluent T75 flask should have around 2 million cells, so each freeze vial will have around 1 million cells. After spinning down, follow the below steps to freeze cells.
      1. Aspirate supernatant, resuspend cell pellet in 3 ml SMC freeze media (see Recipes).
      2. Transfer 1.5 ml of the freeze media with dissolved pellet to each of two cryovials. Cryovials should be labeled with the passage number, date of freezing, and cell line.
      3. Place cryovials into a slow freeze container (either a foam container or an isopropanol based container will work) and freeze in the -80 °C freezer overnight.
      4. Transfer the frozen aliquots to a liquid nitrogen storage system for long-term storage.
    4. When you want to thaw your SMC aliquots, a vial of frozen cells can typically be thawed to one T75 flask.
      1. Prewarm SmBm complete media in the 37 °C water bath for at least 15 min prior to removing cells.
      2. Retrieve the cell vial from the liquid nitrogen freezer. Warm quickly by placing the cryovials in the 37 °C water bath for about 1-2 min.
      3. Add 8 ml of SmBm complete to a 15 ml conical tube. Then transfer the 1.5 ml of thawed cells into the same conical tube.
      4. Spin down for 5 min at 180 x g in a tabletop centrifuge at room temperature.
      5. Remove and discard supernatant, resuspend the cell pellet in 10 ml of SmBm complete and transfer to a T75 flask.

Data analysis

  1. Characterization of SMC phenotype
    In order to confirm the identity of the SMCs, perform immunofluorescence for smooth muscle α-actin, which should be visible in filaments in a differentiated SMC. When the cells are 80% confluent, they are ready to plate. An hour before beginning to work with the cells, place coverslips into a 6-well plate (one coverslip per well, as many as you want to plate, for this experiment we recommend at least two) under the cell culture hood. Turn on the UV in the hood for at least 30 min to sterilize the coverslips. Then to begin, follow steps D2a-D2i for passaging cells above. After spinning down, aspirate supernatant and resuspend the cell pellet in a smaller volume-typically 2 ml of SmBm complete.
    1. Use 10 µl of the resuspended cell mixture to count cells in a hemocytometer.
    2. Calculate the volume needed for 5,000 cells. Add the calculated volume of cell mixture to the well containing the coverslip and add additional SmBm complete media to each well up to 2 ml volume. Any remaining cells can be used for other experiments or put back into flasks as desired.
    3. 18-24 h after plating the cells, change media to low serum SmBm media, which drives differentiation of vascular SMCs. This is SmBM basal media with only 1% FBS and 1x antibiotic/antimycotic added (for 500 ml bottle of SmBm basal media, add 5 ml FBS and 5 ml of 100x antibiotic/antimycotic). Leave low serum SmBm on your cells at least 24 h.
    4. If desired, you can treat some coverslips with rhTGF-β1 to further induce differentiation. 24 h after switching to low serum SmBm media, add 10 ng/ml rhTGF-β1 to low serum SmBm media and add this media to the coverslips. Leave rhTGF-β1 containing media on the cells for 48 h.
    5. When the treatment is done, remove media, wash the cells once with DPBS, and add 1 ml of 4% paraformaldehyde solution to each well to fix the cells. Store at 4 °C until the cells are ready to stain.
    6. For staining, use 50 µl droplets of liquid for each coverslip on Parafilm and invert the coverslip onto the droplet to save antibody. First, incubate the coverslips in blocking buffer for 1 h (blocking buffer is 0.3% Triton-X, 0.5% BSA in DPBS).
    7. Wash coverslips 3 times 5 min in DPBS
    8. Incubate coverslips in 1:500 anti-smooth muscle α-actin antibody made up in blocking buffer at 4 °C overnight.
    9. Wash coverslips 3 times 5 min in DPBS.
    10. Incubate coverslips in 1:500 fluorescent-conjugated anti-mouse IgG secondary antibody made up in blocking buffer for 3 h at room temperature.
    11. Wash coverslips 3 times 5 min in DPBS
    12. Mount coverslips onto microscope slides with mounting media with DAPI (nuclear counterstaining, Vectashield).
    13. Use nail polish or other desired sealant to seal coverslip to the slide.
    14. Assess staining using desired fluorescent microscope. At least 95% of the cells should have smooth muscle α-actin filaments visible in the cells (Figure 6).


      Figure 6. Actin filaments in isolated smooth muscle cells. Immunofluorescence staining for SM α-actin (green) shows polymerized filaments of SM α-actin. Nuclei are counterstained with DAPI (blue).

