Analysis of Mitochondrial Transfer in Direct Co-cultures of Human Monocyte-derived Macrophages (MDM) and Mesenchymal Stem Cells (MSC)
利用细胞源性巨噬细胞(MDM)和间充质干细胞(MSC)共培养体系分析细胞间线粒体的转移   

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Stem Cells
Aug 2016

 

Abstract

Mesenchymal stem/stromal cells (MSC) are adult stem cells which have been shown to improve survival, enhance bacterial clearance and alleviate inflammation in pre-clinical models of acute respiratory distress syndrome (ARDS) and sepsis. These diseases are characterised by uncontrolled inflammation often underpinned by bacterial infection. The mechanisms of MSC immunomodulatory effects are not fully understood yet. We sought to investigate MSC cell contact-dependent communication with alveolar macrophages (AM), professional phagocytes which play an important role in the lung inflammatory responses and anti-bacterial defence. With the use of a basic direct co-culture system, confocal microscopy and flow cytometry we visualised and effectively quantified MSC mitochondrial transfer to AM through tunnelling nanotubes (TNT). To model the human AM, primary monocytes were isolated from human donor blood and differentiated into macrophages (monocyte derived macrophages, MDM) in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF), thus allowing adaptation of an AM-like phenotype (de Almeida et al., 2000; Guilliams et al., 2013). Human bone-marrow derived MSC, were labelled with mitochondria-specific fluorescent stain, washed extensively, seeded into the tissue culture plate with MDMs at the ratio of 1:20 (MSC/MDM) and co-cultured for 24 h. TNT formation and mitochondrial transfer were visualised by confocal microscopy and semi-quantified by flow cytometry. By using the method we described here we established that MSC use TNTs as the means to transfer mitochondria to macrophages. Further studies demonstrated that mitochondrial transfer enhances macrophage oxidative phosphorylation and phagocytosis. When TNT formation was blocked by cytochalasin B, MSC effect on macrophage phagocytosis was completely abrogated. This is the first study to demonstrate TNT-mediated mitochondrial transfer from MSC to innate immune cells.

Keywords: Mesenchymal stem cells (间充质干细胞), Macrophages (巨噬细胞), Mitochondrial transfer (线粒体转移), ARDS (ARDS), Phagocytosis (吞噬作用), Oxidative phosphorylation (氧化磷酸化)

Background

Data from pre-clinical studies, including studies by our group (Xu et al., 2007 and 2008; Nemeth et al., 2009; Gupta et al., 2007 and 2012; Krasnodembskaya et al., 2010 and 2012; Mei et al., 2010; Lee et al., 2013; Jackson et al., 2016) demonstrated strong potential for MSC as a future cell-based therapy for the treatment of ARDS, an injurious hyper-inflammatory condition of the lung. In these studies MSC have displayed regenerative, immune-modulatory and anti-microbial effects which have consequently provided rationale for the design of phase I and phase II clinical trials for MSC in ARDS (Zheng et al., 2014; Wilson et al., 2015). However, despite the rapid translation of MSC into the clinical trials, mechanisms of how MSC alleviate symptoms of ARDS still need to be fully elucidated. Recent studies have reported MSC modulate lung epithelial and endothelial cells through mitochondrial transfer via TNTs, resulting in improvement of the host cell bioenergetics (Islam et al., 2012; Ahmad et al., 2014; Li et al., 2014; Liu et al., 2014). In ARDS, excessive pulmonary inflammation is one of the main characteristics of the disease in which alveolar macrophages (AM) are prominent cells. They orchestrate the inflammatory responses in the alveoli and play an important role in the lung bacterial clearance (Ware and Matthay, 2000; Jackson et al., 2016).

This protocol allowed us to study the functional effects of a TNT mediated process of an organelle transfer between MDMs both in vitro and using the same staining protocol, mouse alveolar macrophages in vivo (Jackson et al., 2016). Although the major focus of our study was mitochondrial transfer, this protocol can be adapted with slight modifications for investigations of transfer of other organelles or even fluorescently labelled molecules.

Materials and Reagents

  1. Extraction of mononuclear cells from human donor Buffy Coats
    1. 50 ml Falcon tubes (SARSTEDT, catalog number: 62.554.502 )
    2. T175 culture flasks (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 178883 )
    3. Sterile Pasteur pipettes (BIOLOGIX GROUP LTD Technical, catalog number: 30-0138A1 )
    4. Cover slip
    5. Tissue culture coated 6 well plate (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 140675 )
    6. Buffy coats are obtained from the Northern Ireland Blood Transfusion Service (NIBTS) following ethical approval by the School Research Ethics Committee of Queen’s University Belfast
    7. Hank’s buffered salt solution (HBSS) No Ca2+ or Mg2+ (Thermo Fisher Scientific, catalog number: 14170138 )
    8. Ficoll-Paque (GE Healthcare, catalog number: 17-5442-03 )
    9. Complete RPMI 1640 (Thermo Fisher Scientific, GibcoTM, catalog number: 21875034 )
    10. Foetal calf serum (FCS) (heat inactivated) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
    11. Granulocyte-macrophage colony stimulating factor (GM-CSF) (PeproTech, catalog number: 300-03 )
    12. Penicillin/streptomycin 10,000 U/ml (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
    13. Trypan blue (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
    14. Complete 1% FBS RPMI (see Recipes)
    15. Complete RPMIGM-CSF (see Recipes)

  2. Culture of human bone marrow-derived mesenchymal stromal cells (MSC)
    1. 50 ml Falcon tubes (SARSTEDT, catalog number: 62.554.502 )
    2. 8-well chamber slides (Sigma-Aldrich, catalog number: C7182 )
    3. Tissue culture coated 6-well and 24-well plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog numbers: 140675 , 142475 )
    4. Pechiney PM999 Parafilm (Bemis, catalog number: PM999 )
    5. Microscope slide
    6. Sterile Eppendorf tubes (SARSTEDT, catalog number: 72.690.001 )
    7. Flow tubes (SARSTEDT, catalog number: 55.475 )
    8. Human bone marrow-derived MSCs are obtained from the NIH repository in Texas A&M Health Science Centre College of Medicine, Institute for Regenerative Medicine (Temple, Texas). The cells meet all the criteria for the classification as MSCs as defined by the International Society of Cellular Therapy (Dominici et al., 2006)
    9. Liquid nitrogen
    10. Dulbecco’s phosphate buffered saline (DPBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190094 )
    11. Complete α-MEM (Thermo Fisher Scientific, GibcoTM, catalog number: 22561021 )
    12. Penicillin/streptomycin 10,000 U/ml (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
    13. L-glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030024 )
    14. Cytochalasin B (Sigma-Aldrich, catalog number: C6762 )
    15. 10x trypsin (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
    16. Foetal calf serum (FCS) (heat inactivated) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
    17. Trypan blue (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
    18. MitoTracker Deep Red FM probe (APC) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: M22426 )
    19. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
    20. Complete α-MEM (see Recipes)
    21. Mitochondrial staining solution (1% FBS α-MEM1%MITO) (see Recipes)
    22. Cytochalasin B solution (1%FBS α-MEM1%CYTO) (see Recipes)

  3. Confocal microscopy
    1. 0.45 µm filter membrane (SARSTEDT, catalog number: 83.1826.001 )
    2. Pechiney PM999 Parafilm (Bemis, catalog number: PM999 )
    3. Paper towel
    4. Microscope slide
    5. Coverslip
    6. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
    7. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: 158127 )
    8. 1 N NaOH (Sigma-Aldrich, catalog number: S2770 )
    9. 1 N HCl (Sigma-Aldrich, catalog number: H9892 )
    10. Normal goat serum (NGS) (Thermo Fisher Scientific, Invitrogen, catalog number: 31872 )
    11. Mouse anti-human CD45 primary antibody (Abcam, catalog number: ab8216 )
    12. Mouse IgG1 isotype (Abcam, catalog number: ab81032 )
    13. Goat anti-rabbit Alexafluor 405 secondary antibody (Abcam, catalog number: ab175655 )
    14. Brightmount/Plus aqueous mounting medium (Abcam, catalog number: ab103748 )
    15. Nail varnish
    16. 1% FBS PBS (see Recipes)
    17. 4% paraformaldehyde (PFA) (see Recipes)
    18. Strong and weak block for immunofluorescent staining (see Recipes)

