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A Co-culture Assay to Determine Efficacy of TNF-α Suppression by Biomechanically Induced Human Bone Marrow Mesenchymal Stem Cells
人骨髓间充质干细胞共培养实验测定生物力学诱导的TNF-α抑制功效   

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Abstract

The beneficial effects of mesenchymal stem cell (MSC)-based cellular therapies are believed to be mediated primarily by the ability of MSCs to suppress inflammation associated with chronic or acute injury, infection, autoimmunity, and graft-versus-host disease. To specifically address the effects of frictional force caused by blood flow, or wall shear stress (WSS), on human MSC immunomodulatory function, we have utilized microfluidics to model WSS at the luminal wall of arteries. Anti-inflammatory potency of MSCs was subsequently quantified via measurement of TNF-α production by activated murine splenocytes in co-culture assays. The TNF-α suppression assay serves as a reproducible platform for functional assessment of MSC potency and demonstrates predictive value as a surrogate assay for MSC therapeutic efficacy.

Keywords: Biomechanical force(生物机械力), Inflammation(炎症), Immunomodulation(免疫调节), Mesenchymal stem cells(间充质干细胞), Potency assay(效能测定), TNF-α(TNF-α)

Background

Immunomodulatory activity of mesenchymal stem cells (MSCs) is mediated by direct cellular interactions and paracrine factors (Singer and Caplan, 2011; English, 2013). MSCs are believed to originate from pericytes that associate with endothelial cells of vasculature within the bone marrow and various tissues (Sacchetti et al., 2007; Crisan et al., 2008). This unique perivascular location positions them in close proximity to inflammatory and other soluble factors in the blood stream, poising them to monitor systemic signals. Indeed, recruitment of mural cells to the endothelium is a key event in vessel maturation, and pericytes play a critical role in vascular maintenance and integrity (Benjamin et al., 1998; Schrimpf et al., 2014). Pericytes likely monitor systemic signals by fluid outflow from arterioles and capillaries through interendothelial clefts or gaps in the basement membrane, which can expose the basolateral surface of endothelial cells outside the vessel to considerable fluid frictional force, or wall shear stress (WSS), that approximates intraluminal forces (Scallan et al., 2010). MSCs and other classes of pericytes might also view the intraluminal environment from openings between vascular endothelial cells by protrusion into the vascular lumen with cytoplasmic projections much like megakaryocytes, though more typically they ensheathe the blood vessel with branching processes (Shepro and Morel, 1993; Murphy et al., 2013). In instances of inflammation or injury, for example due to trauma to the central nervous system, pericytes have been shown to migrate away from microvessels concurrent with perivascular edema and toward injured tissue in association with blood vessel sprouting (Dore-Duffy et al., 2000; Göritz et al., 2011). Cells described as having features of MSCs have been detected circulating in human peripheral blood (Zvaifler et al., 2000), though there is some controversy surrounding evidence for MSCs in the circulation of healthy and even injured individuals (Hoogduijn et al., 2014). In those cases, disruption of endothelial-pericyte interactions could be expected to exacerbate vascular hyperpermeability which could impact migration or intravasation of MSCs (Mills et al., 2013). As MSCs are anchorage-dependent cells, a likely means of motility would include attachment to the vessel wall resulting in direct exposure to intraluminal WSS. In therapeutic applications wherein MSCs are administered intravenously, WSS would be an unavoidable stimulus during handling, infusion, and trafficking (Nitzsche et al., 2017).

We have shown that WSS typical of arterial blood flow promotes signaling through focal adhesion kinase (FAK), NF-κB, and COX2 (Diaz et al., 2017; Lee et al., 2017). Increased COX2 results in elevated prostaglandin E2 (PGE2) biosynthesis. PGE2 secreted by MSCs plays a central role in regulation of innate and adaptive immune cells. Thus, MSCs exposed to WSS more potently suppress immune cell activation in the presence of inflammatory cues (Diaz et al., 2017; Lee et al., 2017). To quantify MSC immunomodulatory activity in cells exposed to fluid flow, we co-cultured MSCs and lipopolysaccharide-activated murine splenocytes in an adaptation of the commonly used mixed lymphocyte reaction (Plumas et al., 2005). TNF-α was measured by species specific ELISA to determine cytokine production from activated murine splenocytes, thus restricting analysis to immune cell activity and enabling separate determinations of cytokine production by human MSCs. Employing this assay as a surrogate measure of MSC potency, we determined that transient exposure of MSCs to fluid shear stress improved their ability to limit activation of immune cells in the presence of inflammatory stimulus. Preconditioning of MSCs by as little as 3 h of WSS in culture was an effective means of enhancing therapeutic efficacy in treatment of a rat traumatic brain injury model. These data demonstrate that WSS enhances the immunomodulatory and neuroprotective function of MSCs. Together with complementary studies implicating PGE2 as a potency marker of MSC therapeutic efficacy (Kota et al., 2017), our studies suggest that mechanotransduction could be leveraged to improve cellular therapies available for patients with neurological injury. This co-culture assay could easily be adapted for analysis of anti-inflammatory potency of MSCs subjected to a variety of treatments, including genetically engineered MSCs.