Notes

  1. It is critical to correctly time the ethanol wash step (step C3 under ‘Explant’ protocol above). Too long a time in the ethanol will kill the cells and dramatically lower success. Too short a time in the ethanol wash may lead to bacterial contamination of the sample.
  2. Although the product numbers for the digestive enzymes listed above have been extensively tested in our hands, we have also had success with other versions of Collagenase and Elastase. The most critical factor is the concentration of active enzyme and the time of digestion. Any alternate enzyme will need to be tested for optimal time of digestion.
  3. Typically, in our hands primary smooth muscle cells retain their phenotype in culture for at least six passages. We always use low passage cells (< P6) for our experiments. However, the cells may be usable beyond passage 6 but we recommend assessing the smooth muscle cell phenotype of higher passage cells before use. Phenotype can be assessed by using the staining protocol described above and/or by expression of smooth muscle myosin heavy chain, encoded by Myh11, which is the most specific marker for differentiated smooth muscle cells.

Recipes

  1. Aorta biopsy storage media
    500 ml of Waymouth’s MB 752/1 medium
    5 ml of 100x antibiotic-antimycotic
    6.25 ml of L-glutamine
    15 ml of sodium bicarbonate
    5 ml of MEM non-essential amino acids
    5 ml of HEPES buffer
  2. Complete smooth muscle media
    500 ml of SmBm basal media
    100 ml of FBS
    1 ml of FGF contained in the bullet kit
    0.5 ml of EGF contained in the bullet kit
    0.5 ml of insulin contained in the bullet kit
    5 ml of antibiotic/antimycotic
  3. Smooth muscle cell freeze media
    45 ml of complete smooth muscle media
    5 ml of filtered FBS
    5 ml of sterile DMSO
  4. Digestive enzyme mix (for 3-4 h digestion time)
    5 ml aorta biopsy storage media
    250 μl collagenase
    100 μl trypsin inhibitor
    324 μl elastase

Acknowledgments

This work was supported by National Institutes of Health: NIH (RO1 HL62594 and P01HL110869-01), John Ritter Foundation, Vivian L. Smith Foundation, and Ehlers Danlos Syndrome Network CARES.
The method was published in Kuang et al. (2016) and it is an adaptation of the methods used in Gordon et al. (1986) and Lemire et al. (1994). Cells explanted using this method were also utilized in the following publications: Cao et al. (2010), Kuang et al. (2012), Kwartler et al. (2014), Papke et al. (2013).

References

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  8. Owens, G. K. (1995). Regulation of differentiation of vascular smooth muscle cells. Physiol Rev 75(3): 487-517.
  9. Owens, G. K., Kumar, M. S. and Wamhoff, B. R. (2004). Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 84(3): 767-801.
  10. Papke, C. L., Cao, J., Kwartler, C. S., Villamizar, C., Byanova, K. L., Lim, S. M., Sreenivasappa, H., Fischer, G., Pham, J., Rees, M., Wang, M., Chaponnier, C., Gabbiani, G., Khakoo, A. Y., Chandra, J., Trache, A., Zimmer, W. and Milewicz, D. M. (2013). Smooth muscle hyperplasia due to loss of smooth muscle α-actin is driven by activation of focal adhesion kinase, altered p53 localization and increased levels of platelet-derived growth factor receptor-β. Hum Mol Genet 22(15): 3132-27.
  11. Wagenseil, J. E. and Mecham, R. P. (2009). Vascular extracellular matrix and arterial mechanics. Physiol Rev 89(3): 957-989.

简介

在升主动脉中的血管平滑肌细胞(SMC)产生于神经嵴细胞,而降主动脉中的SMC来源于presomitic中胚层。 SMC在心血管发育和主动脉瘤形成中起重要作用。该协议描述了从小鼠主动脉组织移出升序和降序的SMC的详细过程。还描述了分离的SMC的维持和传代培养的条件和血管SMC表型的表征。
关键字:组织培养,平滑肌细胞,细胞生物学