  4. Flow cytometry
    1. Cell scrapers (Fisher Scientific, catalog number: 08-100-241 )
    2. Sarstedt 5 ml polystyrene round bottomed flow tubes (SARSTEDT, catalog number: 55.1578 )
    3. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
    4. Human FcR binding inhibitor (Thermo Fisher Scientific, eBioscienceTM, catalog number: 14-9161-73 )
    5. Anti-human CD45 (PE) antibody (Thermo Fisher Scientific, eBioscienceTM, catalog number: 12-9459-41 )
    6. Isotype control (IgG1 kappa) (Thermo Fisher Scientific, eBioscienceTM, catalog number: 12-4714 )
    7. Zombie Aqua Fixable Dye (BioLegend, catalog number: 423101 )

  5. Bacterial culture and phagocytosis assay
    1. Escherichia coli K1 type strain
    2. Phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
    3. LB broth (Lennox) (Sigma-Aldrich, catalog number: L3022 )
    4. LB broth with agar (Lennox) (Sigma-Aldrich, catalog number: L2897 )
    5. Hank’s buffered salt solution (HBSS) No Ca2+ or Mg2+ (Thermo Fisher Scientific, catalog number: 14170138 )
    6. Complete RPMI 1640 (Thermo Fisher Scientific, GibcoTM, catalog number: 21875034 )
    7. Saponin (Sigma-Aldrich, catalog number: 47036 )
    8. Gentamycin (Sigma-Aldrich, catalog number: G1397 )
    9. Complete 1% FBS RPMI (see Recipes)
    10. 0.5% saponin (see Recipes)

Equipment

  1. Water bath (37 °C) (Grant Instruments, model: JBN12 )
  2. Centrifuge (Eppendorf, model: 5810 R )
  3. Laminar flow cabinet (Contained Air Solutions, model: BioMAT 2 safety cabinet class 2 )
  4. Haemocytometer and cover slips (Hawksley Medical and Laboratory Equipment, model: AC1000 Improved Neubauer , catalog number: BS 748)
  5. Incubator (Panasonic Biomedical, model: MCO-170AIC-PE )
  6. Dmi1 microscope (Leica Microsystems, model: DMi1 )
  7. TCS SP5 II Leica confocal microscope (Leica Microsystems, model: TCS SP5 II )
  8. BD FACSCanto II Flow cytometer (BD, model: BD FACSCanto II )
  9. Vortex (Cole-Palmer Instrument, catalog number: UY-86579-20 )
  10. 2-20 µl pipette (Gilson, catalog number: F123600 )
  11. 20-200 µl pipette (Gilson, catalog number: F144565 )
  12. 100-1,000 µl pipette (Gilson, catalog number: F144566 )
  13. Midi PlusTM pipette controller Automatic (Sartorius, catalog number: 710931 )

Software

  1. FlowJo software (FlowJo)
  2. Prism 5 software (GraphPad Software)
  3. LAS-AF software (Leica confocal microscopy)
  4. FACS DIVA software (flow cytometry)

Procedure

  1. Extraction of mononuclear cells from donor Buffy Coats
    1. Single donor leukocyte buffy coats are obtained from the Blood Transfusion Centre in Belfast (NIBTS) or from volunteers following ethical approval (approx. 50 ml/blood bag/volunteer).
    2. Pre-warm all media (HBSS, 1% FBS RPMI and 10% FBS RPMI) in a water bath 1-2 h prior to isolation and pre-warm the centrifuge to 20 °C prior to isolation.
    3. Under a laminar flow cabinet decant the donor blood into a T75 flask and dilute (1:2) using sterile HBSS and mix by inversion.
    4. Pipette 15 ml of Ficoll-Paque into 4 x 50 ml Falcons.
    5. Very gently layer the diluted blood at a 45° angle onto the Ficoll, taking care not to mix the layers. Top up the remaining tube with HBSS to make a final volume of 50 ml.
    6. Centrifuge all tubes at 480 x g, 20 °C for 20 min without brake.
    7. After the spin, locate the pearlescent white layer (buffy layer, shown as lymphocytes, monocytes and platelets layer in Figure 1), which is sedimented through the plasma-density gradient interface. Extract into a new sterile 50 ml Falcon using a Pasteur pipette.


      Figure 1. Schematic diagram to illustrate monocyte isolation from peripheral blood. After blood has been layered onto the Ficoll and centrifuged as described above without brake, blood components are separated into layers. A Pasteur pipette is used to carefully remove the lymphocyte, monocyte and platelet layer to use in downstream processing.

    8. To wash the cells, add 40 ml of HBSS to the mononuclear cells and mix by inversion.
    9. Centrifuge again at 290 x g, 4 °C for 5 min with brake. Aspirate off supernatant slowly (take care as the pellet may become loose and add another 40 ml HBSS). Repeat this wash step 3 times.
    10. After the last wash, resuspend the pellet in 10 ml of complete 1% FBS RPMI and mix thoroughly by either vortexing or inversion.
    11. Place a cover slip on a haemocytometer and load 10 μl of the cell mixture via capillary action through the loading channel.
    12. Incubate in the 37 °C, 5% CO2 incubator for at least 5 min (see Note 1).
    13. After this time, view the cells under a microscope at 20x magnification. Count all mononuclear cells (see Note 1).
    14. Cells are then seeded at density of 1 x 106 cells/well of a 6 well plate using 3 ml complete 1% FBS RPMI (see Table 1 for other seeding densities). Incubate at 37 °C, 5% CO2 for at least 2 h to ensure mononuclear cells have adhered to the plastic of the well.

      Table 1. MSC and MDM (1:20 ratio) seeding densities
      No. of MSC/well
      No. of
      MDM/well
      Type of plate 
      Final volume
      (ml) of 1% FBS RPMI 

      Experiment analysis technique
      5 x 104
      1 x 106
      6 well
      3
      Flow cytometry/Western
      1.5 x 104
      3 x 105
      24 well
      1
      ELISA/bioplex/phagocytosis assays
      2.5 x 103
      5 x 104
      8 well chamber slide
      0.2
      Confocal microscopy


    15. After this time non-adherent cells (lymphocytes) are washed off twice with HBSS (by adding 1 ml HBSS, slight agitation and then aspiration of waste).
    16. To promote monocyte-derived macrophage (MDM) maturation, replace complete 1% FBS RPMI with 3 ml complete RPMIGM-CSF (see Recipes).
    17. Leave cells to rest for 5-7 days at 37 °C, 5% CO2.
    18. After 5-7 days, wash cells twice with HBSS and replace with 3 ml complete 1% FBS RPMI. Leave cells to rest for 1 h prior to stimulation experiments.

  2. Human mesenchymal stromal cell culture
    1. MSC expansion and storage
      1. MSC are shipped from Texas, US in liquid nitrogen in 1 ml cryovials at passage number 1 or 2 (P1-P2) meaning they have undergone at least one or two stages of cell splitting in culture. For experimental use (see section Preparing P3 MSC for Experimental Use) cells are only used from P3-P5.
      2. Shortly after arrival, defrost 1 vial and culture in pre-warmed 15 ml complete 16% FBS α-MEM in a T175 flask for less than 24 h.
      3. Retain the passage number by splitting cells less than 24 h later and expanding them using a density of 21,000 cells per flask. A P2 vial can undergo an additional expansion to be stored at P3. Store cells (1 million cells/vial) at either P2 or P3 in liquid nitrogen storage.
    2. Preparing P3 MSC for experimental use
      Once ready to use, defrost 1x P3 cryovial of MSC in the water bath (37 °C) and place entire 1 ml into 15-20 ml of complete 16% FBS α-MEM, making sure to feed every other day. Cells are maintained in culture until P5-P6.