Materials and Reagents

  1. Falcon culture treated flask, 225 cm2 (Corning, Falcon®, catalog number: 353139 )
  2. Falcon 15 ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352097 )
  3. 5 ml serological pipettes (MIDSCI, catalog number: MWB-5 )
  4. Fisherbrand premium microcentrifuge tubes, 1.5 ml (Fisher Scientific, catalog number: 05-408-129 )
  5. IBIDI µ-Slide VI0.4 ibiTreat, sterile slide (IBIDI, catalog number: 80606 )
  6. Fisherbrand P200 Low Retention Aerosol Barrier pipet tips (Fisher Scientific, catalog number: 02-717-165 )
  7. Falcon Petri Dish 150 x 15 mm (Corning, Falcon®, catalog number: 351058 )
  8. Greiner Petri Dish 35 x 10 mm (Greiner Bio One International, catalog number: 627161 )
  9. 3-Stop silicone tubing, 1.52 mm I.D. (Cole-Parmer, catalog number: SK-07624-36 )
  10. Elbow luer connector (IBIDI, catalog number: 10802 )
  11. Falcon round bottom polypropylene tubes (Corning, Falcon®, catalog number: 352006 )
  12. EASYStrainer, 70 μm cell sieve, sterile (Phenix Research Products, catalog number: TCG-542070 )
  13. Falcon 50 ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352098 )
  14. 1 cc tuberculin syringe plunger
  15. SHARP P1000 Precision Barrier pipet tips (Denville Scientific, catalog number: P1126 )
  16. EASYStrainer, 40 μm cell sieve, sterile (Phenix Research Products, catalog number: TCG-542040 )
  17. 10 ml serological pipettes (MIDSCI, catalog number: MWB-10 )
  18. Fisherbrand Borosilicate glass Pasteur pipettes (Fisher Scientific, catalog number: 13-678-20C )
  19. Paper towel
  20. EMD-Millipore Stericup vacuum filter unit, 500 ml size (EMD Millipore, catalog number: SCGPU05RE )
  21. Parafilm MTM (Bemis, catalog number: PM996 )
  22. Dow Corning silastic laboratory tubing 1.57 mm I.D. x 3.18 mm O.D. (Dow Corning, catalog number: 2415569 )
  23. Human bone marrow (BM) MSC (Whole Bone Marrow aspirates) (AllCells, catalog number: ABM001-0 ) MSCs were isolated from whole bone marrow using a Ficoll gradient followed by plastic adherence and then cultured in MSC media (see Recipes)
    Note: The MSCs used for this work were prescreened for the presence of typical MSC growth, appearance and surface marker expression and expanded for stock cyro-preservation prior to its use (Sekiya et al., 2002; Dominici et al., 2006).
  24. Male C57BL/6 mouse (THE JACKSON LABORATORY, catalog number: 000664 ); recommended age between 2-4 months old
  25. Hyclone Dulbecco’s phosphate buffered saline (DPBS) solution, 500 ml, calcium magnesium free (GE Healthcare, HycloneTM, catalog number: SH30028.FS )
  26. Gibco-Tryp-LE Express enzyme, 1x, 500 ml (Thermo Fisher Scientific, GibcoTM, catalog number: 12604021 )
  27. Gibco-trypan blue solution, 0.4% (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
  28. Atlanta Biological fetal bovine serum (FBS), embryonic stem cell qualified, 500 ml (Atlanta Biologicals, catalog number: S10250 )
  29. Red blood cell lysing buffer hybri-max (Sigma-Aldrich, catalog number: R7767-100ML )
  30. Lipopolysaccharide, BioXtra (Sigma-Aldrich, catalog number: L6529 )
  31. R&D Systems Mouse TNF-alpha Quantikine ELISA kit (R&D Systems, catalog number: MTA00B )
  32. Hyclone MEM alpha modification with glutamine and nucleosides media (GE Healthcare, HycloneTM, catalog number: SH30265.FS )
  33. Gibco Penicillin-streptomycin, 10,000 U/ml (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  34. MSC media (see Recipes)

Equipment

  1. Hettich Rotofix 32A with swing bucket for 15 ml and 50 ml conical tubes (Hettich Lab Technology, model: Rotofix 32A )
  2. Sterile Hood with vacuum suction (The Baker Company, model: SterilGARD® III Advance)
  3. Hausser Scientific Bright-LineTM counting chamber with cover glass (Hausser Scientific, catalog number: 3110V )
  4. P2-20 XL3000i pipettor (Denville Scientific, catalog number: P3950-20A )
    Note: This product has been discontinued.
  5. P20-200 XL3000i pipettor (Denville Scientific, catalog number: P3950-200A )
    Note: This product has been discontinued.
  6. P100-1000 XL3000i pipettor (Denville Scientific, catalog number: P3950-1000A )
    Note: This product has been discontinued.
  7. Sanyo CO2 incubator (SANYO, model: MCO-18AIC )
  8. Ismatec REGLO peristaltic 12 roller pump (Cole-Parmer, catalog number: ISM796B )
  9. Hettich Mikro 200R refrigerated microcentrifuge (Hettich Lab Technology, model: MIKRO 200R )
  10. Colorimetric microplate reader (Molecular Devices, model: SpectraMax M2 )
    Note: This product has been discontinued.
  11. 37 °C water bath (Fisher Scientific, model: Model 210 , catalog number: 15-462-10Q)
    Note: This product has been discontinued.