[背景] 血管平滑肌细胞肌肉内侧动脉层。较大的弹性动脉,例如主动脉,具有由夹在弹性蛋白纤维之间的对齐的平滑肌细胞组成的多个同心薄片。存在于弹性动脉的内侧层中的弹性蛋白和胶原允许它分布由心脏在整个血管壁产生的力(Wagenseil和Mecham,2009)。相比之下,较小的肌肉动脉仅具有限制平滑肌层的内部和外部弹性层。这些动脉位于动脉树的下游,因此受到血流的作用力较小。
   血管平滑肌细胞,不像心脏和骨骼肌细胞,能够调节其表型响应血管损伤或环境线索。在正常生理条件下,静息的,可收缩的SMC填充动脉壁并收缩以调节血管张力并保持血流连续以响应脉动压力。收缩性SMC的特征在于平滑肌特异性收缩基因的高表达,包括平滑肌特异性α-肌动蛋白和肌球蛋白重链(Owens等人,2004)。然而,响应于损伤或促有丝分裂刺激,SMC下调收缩基因的表达,并采取合成表型:它们迅速增殖,迁移到损伤部位,并通过合成基质消化酶和新基质重塑细胞外基质蛋白质。许多血管疾病与合成的SMC表型相关,包括动脉粥样硬化(Owens,1995)。
   位于身体不同区域的SMC实际上源自多种胚胎谱系(Majesky,2007)。例如,填充升胸胸主动脉和脑血管系统的SMC源自神经嵴细胞。然而,下行胸主动脉中的SMC来自中胚层起源。这些区别影响SMC的最终性质,因此当设计实验以使用来自相同发育起源的SMC时,其中感兴趣的表型出现是重要的。
 在本协议中,我们给出详细的说明,从小鼠的升主动脉和下行主动脉中分离和培养SMC。我们先前使用这种技术从遗传修饰的小鼠和它们的野生型同窝幼仔分离SMC,以提供用于观察遗传变化对SMC表型的影响的体外系统(Cao等人,2010; Kuang等人,2012; Kuang等人,2016; Papke等人,2013; Kwartler等人,2014)。

关键字:组织培养, 平滑肌细胞, 细胞生物学

材料和试剂

  1. 对于初始外植体
    1. 解剖材料:2无菌镊子,2无菌镊子,1无菌剪刀,2无菌手术刀
    2. 一次性手术刀(Aspen Surgical,目录号:371621)
    3. 每个样品四个60mm组织培养皿(Corning,目录号:430589)
    4. 注射器,10ml
    5. 注射器过滤器,0.22μm孔
    6. 500ml过滤单元,0.22μm孔(EMD Millipore,目录号:SCGPU01RE)
    7. parafilm
    8. 铝箔
    9. 2.5%的avertin-2,2,2-三溴乙醇(Sigma-Aldrich,目录号:T48402-25g)以粉末形式,通过将25g 2,2,2-三溴乙醇溶解在25ml的2-甲基-2-丁醇(Sigma-Aldrich,目录号:240486-100ML),然后稀释至2.5%的无菌水溶液。储备溶液和稀释液都对光敏感,因此应在4℃的黑暗中储存。
    10. 70%乙醇
    11. 含有钙和镁的Dulbecco's磷酸盐缓冲盐水(DPBS)(GE Healthcare,Hyclone ,目录号:SH30028.02)
    12. 主动脉活检储存介质组分:
      1. Waymouth's MB 752/1培养基,500ml(Thermo Fisher Scientific,Gibco TM ,目录号:11220035)
      2. 抗生素 - 抗真菌的100x(Sigma-Aldrich,目录号:A5955)
      3. L-谷氨酰胺,100x(Sigma-Aldrich,目录号:G7513)
      4. 碳酸氢钠(Sigma-Aldrich,目录号:S8761)
      5. MEM非必需氨基酸(Sigma-Aldrich,目录号:M7145)
      6. HEPES缓冲液(Sigma-Aldrich,目录号:H0887)
    13. 1型胶原酶(Sigma-Aldrich,目录号:C1639)
    14. 弹性蛋白酶,来自猪胰腺的1型胰腺(Sigma-Aldrich,目录号:E1250)
    15. 大豆胰蛋白酶抑制剂(Thermo Fisher Scientific,GibcoTM,目录号:17075-029)
    16. 热灭活的胎牛血清(FBS)(Atlanta Biologicals)
    17. SmBm子弹试剂盒(Lonza,目录号:CC3182)
      1. FGF
      2. EGF
      3. 胰岛素
      4. 庆大霉素和包括FBS - 不要使用!
  2. 继续培养
    1. 500ml过滤单元,0.22μm孔(EMD Millipore,目录号:SCGPU01RE)
    2. 冷冻小瓶(2 ml)
    3. 胎牛血清(FBS,Atlanta Biologicals)
    4. 抗生素 - 抗真菌的100x(Sigma-Aldrich,目录号:A5955)
    5. SmBm子弹试剂盒(Lonza,目录号:CC3182)
      1. FGF
      2. EGF
      3. 胰岛素
      注意:该试剂盒包括一管庆大霉素和25毫升FBS的等分试样,这是本协议的任何步骤不需要。请不要使用它们!有关更多信息,请参阅下面的"制备试剂",了解不使用庆大霉素或提供的FBS的SmBm完全培养基的制备。
    6. TrypLE express(Thermo Fisher Scientific,Gibco TM ,目录号:12604013)
    7. DMSO
  3. 用于免疫荧光以证实SMC身份
    1. 盖玻片(紫外线处理,详情请参阅下面的数据分析部分)
    2. 6孔板
    3. 血细胞计数器
    4. 3-6只小鼠,优选4-6周龄的
    5. SmBm基础培养基(Lonza,目录号:CC3181)
    6. 胎牛血清(FBS)(Atlanta Biologicals)
    7. 重组人TGF-β1(rhTGF-β1,R& D Systems)
    8. 16%甲醛(Thermo Fisher Scientific,Thermo Scientific ,目录号:28906)。在含有钙和镁的DPBS中稀释1至4,最终浓度为4%
    9. 封闭缓冲液(0.3%Triton X,0.5%BSA的DPBS溶液)
    10. DPBS
    11. 平滑肌α-肌动蛋白抗体(Sigma-Aldrich,目录号:A5228)
    12. 抗小鼠第二抗体与荧光结合
    13. 使用DAPI(Vectashield,目录号:H-1200)
  4. 主动脉活检储存介质(见配方)
  5. 完成平滑肌介质(见配方)
  6. 平滑肌细胞冷冻介质(见配方)
  7. 消化酶混合物(见配方)