  3. Direct co-culture of Human Mesenchymal Stromal Cells (MSC and Monocyte-derived  Macrophages (MDM)
    1. Pre-warm DPBS, HBSS, complete 10% FBS RPMI, 1% FBS RPMI, 16.5% FBS α-MEM and 1% FBS α-MEM in the water bath at 37 °C for at least half an hour before the start of the experiment.
      Following work has to be completed in a biosafety cabinet with cabinet light switched off.
    2. At 70-80% confluency, to pre-stain MSC mitochondria, aspirate older 16% FBS α-MEM from the T175 flask and add 20 ml of staining solution (1% FBS α-MEMMITO [see Recipes and Note 2]), incubate for approximately 30 min at 37 °C, 5% CO2.
    3. For MSC being pre-treated with cytochalasin B, 20 ml of 1% FBS α-MEMCYTO is added to cells for 1-2 h at least prior to staining solution (1% FBS α-MEMMITO). As Cyto B is soluble in DMSO, make sure to have a control flask of cells for DMSO vehicle control.
    4. Aspirate 1% FBS α-MEMCYTO from the flask and replace with 1% FBS α-MEMMITO, and incubate for 30 min at 37 °C, 5% CO2.
    5. After this time, the staining solution is aspirated from MSC and the cells are washed at least three times in 5 ml DPBS.
    6. Aspirate waste DPBS and add 7 ml 1x trypsin (diluted in DPBS) and leave in the incubator for approximately 2-5 min.
    7. Once the cells are suitably detached, add 7 ml of complete α-MEM to counteract trypsin action and pipette the cells into a 50 ml Falcon.
    8. Centrifuge the cells at 290 x g for 5 min.
    9. During this time, prepare the MDMs by aspirating older culture medium and washing twice with 1-2 ml of HBSS.
    10. Aspirate the remaining HBSS and leave the MDMs to rest in the incubator in 200 μl (chamber slide), 1 ml (24 well plate) or 3 ml (6 well plate) of 1% FBS RPMI.
    11. After MSC centrifugation, aspirate the supernatant and resuspend the pellet in 1 ml 1% FBS RPMI (see Note 3).
    12. Count cells using a haemocytometer, using trypan blue exclusion and depending on well size needed for experiment, seed corresponding cell densities and co-culture with MDMs for 24 h (see Table 1).
    13. Proceed to either confocal microscopy or flow cytometry procedures.

  4. Confocal microscopy staining procedure
    1. After co-culture on a chamber slide, carefully aspirate off culture medium, and wash cells twice using 200 μl of ice cold PBS.
    2. Aspirate the PBS and fix the cells using 100 μl of 4% PFA solution.
    3. Incubate at room temperature with gentle agitation for approximately 15 min.
    4. After washing twice again with PBS, proceed to blocking the cells with 100 μl strong block for at least 1 h.
    5. Wash twice with PBS and add 50 μl of primary antibody mouse anti-human CD45 primary antibody or isotype control Mouse IgG1 (diluted 1:200) in weak block. Seal slide with Parafilm and incubate slide at 4 °C overnight with agitation.
    6. The next day, repeat the PBS wash step as before and then add 50 μl of secondary antibody goat anti-rabbit Alexafluor 405 secondary antibody (diluted 1:1,000 in weak block) for one hour. Incubate in the dark at room temperature for one hour.
    7. Immediately after, repeat the PBS wash step, taking care not to expose slide to light for longer than 10 min.
    8. Gently tap the chamber slide onto a paper towel to remove any excess PBS wash and then carefully dislodge the plastic chamber portion to separate the underlying microscope slide.
    9. Add approximately 2-3 drops of mounting medium and place a coverslip over the area of stained cells ensuring all air bubbles are removed. Allow to air dry for 30-45 min in the dark.
    10. When dry, use commercial nail varnish around the edges to act as glue and fix the coverslip in place.
    11. Leave nail varnish to dry in the dark at room temperature. The slide may also be stored at 4 °C long-term sealed with Parafilm in the dark for future analysis.
    12. Proceed to viewing using a confocal microscope (Figure 2).


      Figure 2. Visualization of transfer of MSC mitochondria to MDM by confocal microscopy. A. MDM express surface CD45 marker (blue); B. MSC mitochondria are stained with MitoTracker probe (red); C. After 24 h of co-culture mitochondrial positive TNT are observed, protruding from the MSC (arrows) ranging up to 200 µm in length. Mitochondrial transfer from MSC to MDM is evidenced by co-localisation of blue and red staining (pink). (Images were taken at magnification 10 x 63, scale bar = 75 µm). (Adapted from Jackson et al., Stem Cells. 2016 Aug; 34(8): 2210-23)

  5. Flow cytometry staining procedure
    1. Immediately after co-culture place the 6 well plate on ice and aspirate off culture medium.
    2. Add 1 ml of ice cold 1% FBS PBS to the cell co-culture and lightly scrape using a cell scraper all over the well, so as to gently detach cells from the plastic surface.
    3. Once detached, place the cells into 5 ml polystyrene round bottomed flow tubes and centrifuge the cells at 800 x g for 5 min.
    4. In order to test viability of the cells after culture and detachment, it is necessary to include a live/dead control. This requires a separate tube of unstained cells and the addition of a live/dead marker stain Zombie Aqua (use according to manufacturer’s protocol).
    5. Decant off the supernatant, leaving the pellet submerged in approximately 100 µl of 1% FBS PBS.
    6. Vortex vigorously and add 2.5 μg/1 x 106 cells of FcR binding inhibitor to the cells and vortex this again.
    7. Leave in the dark and on ice for 30 min.
    8. After this time directly add 5 μl/1 x 106 cells of anti-human CD45 (PE) or Isotype control (IgG1 kappa) to the cell suspension and vortex again to mix thoroughly.
    9. Leave in the dark and on ice for 20 min.
    10. Following this, add an additional 1 ml of 1% FBS PBS to wash the cells and dispose of any unbound antibody. Vortex thoroughly.
    11. Centrifuge the suspension as before at 800 x g for 5 min.
    12. Decant the supernatant and repeat this wash step a final time. Resuspend the pellet in 200 μl of 1% FBS PBS and place the cells on ice in preparation for flow cytometry.
    13. Use FACS DIVA software to perform analysis. To rule out MSC-macrophage aggregates, single cells were gated as W-FSC vs. A-FSC and from this we defined CD45-PE+MitoRed-APC- population as MDM and also MitoRed-APC+CD45-PE- population as MSC (Figure 3).


      Figure 3. Quantification of mitochondrial transfer from MSC to MDM by flow cytometry. A-B. Live single cells were gated as W-FSC vs. A-FSC and from this we defined C. C. CD45-PE+ MDM population without co-culture with MSC is MitoRed-APC negative. D. APC+ MSC population alone is positive for mitochondria but negative for macrophage marker, CD45-PE-. E. After 4 h in co-culture MDM had acquired 92% of MSC mitochondria shown by the MitoRed fluorescence marker APC. F. Histogram illustrates a reduction in MitoRed fluorescence in the MSC population indicating robust donation of mitochondria to the MDM population. (Adapted from Jackson et al., Stem Cells. 2016 Aug; 34(8): 2210-23)

  6. Bacterial culture and phagocytosis assay
    1. Inoculate 15 ml of LB broth with a stored aliquot of E.coli K1 type strain (sterile LB broth/15% glycerol) and incubate at 37 °C to grow an overnight culture.
    2. Use a 24-well plate to set up a direct co-culture with MDM and MSC (see Table 1 for seeding densities).
    3. For E. coli, a total viable count of 1 x 108 colony forming units (CFU/ml) equates to an optical density (OD) of approximately 0.25-0.3 at 600 nm.
    4. Spin bacteria at 15,000 x g, for 5 min and resuspend the pellet using sterile 1 ml HBSS.
    5. After 24 h of MDM and MSC co-culture infect cells using a Multiplicity of Infection of 10 (MOI 10, see Note 4 for MOI calculation), using HBSS as a vehicle control.
    6. Incubate for 4 h at 37 °C, 5% CO2.
    7. After this time, retrieve the supernatant from the cells for quantification of extracellular bacteria and make a dilution series of 10-1 to 10-6 in sterile PBS.
    8. Pipette 3 x 20 μl spots of each dilution out onto LB agar and incubate overnight at 37 °C.
    9. For intracellular bacteria wash the cells in 1 ml sterile PBS 3 times, aspirate wash and then add 300 μg/ml of gentamycin (diluted in 1% FBS RPMI), 100 μl/well and incubate at 37 °C for 1 h to kill adherent bacteria.
    10. After this time wash the cells in 1 ml sterile PBS 3 times, aspirate wash and add 100 μl of 0.5% saponin to lyse the cells and leave the bacteria intact.
    11. After 5 min, add an additional 900 μl of sterile PBS to each well to dilute saponin, and repeat the dilution series again, plating out each dilution. Incubate plates at 37 °C.
    12. Count CFU/ml within 24 h (Figures 4D-4G).


      Figure 4. Cytochalasin B blocks TNT-mediated mitochondrial transfer thereby preventing MSC functional effects on MDM. A. Confocal microscopy shows distinctive spindle MSC shape (red) with MDM (blue) in co-culture. TNT is shown to be present with robust mitochondrial transfer to MDM. B. Cytochalasin B pre-treatment of MSC inhibited actin filament elongation, causing them to appear rounded with loss of TNT structures. Mitochondrial transfer is still however evident due to probable microvesicle secretion from MSC to MDM (magnification 10 x 63, scale bar = 50 µm). C. Cytochalasin B treatment of MSC showed that MitoRed mean fluorescence intensity (MFI) decreased by 50% in MDMs in co-culture (*P < 0.05 Mann Whitney U test). D. After 4 h of live E.coli infection of the co-culture untreated MSC significantly reduced extracellular CFU, whilst simultaneously elevated intracellular CFU in macrophages, an effect recapitulated by stimulation with isolated MSC mitochondria (F + G). H. This effect was completely abrogated with cytochalasin B-treated MSC as even in their presence MDM phagocytosis remained unaffected. (Adapted from Jackson et al., Stem Cells. 2016 Aug; 34(8): 2210-23)

Data analysis

In vitro experiments using 96-well and 24-well plates were performed in triplicate, of which means ± SD were calculated for at least 3 independent experiments. For flow cytometry median fluorescence intensity was calculated using FlowJo software version 7. Data were tested for normality by plotting histograms of frequency distribution and using the D’Agostino and Pearson Omnibus normality test in GraphPad Prism 5. Comparisons of parametric data were analysed by Student’s t-test, one-way or two-way ANOVA for multiple groups. Post hoc analysis using the Bonferroni method was then used to test where significance lay. For non-parametric data the Mann Whitney U and Kruskal Wallis test were used with the Dunn’s method as a post test. Statistical significance was considered when P < 0.05 and all data are displayed as mean ± SD. All Statistical analysis was performed using GraphPad Prism version 5. All of the above statistical information and supporting documentation can be found in Jackson et al., Stem Cells. 2016 Aug; 34(8):2210-23 with URL: http://onlinelibrary.wiley.com/doi/10.1002/stem.2372/full.

Notes

  1. The 5 min incubation step allows mononuclear cells to adhere to the haemocytometer and are easily distinguished next to lymphocytes. Monocytes appear ‘blurry’, ‘splodgy’ or ‘ghost-like’ whereas lymphocytes appear much clearer and sharper. In the case where the original suspension is too dense with cells, a 1:10 or 1:20 dilution is required to properly visualise mononuclear cells.
  2. As the MitoTracker probe is supplied in a reduced form, it may be susceptible to oxidases in serum, and therefore the staining solution consists of up to 1% FCS content to allow for maximum staining potential (https://www.thermofisher.com/order/catalog/product/M22426)
  3. All stimulation experiments with co-culture of MSC and MDMs are performed in 1% FBS RPMI media.
  4. Calculation of multiplicity of infection MOI
    For E. coli K1, an optical density of 0.25-0.3 at 600 nm equates to approximately 1 x 108 CFU/ml of bacteria. This varies for each strain of E.coli and indeed for different types of bacteria. Therefore beginning bacterial work, a growth curve (CFU/ml vs. time) and also absorbance (OD) vs. CFU/ml must be performed to accurately estimate bacterial rates of growth and also how OD varies with bacterial concentration. Once this is established, MOI can then be estimated. This is the number of bacterial cells added per MDM in each infection. See example below.
    Initial MDM seeding density = 3 x 105 cells (24 well plate)
    Starting OD600 nm (0.25-0.3) = 1 x 108 CFU/ml of E. coli
    Therefore MOI 1 = 1 x 105 CFU/ml of E. coli (1 bacterial cell:1 MDM cell)
                    MOI 10 = 1 x 106 CFU/ml of E. coli (10:1)
                    MOI 100 = 1 x 107 CFU/ml of E. coli (100:1)

Recipes

  1. Complete 1%FBS RPMI
    500 ml RPMI 1640
    5 ml of FCS (1%) or 50 ml FCS (10%)
    5 ml Pen-Strep (1%)
  2. Complete RPMIGM-CSF
    500 ml RPMI 1640
    50 ml FCS (10%)
    5 ml Pen-Strep (1%)
    10 ng/ml GM-CSF
  3. Complete α-MEM
    500 ml α-MEM
    5 ml of FCS (1%) or 80 ml FCS (16.5%)
    5 ml Pen-Strep (1%)
    5 ml L-glutamine (1%)
  4. Mitochondrial staining solution (1% FBS α-MEMMITO)
    500 ml α-MEM
    5 ml of FCS (1%)
    5 ml Pen-Strep (1%)
    5 ml L-glutamine (1%)
    200 nM MitoTracker Deep Red FM probe
  5. Cytochalasin B solution (1% FBS α-MEMCYTO)
    500 ml α-MEM
    5 ml of FCS (1%)
    5 ml Pen-Strep (1%)
    5 ml L-glutamine (1%)
    500 nM cytochalasin B or same volume for DMSO (vehicle control)
  6. 1% FBS PBS
    50 ml sterile PBS
    0.5 ml of FCS (1%)
  7. 4% paraformaldehyde (PFA)
    a. Add 4 g of paraformaldehyde to 50 ml of double distilled H2O (ddH2O)
    b. Add 1 ml 1 N NaOH to the solution and heat on the heating block at 60 °C until PFA is dissolved
    c. Add 10 ml of 10x PBS to the mixture and allow to cool to room temperature
    d. The pH is then adjusted to 7.4 with 1 N HCl
    e. Adjust the final volume of the mixture to 100 ml using ddH2O
    f. Use a 0.45 µM membrane to filter the solution and remove debris. Aliquots may be stored up to 3-6 months at -20 °C
  8. Strong and weak block for immunofluorescent staining
    Strong block: 45 ml PBS + 5 ml of NGS (10%)
    Weak block: 49 ml PBS + 1 ml of NGS (2%)
  9. 0.5% saponin
    50 mg of saponin powder
    10 ml sterile PBS

Acknowledgments

This work was funded by Medical Research Council of the UK MR/L017229/1 (ADK), NHLBI HL51854 (MAM). Some of the materials employed in this work were provided by the Texas A&M Health Science Center College of Medicine Institute for Regenerative Medicine at Scott & White through a grant from NCRR of the NIH, Grant # P40RR017447. This protocol was adapted from our publication (Jackson et al., 2016).