设备

注意:实验的再现性不取决于这些设备的具体品牌或型号。任何显微镜,组织培养罩/孵化器,离心机等,将给出类似的结果。

  1. 磁力搅拌器
  2. 解剖显微镜
  3. 无菌组织培养罩
  4. 无菌组织培养孵化器
  5. 10ml移液管
  6. 水浴
  7. 台式离心机
  8. 200μl移液器
  9. T25烧瓶
  10. T75瓶
  11. 缓慢冷冻容器(泡沫容器或异丙醇容器将工作)
  12. 血细胞计数器
  13. 倒置显微镜
  14. 热珠灭菌器

程序

  1. 试剂的制备
    1. 要制作主动脉活检储存介质,加入以下瓶500毫升。 Waymouth的MB 752/1中等。
      1. 5ml抗生素/抗真菌剂
      2. 6.25ml L-谷氨酰胺
      3. 15ml碳酸氢钠
      4. 5ml MEM非必需氨基酸
      5. 5ml HEPES缓冲液
      注意:请注意,我们不会删除任何基础培养基以补偿添加剂的添加。因此,主动脉活检储存介质的最终体积为536.25ml。介质可以在4°C下储存长达6个月,或者直到瓶子上的制造商到期日期,以较早者为准。
    2. avertin溶液的制备
      1. 将15.5ml叔戊醇加入到25g avertin(2-2-2-三溴乙醇)中,并在磁力搅拌器上搅拌溶液,直到甲壳蛋白完全溶解。这可能需要一夜。该物料应在室温下储存在黑暗的瓶子中,并盖紧。
      2. 在50ml量筒中混合0.5ml avertin原液和39.5ml生理盐水或PBS
      3. 放在磁力搅拌棒,密封刻度缸与石蜡膜和完全包裹缸铝箔,以排除所有的光。
      4. 搅拌过夜以溶解。
      5. 将工作溶液通过0.2微米过滤器过滤到深色瓶中。保持工作溶液在4°C。
      6. 库存和工作溶液容器将被正确标记(名称,日期,实验室成员的浓度和首字母)
      7. 储存溶液应在四个月后丢弃,工作溶液应在2周后丢弃
      8. 如果检测到沉淀或变色,则不应使用所有溶液
    3. 为了制备完全平滑肌肉培养基(SmBm完全),通过0.22μM真空过滤器过滤以下,并加入到一瓶500ml来自Lonza的SmBm基础培养基中。该培养基可以在4℃下储存长达2个月,或者直到瓶子上的制造商的有效期,以较早者为准。重要的是要注意,不应该添加包含在SmBm弹头套件中的庆大霉素的等分试样。
      1. 100 ml FBS
      2. 子弹试剂盒中含有的FGF的等分试样(1ml)
      3. 子弹试剂盒中含有的EGF的等分试样(0.5ml)
      4. 子弹试剂盒中含有的胰岛素的等分试样(0.5ml)
      5. 5ml抗生素/抗真菌剂
      注意:我们不删除任何基础培养基以补偿添加剂的添加。因此,SmBm完全的最终体积为607ml。
    4. 消化酶和其他试剂的制备
      1. 解冻并过滤一个500毫升的FBS瓶,并存储在4℃下,50毫升等分
      2. 为了制备平滑肌细胞冷冻培养基,将45ml SmBm完全与5ml过滤的FBS和5ml无菌DMSO组合。
      3. 胶原酶1型作为粉末接受。 3-4小时消化所需的最终浓度为1.00mg/ml。为了准备储备溶液,添加2.5毫升主动脉活检存储介质重新悬浮50毫克胶原酶。混合好。使500μl等分试样,并存储在-20°C
      4. 接收大豆胰蛋白酶抑制剂为粉末。 3-4小时消化所需的最终浓度为0.25mg/ml。为了制备储备溶液,取2.5mg并溶解在10ml主动脉活检存储介质中,然后制成10等分,1ml /等分
      5. 1型弹性蛋白酶作为液体接受。该瓶可以在4℃下储存。不等分,因为试剂等分试样时不稳定,并储存在-20℃。 3-4小时消化所需的最终浓度为0.1875mg/ml。