References

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  2. de Almeida, M. C., Silva, A. C., Barral, A. and Barral Netto, M. (2000). A simple method for human peripheral blood monocyte isolation. Mem Inst Oswaldo Cruz 95(2): 221-223.
  3. Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F., Krause, D., Deans, R., Keating, A., Prockop, D. and Horwitz, E. (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. The international society for cellular therapy position statement. Cytotherapy 8(4): 315-317.
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  5. Gupta, N., Krasnodembskaya, A., Kapetanaki, M., Mouded, M., Tan, X., Serikov, V. and Matthay, M. A. (2012). Mesenchymal stem cells enhance survival and bacterial clearance in murine Escherichia coli pneumonia. Thorax 67(6): 533-539.
  6. Gupta, N., Su, X., Popov, B., Lee, J. W., Serikov, V. and Matthay, M. A. (2007). Intrapulmonary delivery of bone marrow-derived mesenchymal stem cells improves survival and attenuates endotoxin-induced acute lung injury in mice. J Immunol 179(3): 1855-1863.
  7. Islam, M. N., Das, S. R., Emin, M. T., Wei, M., Sun, L., Westphalen, K., Rowlands, D. J., Quadri, S. K., Bhattacharya, S. and Bhattacharya, J. (2012). Mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury. Nat Med 18(5): 759-765.
  8. Jackson, M. V., Morrison, T. J., Doherty, D. F., McAuley, D. F., Matthay, M. A., Kissenpfennig, A., O'Kane, C. M. and Krasnodembskaya, A. D. (2016). Mitochondrial transfer via tunneling nanotubes is an important mechanism by which mesenchymal stem cells enhance macrophage phagocytosis in the in vitro and in vivo models of ARDS. Stem Cells 34(8): 2210-2223.
  9. Krasnodembskaya, A., Samarani, G., Song, Y., Zhuo, H., Su, X., Lee, J. W., Gupta, N., Petrini, M. and Matthay, M. A. (2012). Human mesenchymal stem cells reduce mortality and bacteremia in gram-negative sepsis in mice in part by enhancing the phagocytic activity of blood monocytes. Am J Physiol Lung Cell Mol Physiol 302(10): L1003-1013.
  10. Krasnodembskaya, A., Song, Y., Fang, X., Gupta, N., Serikov, V., Lee, J. W. and Matthay, M. A. (2010). Antibacterial effect of human mesenchymal stem cells is mediated in part from secretion of the antimicrobial peptide LL-37. Stem Cells 28(12): 2229-2238.
  11. Lee, J. W., Krasnodembskaya, A., McKenna, D. H., Song, Y., Abbott, J. and Matthay, M. A. (2013). Therapeutic effects of human mesenchymal stem cells in exvivo human lungs injured with live bacteria. Am J Respir Crit Care Med 187(7): 751-760.
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  13. Liu, K., Ji, K., Guo, L., Wu, W., Lu, H., Shan, P. and Yan, C. (2014). Mesenchymal stem cells rescue injured endothelial cells in an in vitro ischemia-reperfusion model via tunneling nanotube like structure-mediated mitochondrial transfer. Microvasc Res 92: 10-18.
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简介

间充质干/基质细胞(MSC)是成年干细胞,已被证明可以改善急性呼吸窘迫综合征(ARDS)和败血症临床前模型中的存活,增强细菌清除和减轻炎症。这些疾病的特征在于通常受细菌感染支持的不受控制的炎症。 MSC免疫调节作用的机制尚未完全了解。我们试图研究与肺泡巨噬细胞(AM),在肺炎症反应和抗菌防御中起重要作用的专业吞噬细胞的MSC细胞接触依赖性通信。通过使用基本的直接共培养系统,共聚焦显微镜和流式细胞术,我们通过隧道纳米管(TNT)显现并有效量化了MSC线粒体转移到AM。为了模拟人类AM,在粒细胞巨噬细胞集落刺激因子(GM-CSF)存在下,从人供体血液中分离原代单核细胞并分化成巨噬细胞(单核细胞衍生的巨噬细胞,MDM),从而允许适应AM样表型(de Almeida等人,2000; Guilliams等人,2013)。人骨髓衍生的MSC用线粒体特异性荧光染色标记,广泛洗涤,以1:20(MSC / MDM)的比例接种到具有MDM的组织培养板中并共培养24小时。通过共焦显微镜观察TNT形成和线粒体转移,并通过流式细胞术半定量。通过使用我们在这里描述的方法,我们确定MSC使用TNT作为将线粒体转移到巨噬细胞的手段。进一步的研究表明,线粒体转移增强巨噬细胞氧化磷酸化和吞噬作用。当TNT形成被细胞松弛素B阻断时,MSC对巨噬细胞吞噬作用的影响被完全消除。这是第一个研究表明TNT介导的线粒体转移从MSC到先天免疫细胞。

背景 来自临床前研究的数据,包括我们集团的研究(Xu et al。,2007和2008; Nemeth等人,2009; Gupta等人& 2010年和2012年; Krasnodembskaya等人,2010年和2012年; Mei等人,2010; Lee等人。 ,2013年;杰克逊等人,2016)证明了MSC作为未来基于细胞的治疗ARDS(肺的有害的高热症状)的治疗的强大潜力。在这些研究中,MSC已经展示了再生,免疫调节和抗微生物作用,因此为ARDS中的MSC I期和II期临床试验设计提供了依据(Zheng等,2014) ; Wilson等,,2015)。然而,尽管将MSC快速翻译成临床试验,但MSC缓解ARDS症状的机制仍然需要充分阐明。最近的研究报道了MSC通过TNT通过线粒体转移调节肺上皮细胞和内皮细胞,导致宿主细胞生物能量的改善(伊斯兰教,2012; Ahmad等人, 2014年; Li等人,2014; Liu等人,2014)。在ARDS中,过多的肺部炎症是其中肺泡巨噬细胞(AM)是突出细胞的疾病的主要特征之一。他们编排肺泡中的炎症反应,并在肺细菌清除中发挥重要作用(Ware和Matthay,2000; Jackson等人,2016)。
&NBSP;该协议允许我们研究TNT介导的体外MDM之间的细胞器转移过程的功能效应,并使用相同的染色方案,体内小鼠肺泡巨噬细胞 (Jackson等人,2016)。虽然我们研究的主要焦点是线粒体转移,但是该方案可以适应于对其他细胞器或甚至荧光标记分子的转移的研究的轻微修改。

关键字:间充质干细胞, 巨噬细胞, 线粒体转移, ARDS, 吞噬作用, 氧化磷酸化

材料和试剂

  1. 提取人源供体的单核细胞Buffy Coats
    1. 50ml Falcon管(SARSTEDT,目录号:62.554.502)
    2. T175培养瓶(Thermo Fisher Scientific,Thermo Scientific TM,目录号:178883)
    3. 无菌巴斯德移液器(BIOLOGIX GROUP LTD技术,目录号:30-0138A1)
    4. 封面
    5. 组织培养包被的6孔板(Thermo Fisher Scientific,Thermo Scientific TM,目录号:140675)
    6. Buffy外套是从北爱尔兰输血服务(NIBTS)获得的,遵循贝尔法斯特女王大学学校研究伦理委员会的伦理批准
    7. Hank's缓冲盐溶液(HBSS)No Ca 2 + 或Mg 2 + (Thermo Fisher Scientific,目录号:14170138)
    8. Ficoll-Paque(GE Healthcare,目录号:17-5442-03)
    9. 完成RPMI 1640(Thermo Fisher Scientific,Gibco TM ,目录号:21875034)
    10. 胎牛血清(FCS)(热灭活)(Thermo Fisher Scientific,Gibco TM,目录号:10270106)
    11. 粒细胞巨噬细胞集落刺激因子(GM-CSF)(PeproTech,目录号:300-03)
    12. 青霉素/链霉素10,000U/ml(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
    13. 台an蓝(Thermo Fisher Scientific,Gibco TM ,目录号:15250061)
    14. 完成1%FBS RPMI(见配方)
    15. 完整的RPM子IGM-CSF (见配方)

  2. 人骨髓间充质干细胞培养(MSC)
    1. 50ml Falcon管(SARSTEDT,目录号:62.554.502)
    2. 8孔室玻片(Sigma-Aldrich,目录号:C7182)
    3. 组织培养包被的6孔和24孔板(Thermo Fisher Scientific,Thermo Scientific TM,目录号:140675,142475)
    4. Pechiney PM999 Parafilm(Bemis,目录号:PM999)
    5. 显微镜幻灯片
    6. 无菌Eppendorf管(SARSTEDT,目录号:72.690.001)
    7. 流量管(SARSTEDT,目录号:55.475)
    8. 人骨髓来源的MSC从得克萨斯A& M健康科学中心医学院的再生医学研究所(德克萨斯州Temple)的NIH储存库获得。细胞符合国际细胞治疗学会(Dominici等人,2006)定义的MSCs分类的所有标准。
    9. 液氮
    10. Dulbecco的磷酸盐缓冲盐水(DPBS)(Thermo Fisher Scientific,Gibco TM,目录号:14190094)
    11. 完整的α-MEM(Thermo Fisher Scientific,Gibco TM,目录号:22561021)
    12. 青霉素/链霉素10,000U/ml(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
    13. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:25030024)
    14. 细胞松弛素B(Sigma-Aldrich,目录号:C6762)
    15. 10x胰蛋白酶(Thermo Fisher Scientific,Gibco TM ,目录号:15250061)
    16. 胎牛血清(FCS)(热灭活)(Thermo Fisher Scientific,Gibco TM,目录号:10270106)
    17. 台盼蓝(Thermo Fisher Scientific,Gibco TM ,目录号:15250061)
    18. MitoTracker深红色FM探针(APC)(Thermo Fisher Scientific,Molecular Probes TM,目录号:M22426)
    19. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418)
    20. 完成α-MEM(参见食谱)
    21. 线粒体染色溶液(1%FBSα-MEM 1%MITO )(参见食谱)
    22. 细胞松弛素B溶液(1%FBSα-MEM <1%CYTO )(参见食谱)