  2. 解剖小鼠以去除主动脉组织
    1. 通过腹膜内(IP)注射2.5%的avertin麻醉实验小鼠(3-6只小鼠/外植体)。每个小鼠使用0.5毫升的溶液。要确定何时麻醉有效,捏紧鼠标的脚趾之间你的指甲之间。如果鼠标踢或反应根本,麻醉是不完整的。等待几分钟,再次测试,直到没有对脚趾捏的反应。如果麻醉在5-7分钟后仍然不完全,您可以再次给小鼠再次加入0.3ml的阿佛丁。
    2. 将鼠标放在光源下进行解剖。
    3. 将鼠标的皮肤从腹部切到胸部顶部。
    4. 用镊子提起胸骨,切开膈肌。然后切下肋骨的下部暴露心脏,去除肺和胸腺,并切除食道,暴露沿脊柱运行的主动脉。请参见图1和视频1.


      图1.鼠标胸腔。图片显示胸部打开后的鼠标。蓝色表示肺组织,绿色表示胸腺组织,为了更好地接近心脏和主动脉,必须将两者去除。

      <! - flashid2045v132开始 - >
      视频1.打开小鼠胸腔和切除组织。此视频显示从小鼠胸腔开口到清除肺和气管的解剖的第一部分。
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    5. 将鼠标放在解剖显微镜下。
    6. 取出剩余的组织,并使用显微切割剪刀或镊子清除心脏,升主动脉及其分支顶部周围的所有脂肪。见图2.


      图2.鼠标中暴露的心脏和降主动脉。图片显示胸部打开并且肺,胸腺和其他组织被移除后的鼠标。红色箭头表示心脏,应该被轻轻拉起以暴露升主动脉和颈动脉分支。红色箭头表示降主动脉,应该夹在隔膜下面。

    7. 用镊子轻轻拉起心脏,不打破升主动脉。使用镊子或剪刀轻轻地分离降主动脉从脊柱。夹住膜片下方的降主动脉。夹住颈动脉释放升主动脉。参见视频2.将心脏,升序和降主动脉放在一个60毫米的培养皿中,连接无菌DPBS。

      <! - flashid2045v133开始 - >
      视频2.删除鼠标心脏和主动脉。 此视频显示解剖的第二部分:去除心脏和主动脉。
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    8. 将盘放在解剖显微镜下,并从升主动脉和降主动脉中删除任何脂肪或结缔组织。参见图3.通过在左颈总动脉的原点的远端切割来分离升主动脉和降主动脉。根据主动脉的区域和小鼠的基因型,将升高和降主动脉分别置于新的具有DPBS标记的培养皿中。
    9. 工作快速但仔细,重复每个实验鼠,直到所有的组织已经分离。每个外植体的升主动脉升主动脉和降主动脉。
    10. 将所有的主动脉组织皿移至组织培养罩。


      图3.解剖主动脉的外观。整个安装图像显示主动脉组织,它已从鼠标中移除并完全清洁。升主动脉标有红色箭头。

  3. 解剖组织
    注意:请注意,协议其余部分的所有步骤都应在组织培养罩下使用标准无菌组织培养技术进行。
    1. 准备酶混合物
      1. 在开始解剖小鼠之前,在4℃下将胶原酶和胰蛋白酶抑制剂各解冻一等份。
      2. 对于每个外植体,使用5ml主动脉活检存储介质。如果你在白天进行3-4小时消化,添加250微升胶原酶,100微升胰蛋白酶抑制剂和324微升*弹性蛋白酶为每5毫升主动脉活检存储介质。对于过夜(15-18小时)消化,使用1:10稀释因此25μl胶原酶,10μl胰蛋白酶抑制剂和32.4μl*弹性蛋白酶。
        注意:添加的弹性蛋白酶的量基于瓶的质量和体积计算。如果您从不同来源订购弹性蛋白酶,则需要重新计算要添加的酶的量。
    2. 将70%乙醇,DPBS和主动脉活检存储介质倒入三个单独的60mm组织培养皿中。
    3. 使用镊子,从每个隔离的主动脉从其存储管和洗涤组织在乙醇,然后DPBS,然后活检介质。不要将组织留在乙醇中超过约10-15秒,但留在DPBS或介质中更长时间是可以的。
    4. 将每片组织放入60毫米组织培养皿的盖子。使用手术刀和镊子,将组织切成小块 - 确保沿周向和纵向切割组织,以增加暴露于消化混合物的表面积。这些块的尺寸可以变化,但是平均尺寸应该是1-2mm的直径。
    5. 将切碎的组织片放入一个新的60毫米组织培养皿,其中含有5毫升培养基加上消化酶混合物在步骤C1中制成。标记菜肴。
    6. 将培养皿放入培养箱(37℃)。如果进行3-4小时消化,在2小时和之后每30分钟检查。如果做过夜消化,离开过夜,在15小时和之后每小时检查。
    7. 当组织看起来大部分消化与几个浮动单细胞,但仍然一些大块组织,吸管上下组织和媒体5-10次使用10毫升移液管帮助释放组织细胞。放回孵育器30多分钟。另外,从冰箱中取出过滤的FBS以升温至室温,将完全SmBM培养基置于水浴中,升温至37℃。
    8. 在孵育时间结束时,在显微镜下观察组织,以确保细胞没有组织。参见图4.如果组织已经被良好消化(应当仍然有一些完整的组织片),加入2.5ml血清和移液管上下约5次。接下来加入2.5ml完全SmBM培养基和吸移管上下〜5次。


      图4.消化组织的外观。消化后的菜的照片,有一些"网状"组织漂浮在其中(黑色箭头),但大部分组织片被消化。

    9. 转移组织培养皿(应总共10毫升)的整个内容到一个15毫升管。
    10. 在室温下以180×g离心5分钟(在Sorvall Legend T +台式离心机中约700rpm)5分钟。
    11. 仔细检查,看看是否有任何浮动的组织片在管中。如果是这样,使用200μl移液器取出组织片,并将其直接放入带有完整SmBm的标记T25烧瓶。一旦没有组织块漂浮,删除上清液,并将沉淀重悬在5ml的完整的SmBM培养基中。
      注意:如果上清液混浊,重复步骤C10和C11,直到上清液澄清。如果上清液清澈,则进行步骤C12。
    12. 转移5毫升完全SmBm与重悬细胞到标记的T25烧瓶。将烧瓶置于培养箱中。
    13. 让细胞沉降并粘附在烧瓶上。不要更换介质至少48小时。如果细胞在这一点仍然没有沉降,不要删除任何介质,并加入2毫升培养基的烧瓶。让细胞再定居1到2天。

  4. 小鼠血管平滑肌细胞的维持和传代培养
    1. 一旦SMC成长,每3-4天给细胞补充新鲜培养基。
      1. 将SmBm完全培养基瓶在37°C水浴中预温至少15分钟,然后喂养细胞。
      2. 从每个烧瓶中吸出培养基并丢弃。
      3. 为每个T25烧瓶添加5毫升培养基,而不干扰细胞层。
      4. 将烧瓶放回培养箱。
    2. 当细胞达到约70%汇合时,或当一些区域开始变得非常紧密包装并且几乎过度生长时,细胞准备分裂。参见图5.将SMC从一个T25烧瓶传送到一个T75烧瓶。
      1. 在37°C水浴中预热SmBm完全培养基瓶,并允许TrypLE Express试剂在分裂细胞前在室温下平衡至少15分钟。
      2. 从每个烧瓶中取出培养基并丢弃
      3. 用4ml DPBS洗涤每个烧瓶一次,使其覆盖细胞层,然后吸出并弃去DPBS。
      4. 向每个烧瓶中加入1 ml TrypLE Express试剂。确保胰蛋白酶均匀地覆盖烧瓶/细胞层的底部。
      5. 将烧瓶置于37℃培养箱中3分钟。
      6. 在显微镜下确认消化。如果大多数细胞是圆的,在两边敲两次烧瓶。如果大多数细胞仍然连接在室温下孵育1分钟,然后确认消化和烧瓶两侧两次。
      7. 向每个烧瓶中加入4ml SmBm完全以中和胰蛋白酶。上下移动以分离所有细胞并完全中和酶。
      8. 将所有5ml液体加细胞转移到15ml锥形管中
      9. 在室温下在台式离心机中以180×g离心5分钟
      10. 吸出上清液,重悬细胞沉淀在10ml的SmBm完全,并将所有10毫升转移到标记的T75烧瓶。标记分割的日期和通过编号(第一次传代的P1)。