  3. 共焦显微镜
    1. 0.45μm滤膜(SARSTEDT,目录号:83.1826.001)
    2. Pechiney PM999 Parafilm(Bemis,目录号:PM999)
    3. 纸巾
    4. 显微镜幻灯片
    5. 盖子
    6. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:10010023)
    7. 多聚甲醛(PFA)(Sigma-Aldrich,目录号:158127)
    8. 1N NaOH(Sigma-Aldrich,目录号:S2770)
    9. 1N HCl(Sigma-Aldrich,目录号:H9892)
    10. 正常山羊血清(NGS)(Thermo Fisher Scientific,Invitrogen,目录号:31872)
    11. 小鼠抗人CD45一抗(Abcam,目录号:ab8216)
    12. 小鼠IgG1同种型(Abcam,目录号:ab81032)
    13. 山羊抗兔子Alexafluor 405二抗(Abcam,目录号:ab175655)
    14. Brightmount/Plus水性安装介质(Abcam,目录号:ab103748)
    15. 指甲油
    16. 1%FBS PBS(参见食谱)
    17. 4%多聚甲醛(PFA)(见配方)
    18. 强和弱块免疫荧光染色(见配方)

  4. 流式细胞仪
    1. 细胞刮刀(Fisher Scientific,目录号:08-100-241)
    2. Sarstedt 5毫升聚苯乙烯圆底流量管(SARSTEDT,目录号:55.1578)
    3. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:10010023)
    4. 人FcR结合抑制剂(Thermo Fisher Scientific,eBioscience TM,目录号:14-9161-73)
    5. 抗人CD45(PE)抗体(Thermo Fisher Scientific,eBioscience TM,目录号:12-9459-41)
    6. 同种型对照(IgG1 kappa)(Thermo Fisher Scientific,eBioscience TM,目录号:12-4714)
    7. Zombie Aqua Fixable Dye(BioLegend,目录号:423101)

  5. 细菌培养和吞噬分析
    1. 大肠杆菌K1型菌株
    2. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:10010023)
    3. LB肉汤(Lennox)(Sigma-Aldrich,目录号:L3022)
    4. LB琼脂培养液(Lennox)(Sigma-Aldrich,目录号:L2897)
    5. Hank's缓冲盐溶液(HBSS)No Ca 2 + 或Mg 2 + (Thermo Fisher Scientific,目录号:14170138)
    6. 完成RPMI 1640(Thermo Fisher Scientific,Gibco TM ,目录号:21875034)
    7. 皂苷(Sigma-Aldrich,目录号:47036)
    8. 庆大霉素(Sigma-Aldrich,目录号:G1397)
    9. 完成1%FBS RPMI(见配方)
    10. 0.5%皂角苷(参见食谱)

设备

  1. 水浴(37°C)(Grant仪器,型号:JBN12)
  2. 离心机(Eppendorf,型号:5810 R)
  3. 层流柜(包含空气解决方案,型号:BioMAT 2安全柜2类)
  4. 血细胞计数器和盖玻片(Hawksley Medical and Laboratory Equipment,型号:AC1000 Improved Neubauer,catalog number:BS 748)
  5. 孵化器(Panasonic Biomedical,型号:MCO-170AIC-PE)
  6. Dmi1显微镜(Leica Microsystems,型号:DMi1)
  7. TCS SP5 II Leica共聚焦显微镜(Leica Microsystems,型号:TCS SP5 II)
  8. BD FACSCanto II流式细胞仪(BD,型号:BD FACSCanto II)
  9. 涡旋(Cole-Palmer仪器,目录号:UY-86579-20)
  10. 2-20微升移液管(Gilson,目录号:F123600)
  11. 20-200μl移液器(Gilson,目录号:F144565)
  12. 100-1,000μl移液器(Gilson,目录号:F144566)
  13. Midi Plus TM 移液管控制器自动(Sartorius,目录号:710931)

软件

  1. FlowJo软件(FlowJo)
  2. Prism 5软件(GraphPad软件)
  3. LAS-AF软件(Leica共焦显微镜)
  4. FACS DIVA软件(流式细胞仪)

程序

  1. 从供体Buffy Coats中提取单核细胞
    1. 单一供体白细胞血沉棕黄层是从贝尔法斯特血液输血中心(NIBTS)获得的,或者遵循伦理批准(大约50毫升/血袋/志愿者)的志愿者。
    2. 在分离前1-2小时将所有培养基(HBSS,1%FBS RPMI和10%FBS RPMI)预热至1-2小时,然后分离前将离心机预温至20℃。
    3. 在层流柜下,将供体血液倒入T75烧瓶中,并使用无菌HBSS稀释(1:2),并通过倒置混合。
    4. 将15 ml Ficoll-Paque吸入4 x 50ml的猎鹰。
    5. 将稀释的血液以45度角轻轻地分层到Ficoll上,注意不要混合各层。用HBSS补充剩余的管,使最终体积达到50ml。
    6. 将所有管以480 x g,20℃离心20分钟而不用制动。
    7. 旋转后,将珠光白色层(血沉棕黄层,如图1所示淋巴细胞,单核细胞和血小板层)定位,通过等离子体密度梯度界面沉淀。使用巴斯德吸管将其提取成新的无菌50ml隼

      图1.用于说明从外周血中分离单核细胞的示意图将血液分层到Ficoll上,如上所述离心分离而不用制动器,将血液成分分成多层。巴斯德吸管用于小心地去除下游加工中使用的淋巴细胞,单核细胞和血小板层。

    8. 为了洗涤细胞,将40ml HBSS加入到单核细胞中,并通过倒置混合
    9. 再次以290 x g,4℃离心5分钟制动。缓慢吸出上清液(注意颗粒可能变松,再加入40 ml HBSS)。重复此洗涤步骤3次。
    10. 最后一次洗涤后,将沉淀重悬在10ml完整的1%FBS RPMI中,并通过涡旋或倒置充分混合。
    11. 在血细胞计数器上放置盖子,并通过毛细作用将10μl细胞混合物装入装载通道。
    12. 在37℃,5%CO 2培养箱中孵育至少5分钟(见注1)。
    13. 此后,在20倍放大倍率的显微镜下观察细胞。计数所有单核细胞(见注1)。
    14. 然后使用3ml完全1%FBS RPMI(参见表1其他种子密度),将细胞以6孔板的1×10 6个细胞/孔的密度接种。在37℃,5%CO 2孵育至少2小时以确保单核细胞已经粘附到该孔的塑料上。

      表1. MSC和MDM(1:20比率)播种密度
      否。的MSC /井
      否。的
      MDM /井
      牌类型
      最终音量
      (ml)的1%FBS RPMI<

      实验分析技术
      5 x 10 4
      1 x 10 6
      6井
      3
      流式细胞仪/Western
      1.5 x 10 4
      3 x 10 5
      24井
      1
      ELISA/bioplex /吞噬分析
      2.5 x 10 3
      5 x 10 4
      8孔室幻灯片
      0.2
      共焦显微镜


    15. 此后,用HBSS洗涤非贴壁细胞(淋巴细胞)两次(通过加入1ml HBSS,轻微搅动,然后抽吸废物)。
    16. 为了促进单核细胞衍生的巨噬细胞(MDM)成熟,用3ml完整的RPM IGM-CSF 替换完整的1%FBS RPMI(参见食谱)。
    17. 使细胞在37℃,5%CO 2下静置5-7天。
    18. 5-7天后,用HBSS洗涤细胞两次,并用3ml完整的1%FBS RPMI代替。在进行刺激实验之前让细胞休息1小时。