        图5.融合的SMC的外观。左图显示平滑肌细胞的100%汇合区。这些细胞需要在24小时内传代。右图显示约70%汇合的平滑肌细胞。注意主轴形态是SMC培养的特征
    3. 当SMC已经扩增时,可以冷冻细胞等分试样。按照上述用于传代SMC的方案中的步骤D2a-D2i。一个T75瓶可以分成2个冷冻细胞等分试样。一个融合的T75瓶应该有大约200万个细胞,所以每个冷冻瓶将有大约1百万个细胞。离心后,按照以下步骤冻结细胞。
      1. 吸出上清液,重悬细胞沉淀在3毫升SMC冷冻介质(见Recipes)。
      2. 转移1.5毫升具有溶解沉淀的冷冻介质到两个冷冻管中的每一个。冷冻库应该用通道数,冷冻日期和细胞系标记。
      3. 将冷冻管放入缓慢冷冻的容器(泡沫容器或异丙醇容器将工作),并在-80℃冰箱过夜冷冻。
      4. 将冷冻的等分试样转移到液氮储存系统中长期储存。
    4. 当您想解冻SMC等分试样时,通常可将一小瓶冷冻细胞解冻至一个T75烧瓶。
      1. 在37°C水浴中预热SmBm完全培养基至少15分钟,然后取出细胞。
      2. 从液氮冷冻箱中取出细胞瓶。通过将冷冻管置于37℃水浴中约1-2分钟快速加热
      3. 加入8毫升SmBm完成到15毫升锥形管。然后将1.5毫升解冻的细胞转移到同一个锥形管中
      4. 在室温下在台式离心机中以180×g离心5分钟。
      5. 取出并弃去上清液,将细胞沉淀重悬于10ml SmBm完全培养基中,转移至T75烧瓶中。

数据分析

  1. SMC表型的表征
    为了确认SMC的身份,执行平滑肌α-肌动蛋白的免疫荧光,应在分化SMC的丝中可见。当细胞是80%汇合时,它们准备平板。在开始使用细胞前一小时,将盖玻片放入6孔板(每孔一个盖玻片,您想要盖板的数量,对于本实验,我们建议至少两个)在细胞培养罩下。打开罩在罩中的UV至少30分钟,以消毒盖玻片。然后开始,按照步骤D2a-D2i传代上面的细胞。离心后,吸出上清液,并在较小体积(通常为2ml的SmBm完全)中重悬细胞沉淀。
    1. 使用10μl重悬细胞混合物计数血细胞计数器中的细胞
    2. 计算5,000个单元格所需的体积。将计算体积的细胞混合物加入含有盖玻片的孔中,并向每孔加入额外的SmBm完全培养基至2ml体积。任何剩余的细胞可用于其他实验或根据需要放回烧瓶中
    3. 18-24小时后,电镀细胞,更换培养基到低血清SmBm培养基,其驱动血管SMC的分化。这是SmBM基础培养基,仅添加1%FBS和1x抗生素/抗真菌剂(对于500ml的SmBm基础培养基瓶,加入5ml FBS和5ml的100x抗生素/抗真菌剂)。在细胞上留下低血清SmBm至少24小时
    4. 如果需要,您可以用rhTGF-β1处理一些盖玻片以进一步诱导分化。切换至低血清SmBm培养基24小时后,向低血清SmBm培养基中加入10ng/ml rhTGF-β1,并将该培养基加入盖玻片中。将含有rhTGF-β1的培养基保留在细胞上48小时
    5. 当进行处理时,除去培养基,用DPBS洗涤细胞一次,并向每个孔中加入1ml的4%多聚甲醛溶液以固定细胞。储存在4°C,直到细胞准备染色。
    6. 对于染色,使用50μl液体的每个盖玻片在Parafilm上,并将盖玻片倒转到液滴以节省抗体。首先,在封闭缓冲液中孵育盖玻片1小时(封闭缓冲液是0.3%Triton-X,0.5%BSA在DPBS中)。
    7. 在DPBS中洗涤盖玻片3次5分钟
    8. 孵育盖玻片在1:500抗平滑肌α-肌动蛋白抗体在封闭缓冲液中在4℃过夜。
    9. 在DPBS中洗涤盖玻片3次5分钟。
    10. 孵育盖玻片在1:500荧光缀合的抗小鼠IgG二抗在封闭缓冲液在室温下3小时。
    11. 在DPBS中洗涤盖玻片3次5分钟
    12. 使用DAPI(核复染,Vectashield)的安装介质将盖玻片安装到显微镜载玻片上
    13. 使用指甲油或其他所需的密封剂将盖玻片密封到载玻片上。
    14. 使用所需的荧光显微镜评估染色。至少95%的细胞应该具有在细胞中可见的平滑肌α-肌动蛋白丝(图6)