  2. 人类间充质基质细胞培养
    1. MSC扩展和存储
      1. MSC在美国得克萨斯州从1号或2号(P1-P2)的1毫升细胞液中以液氮的形式运输,这意味着它们在培养物中经历了至少一个或两个阶段的细胞分裂。对于实验用途(参见"准备P3 MSC实验用途")细胞仅用于P3-P5。
      2. 抵达后不久,将1个小瓶除霜,并在T175烧瓶中预热的15 ml完全16%FBSα-MEM中培养不超过24 h。
      3. 通过在24小时后分裂细胞保留通道数,并使用每个烧瓶21,000个细胞的密度扩增它们。 P2小瓶可以进行额外的扩张以在P3存储。在P2或P3的液氮储存中储存细胞(100万个细胞/小瓶)。
    2. 准备P3 MSC进行实验用途
      一旦准备好使用,在水浴(37°C)中除冰1×3 3的MSC冷冻机,并将全部1ml置于15-20ml的完全16%FBSα-MEM中,确保每隔一天进食。细胞保持培养直至P5-P6。

  3. 人间质基质细胞(MSC和单核细胞衍生的巨噬细胞(MDM))的直接共培养
    1. 预热DPBS,HBSS,在37℃的水浴中,在开始前至少半小时,完成10%FBS RPMI,1%FBS RPMI,16.5%FBSα-MEM和1%FBSα-MEM实验。
      以下工作必须在机柜灯关闭的生物安全柜内完成。
    2. 在70-80%汇合时,为了预先染色MSC线粒体,从T175烧瓶中吸出较老的16%FBSα-MEM,并加入20ml染色溶液(1%FBSα-MEM MITO 食谱和注2]),37℃,5%CO 2孵育约30分钟。
    3. 对于MSC用细胞松弛素B进行预处理,至少在染色溶液(1%FBSα-MEM 子> MITO )。由于Cyto B可溶于DMSO,因此请确保有一个控制瓶的细胞用于DMSO载体对照
    4. 从烧瓶中吸取1%FBSα-MEM CYTO ,并用1%FBSα-MEM MITO替代,并在37℃,5%CO 2下孵育30分钟, sub> 2 。
    5. 此后,染色溶液从MSC吸出,细胞在5ml DPBS中洗涤至少三次。
    6. 吸入DPBS并加入7ml 1x胰蛋白酶(DPBS中稀释),并在培养箱中放置约2-5分钟。
    7. 一旦细胞被适当地分离,加入7ml完整的α-MEM以抵抗胰蛋白酶作用并将细胞移液到50ml的猎鹰中。
    8. 将细胞离心离心分离5分钟。
    9. 在此期间,通过吸取较老的培养基并用1-2ml HBSS洗涤两次来制备MDM。
    10. 吸出剩余的HBSS,并将MDM在培养箱中放置200μl(室载玻片),1 ml(24孔板)或3 ml(6孔板)1%FBS RPMI。
    11. MSC离心后,吸出上清液,并将沉淀重悬于1 ml 1%FBS RPMI(见注3)。
    12. 使用血细胞计数器计数细胞,使用台盼蓝排除,取决于实验所需的孔尺寸,种子相应的细胞密度和与MDM共培养24小时(见表1)。
    13. 进行共焦显微镜或流式细胞术程序。

  4. 共焦显微镜染色程序
    1. 在室载玻片上共培养后,小心地吸出培养基,并使用200μl冰冷的PBS洗涤细胞两次。
    2. 吸出PBS并用100μl的4%PFA溶液固定细胞。
    3. 在室温下温和搅拌约15分钟。
    4. 再次用PBS洗涤两次后,继续用100μl强阻断细胞至少1小时。
    5. 用PBS洗涤两次,并在弱块中加入50μl一抗抗体小鼠抗人CD45一抗或同种型对照小鼠IgG1(稀释1:200)。用Parafilm密封载玻片,并在4℃下孵育载玻片,搅拌过夜。
    6. 第二天重复PBS清洗步骤,然后再加入50μl二抗山羊抗兔亚硝酸405二抗(在弱块中稀释1:1,000)1小时。在室温黑暗中孵育一小时。
    7. 紧接着,重复PBS洗涤步骤,注意不要将载玻片暴露在光照下超过10分钟。
    8. 轻轻地将腔室滑块轻轻敲打到纸巾上,以除去任何多余的PBS清洗液,然后小心地取出塑料腔室部分以分离下面的显微镜载玻片。
    9. 加入约2-3滴安装介质,并将盖玻片放在染色细胞的区域上,确保除去所有气泡。允许在黑暗中风干30-45分钟。
    10. 当干燥时,使用边缘周围的商业指甲油作为胶水并将盖玻片固定到位。
    11. 使指甲油在室温下在黑暗中干燥。幻灯片还可以在黑暗中用Parafilm长时间密封保存在4°C,以备将来分析。
    12. 使用共聚焦显微镜进行观察(图2)

      Fi gure 2。通过共焦显微镜观察MSC线粒体转移到MDM的可视化 。A.MDM表达CD45标记(蓝色); B.线粒体用MitoTracker探针(红色)染色; C.共培养24 h后,观察到线粒体阳性TNT,从MSC(箭头)突出长达200μm。从MSC到MDM的线粒体转移通过蓝色和红色染色(粉红色)的共定位来证明。 (以10×63放大倍率拍摄图像,比例尺=75μm)。 (改编自Jackson e 干细胞。2016年8月; 34(8):2210-23)

  5. 流式细胞仪染色程序
    1. 共培养后立即将6孔板放在冰上,然后从培养基中取出
    2. 将1ml冰冷的1%FBS PBS加入到细胞共培养物中,并使用细胞刮刀轻轻刮擦整个孔,以便从塑料表面轻轻地分离细胞。
    3. 一旦分离,将细胞置于5ml聚苯乙烯圆形底部的流动管中,并将细胞以800×g离心5分钟。
    4. 为了在培养和分离后测试细胞的活力,需要包括活/死控制。这需要一个单独的未染色细胞管,并添加活/死标记染色Zombie Aqua(根据制造商的协议使用)。
    5. 滗出上清液,将沉淀物浸入约100μl的1%FBS PBS中。
    6. 剧烈涡旋,并向细胞中加入2.5μg/1×10 6个细胞的FcR结合抑制剂,并再次旋转。
    7. 在黑暗中和冰上休息30分钟。
    8. 此后,向细胞悬液中直接加入5μl/1×10 6细胞的抗人CD45(PE)或同型对照(IgG1 kappa),再次涡旋以彻底混合。
    9. 在黑暗中和冰上休息20分钟。
    10. 然后加入1ml 1%FBS PBS洗涤细胞并处理任何未结合的抗体。彻底旋涡。
    11. 离心悬浮液,如前所述,800 x g 5分钟。
    12. 弃上清,最后重复洗涤步骤。将沉淀重悬于200μl的1%FBS PBS中,并将细胞置于冰上以准备流式细胞术。
    13. 使用FACS DIVA软件进行分析。为了排除MSC-巨噬细胞聚集体,将单个细胞作为W-FSC与A-FSC进行门控,并从中我们将CD45-PE 个体定义为MitoRed-APC MDM和MitoRed-APC + CD45-PE - 群体作为MSC(图3)。


      Figu re 3。通过流式细胞术将线粒体 从MSC转移到 MDM。 A-B。活细胞被门控为W-FSC对A-FSC,并且由此我们定义C.C.CD45-PE不与MSC共同培养的MDM群体为MitoRed-APC阴性。 D. APC + 单独MSC铺线对于线粒体是阳性的,但对于巨噬细胞标志物CD45-PE
      是阴性的。 E.共培养4小时后,MDM获得了由MitoRed荧光标记APC显示的92%的MSC线粒体。 F.直方图阻碍了MSC群体中MitoRed荧光的减少,表明线粒体对MDM群体的强烈捐赠。 (改编自Jackson e 干细胞。 2016年8月34(8):2210-23)

  6. 细菌培养和吞噬分析
    1. 用储存的大肠杆菌K1型菌株(无菌LB肉汤/15%甘油)的等分试样接种15ml LB肉汤,并在37℃下孵育以生长过夜培养物。
    2. 使用24孔板与MDM和MSC建立直接共培养(见表1的种子密度)。
    3. 对于E。大肠杆菌,1×10 8个菌落形成单位(CFU/ml)的总活菌数等于600nm处的约0.25-0.3的光密度(OD)。 >
    4. 旋转15,000 x g的细菌5分钟,并使用无菌的1 ml HBSS重新悬浮沉淀。
    5. MDM和MSC共培养24小时后,使用感染的多重感染10(MOI 10,参见注释4进行MOI计算)感染细胞,使用HBSS作为载体对照。
    6. 在37℃,5%CO 2孵育4小时。
    7. 此后,从细胞中取出上清液,用于定量细胞外细菌,并在无菌PBS中稀释10倍至10"-6"。
    8. 将3 x 20μl每次稀释的斑点移至LB琼脂上,并在37℃下孵育过夜
    9. 细胞内细菌用1 ml无菌PBS洗涤细胞3次,抽吸洗涤,然后加入300μg/ml庆大霉素(稀释于1%FBS RPMI),每孔100μl,37℃孵育1小时以杀死粘附细菌
    10. 此后,将细胞在1 ml无菌PBS中洗涤3次,抽吸洗涤,加入100μl0.5%皂角蛋白溶解细胞,使细菌完好无损。
    11. 5分钟后,向每个孔中加入900μl无菌PBS稀释皂苷,再次重复稀释,每次稀释。 37℃培养板。
    12. 24小时内计算CFU/ml(图4D-4G)。


      图4.细胞松弛素B阻断TNT介导的线粒体转移,从而阻止MSC对MDM的功能影响。A.共焦显微镜显示共培养中具有MDM(蓝色)的独特的心轴MSC形状(红色)。 TNT显示具有强大的线粒体转移到MDM。 B.细胞松弛素B预处理MSC抑制肌动蛋白丝延长,导致TNT结构丧失而呈现圆形。由于MSC到MDM的可能的微泡分泌(放大倍率为10×63,比例尺=50μm),线粒体转移仍然是明显的。 C.细胞松弛素B处理MSC显示,在共培养的MDM中,MitoRed平均荧光强度(MFI)降低了50%(* Mann Whitney U检验)。 D.共培养的大肠杆菌感染4小时后,未经处理的MSC显着减少细胞外CFU,同时在巨噬细胞中同时升高细胞内CFU,通过用分离的MSC线粒体刺激(F + G)。 H.这种作用与细胞松弛素B处理的MSC完全消除,因为即使在其存在下,MDM吞噬作用仍然不受影响。 (改编自Jackson等人,Stem Cells,2016 Aug; 34(8):2210-23)

数据分析

使用96孔和24孔板进行体外实验,一式三份进行,其中计算了至少3次独立实验的±SD。对于流式细胞术,使用FlowJo软件版本7计算中值荧光强度。通过绘制频率分布的直方图并使用GraphPad Prism 5中的D'Agostino和Pearson Omnibus正态性测试来测试数据的正常性。参数数据的比较由Student's em> t -test,多组的单向或双向ANOVA。然后使用Bonferroni方法进行事后分析,检验其中的显着性。对于非参数数据,Mann Whitney U和Kruskal Wallis测试与Dunn's方法一起用于后测试。当P 时考虑统计学意义0.05,所有数据显示为平均值±SD。所有统计分析均使用GraphPad Prism版本5进行。以上所有统计信息和支持文档均可在Jackson等人的中找到,干细胞。 2016年8月34(8):2210-23,URL: http ://onlinelibrary.wiley.com/doi/10.1002/stem.2372/full

笔记

  1. 5分钟的孵育步骤允许单核细胞粘附到血细胞计数器,并且容易区分淋巴细胞。单核细胞出现"模糊","splodgy"或"鬼样",而淋巴细胞看起来更清晰和更清晰。在原始悬浮液细胞太密的情况下,需要1:10或1:20的稀释度来正确地观察单核细胞。
  2. 由于MitoTracker探针以还原的形式提供,它可能对血清中的氧化酶敏感,因此染色溶液由高达1%的FCS含量组成,以允许最大染色电位( https://www.thermofisher.com/order/catalog/product/M22426
  3. 在1%FBS RPMI培养基中进行MSC和MDM共培养的所有刺激实验。
  4. 计算感染的多重性MOI
    对于E。大肠杆菌K1,600nm处的光密度为0.25-0.3等于大约1×10 8 CFU/ml细菌。这对于大肠杆菌的每个菌株以及对于不同类型的细菌而言是不同的。因此,开始细菌工作时,必须进行生长曲线(CFU/ml对时间)和吸光度(OD)对CFU/ml的准确估计细菌生长速率,以及OD如何随细菌浓度而变化。一旦建立,可以估计MOI。这是每个感染中每MDM添加的细菌细胞数。见下面的例子 初始MDM接种密度= 3×10 5个细胞(24孔板)
    起始OD 600nm(0.25-0.3)= 1×10 8 cfu/ml的E。大肠杆菌
    因此,MOI 1 = 1×10 5。大肠杆菌(1个细菌细胞:1个MDM细胞)
                         MOI 10 = 1 x 10 6 CFU/电子。大肠杆菌(10:1)
                         MOI 100 = 1 x 10 7 CFU/电子。大肠杆菌(100:1)

食谱

  1. 完成1%FBS RPMI
    500毫升RPMI 1640
    5ml FCS(1%)或50ml FCS(10%)
    5毫升Pen-Strep(1%)
  2. 完成RPMI GM-CSF
    500毫升RPMI 1640
    50ml FCS(10%)
    5毫升Pen-Strep(1%)
    10 ng/ml GM-CSF
  3. 完成α-MEM
    500 mlα-MEM
    5ml FCS(1%)或80ml FCS(16.5%)
    5毫升Pen-Strep(1%)
    5 ml L-谷氨酰胺(1%)
  4. 线粒体染色溶液(1%FBSα-MEM MITO )
    500 mlα-MEM
    5 ml FCS(1%)
    5毫升Pen-Strep(1%)
    5 ml L-谷氨酰胺(1%)
    200 nM MitoTracker深红色FM探头
  5. 细胞松弛素B溶液(1%FBSα-MEM CYTO )
    500 mlα-MEM
    5 ml FCS(1%)
    5毫升Pen-Strep(1%)
    5 ml L-谷氨酰胺(1%)
    500nM细胞松弛素B或相同体积的DMSO(载体对照)
  6. 1%FBS PBS
    50ml无菌PBS
    0.5ml FCS(1%)
  7. 4%多聚甲醛(PFA)
    一个。向50ml蒸馏的H 2 O(ddH 2 O)中加入4g多聚甲醛
    湾向溶液中加入1ml 1N NaOH,并在60℃加热块加热至PFA溶解 C。向混合物中加入10毫升10倍的PBS,并使其冷却至室温 天。然后用1N HCl调节pH至7.4 即使用ddH 2 O
    将混合物的最终体积调整至100ml F。使用0.45μM膜过滤溶液并清除碎屑。等分试样可在-20°C下储存3-6个月
  8. 强和弱块免疫荧光染色
    强阻滞:45ml PBS + 5ml NGS(10%)
    弱块:49ml PBS + 1ml NGS(2%)
  9. 0.5%皂角苷
    50毫克皂苷粉
    10ml无菌PBS

致谢

这项工作由英国医学研究理事会MR/L017229/1(ADK),NHLBI HL51854(MAM)资助。这项工作中使用的一些材料由德克萨斯A& M健康科学中心医学院提供,用于再生医学研究所Scott&白人通过NCRR的NIH授予,授予#P40RR017447。这个协议是从我们的出版物(杰克逊等人,2016年)改编而来。

参考

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引用:Jackson, M. V. and Krasnodembskaya, A. D. (2017). Analysis of Mitochondrial Transfer in Direct Co-cultures of Human Monocyte-derived Macrophages (MDM) and Mesenchymal Stem Cells (MSC). Bio-protocol 7(9): e2255. DOI: 10.21769/BioProtoc.2255.
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