      图6.在分离的平滑肌细胞中的肌动蛋白丝。SMα-肌动蛋白(绿色)的免疫荧光染色显示SMα-肌动蛋白的聚合丝。核用DAPI(蓝色)复染色

笔记

  1. 正确计时乙醇洗涤步骤(上述"外植体"方案下的步骤C3)是关键的。太长时间在乙醇会杀死细胞,并大大降低成功。在乙醇洗涤中的时间过短可能导致样品的细菌污染。
  2. 虽然上面列出的消化酶的产品编号已经在我们手中广泛测试,我们也已经成功与其他版本的胶原酶和弹性蛋白酶。最关键的因素是活性酶的浓度和消化的时间。任何替代酶将需要测试最佳消化时间
  3. 通常,在我们的手中,初级平滑肌细胞在培养中保持其表型至少6代。我们总是使用低通过细胞(

食谱

  1. 主动脉活检存储介质
    500ml Waymouth's MB 752/1培养基
    5ml的100×抗生素 - 抗真菌剂
    6.25ml L-谷氨酰胺 15ml碳酸氢钠 5ml MEM非必需氨基酸
    5ml HEPES缓冲液
  2. 完成平滑肌介质
    500 ml SmBm基础培养基
    100ml FBS
    1ml的FGF包含在子弹套件
    中 0.5ml包含在子弹套件
    中的EGF 0.5ml包含在子弹套件中的胰岛素
    5ml抗生素/抗真菌剂
  3. 平滑肌细胞冷冻介质
    45 ml完全平滑肌介质
    5ml过滤的FBS 5ml无菌DMSO
  4. 消化酶混合物(3-4小时消化时间)
    5ml主动脉活检储存介质
    250μl胶原酶
    100μl胰蛋白酶抑制剂
    324μl弹性蛋白酶

致谢

这项工作得到国家卫生研究院的支持:NIH(RO1 HL62594和P01HL110869-01),John Ritter基金会,Vivian L. Smith基金会和Ehlers Danlos综合征网络。 该方法在Kuang等人中公开。 (2016),并且它是Gordon等人使用的方法的改编。 (1986)和Lemire等人。 (1994)。使用该方法移出的细胞也用于以下出版物中:Cao等人。 (2010),Kuang等人。 (2012),Kwartler等人。 (2014),Papke等人。 (2013年)。

参考文献

  1. Cao,J.,Gong,L.,Guo,DC,Mietzsch,U.,Kuang,SQ,Kwartler,CS,Safi,H.,Estrera,A.,Gambello,MJ和Milewicz,DM(2010) 结节性硬化综合征中的胸主动脉疾病:分子发病机制和潜在疗法Tsc2 +/- mice。 Hum Mol Genet 19(10):1908-1920。
  2. Gordon,D.,Mohai,LG和Schwartz,SM(1986)。  在新生大鼠主动脉平滑肌细胞培养物中诱导多倍体。 Circ Res 59(6):633-644。
  3. Kuang,SQ,Kwartler,CS,Byanova,KL,Pham,J.,Gong,L.,Prakash,SK,Huang,J.,Kamm,KE,Stull,JT,Sweeney,HL和Milewicz,DM(2012)。   平滑肌特异性同种型中的罕见,非同义变体肌球蛋白重链,MYH11,R247C,改变主动脉中的力产生和平滑肌细胞的表型。 Circ Res 110(11):1411-1422。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kwartler, C. S., Zhou, P., Kuang, S., Duan, X., Gong, L. and Milewicz, D. M. (2016). Vascular Smooth Muscle Cell Isolation and Culture from Mouse Aorta. Bio-protocol 6(23): e2045. DOI: 10.21769/BioProtoc.2045.
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