Procedure

  1. Seeding the MSCs
    1. Prescreened human bone marrow (BM) MSC frozen stock at 2 million per cryo-vial, are thawed and seeded in a Falcon T225 cm2 flask with vented seal cap at 37 °C with 5% CO2. Ideal passage is between 1-5. After 2 to 3 days, 80% adherent cell confluency should be achieved for experimental use.
    2. Remove media completely and add 5 ml of room temperature sterile DPBS to rinse the cell monolayer. Aspirate out completely.
    3. Add 5 ml of Tryp-LE Express dissociation reagent. Allow volume to spread throughout monolayer and place the flask inside the tissue culture incubator for 5-8 min.
    4. Collect the cell suspension by adding 5 ml of MSC media (see Recipes) to curtail the Tryp-LE effects. Add contents into a clean 15 ml conical centrifuge tube.
    5. Re-rinse the flask with 2-3 ml of MSC media to collect remnant cells.
    6. Add contents into the same 15 ml centrifuge tube.
    7. Place the centrifuge tube into a swing bucket tabletop centrifuge (Hettich Rotofix 32A) and spin down suspension at 617 x g (2,000 rpm) for 5 min.
    8. Aspirate out the media mix using a vacuum suction, without disturbing the cell pellet.
    9. Gently tap with fingers the conical end of the 15 ml centrifuge tube, to aid in pellet disbursal. Re-suspend the cell pellet with 2 ml of MSC media by gently mixing with a 5 ml serological pipette.
    10. Once mixed, quickly take a 10 μl aliquot of cell suspension for cell counting, and place it into a fresh 1.5 ml microcentrifuge tube. Add to the suspension, 10 μl of trypan blue. Mix by pipetting 5-8 times.
    11. Take a final 10 μl aliquot of this solution and place into a hemocytometer (Hausser Scientific) with cover glass (Figure 1).


      Figure 1. Cell counting profile for MSCs. Example of one of the four quadrants at 200 μm.

    12. Count the four outer quadrants. Cell numbers should be close to and not fewer than approximately 30 cells per quadrant (Figure 1).
    13. Calculate the cell concentration (number of cells per ml) by the sum of 4 quadrant counts multiplied by the dilution factor of 2 and multiplied by the factor 2,500. This will equal cells per ml.
    14. Once the cell number per ml is calculated, take the required aliquot of cell suspension needed to obtain a 2 x 105 cells per ml.
    15. The slides utilized are IBIDI VI0.4 six channel slides with a channel volume of 30 μl. Add 32 μl of your cell suspension into each channel using a P200 pipettor and tip. Insert the pipette tip into the slide reservoir and inject cells directly into the channel (Figure 2A).


      Figure 2. Seeding method for the IBIDI VI0.4 slide. A. Pipet in cell suspension directly into the slide channel inside reservoir; B. All channels filled; C. Humidified chamber setup.

    16. If there are bubbles that have formed after adding the cells, pick up the IBIDI slide and gently tap the edge to allow the bubbles to move toward the reservoirs of the channel. Place the slide cover over the IBIDI slide (Figure 2B).
    17. Place the slide into a 15 cm Petri dish, containing a 35 mm Petri dish filled ~3 ml of DPBS (Figure 2C). Place the lid back on the 15 cm dish with the lid.
      Note: This setup provides a ‘humidified chamber’ for the IBIDI slide, preventing media evaporation while inside a tissue culture incubator.
    18. Place the dish inside a 37 °C incubator and allow the cells to attach for 30 min.
    19. After 30 min, take the slide out and fill the individual channel by pipet dispensing ~2 drops between each channel reservoir until the full 125 μl of MSC media is dispensed.
      Note: This alternating drop-wise dispensing of media for each channel helps to minimize flow disruption, which allows non-attached MSCs to attach uniformly through the channel.
    20. Leave the slide overnight at 37 °C.

  2. Setting up IBIDI slides for WSS
    1. Prior to the WSS, the media must be replenished in each channel of the IBIDI slide. Duplicate channels for each treatment group (static, WSS, and other conditions) are required to obtain sufficient media for analysis in subsequent ELISA assays. Each channel has a reservoir port. From one end, draw out 125 μl of the old media without drawing out the 30 μl volume in the channel (see Video 1, start to 1:21 min).

      Video 1. Demonstration of steps 1-5 for setting up IBIDI slide for WSS

    2. To the opposite reservoir port, add 125 μl of fresh MSC media. Repeat this one more time to ensure complete change of media.
    3. Wait for one hour prior to start of WSS. During that time, set up the REGLO peristaltic pump along with the 3-stop tubing, slide elbow connectors and round bottom 14 ml centrifuge tube, prefilled with 6 ml of MSC media. This is the flow reservoir that will supply individual channels of the IBIDI slide for recirculation. Each channel is provided with a separate 6 ml reservoir for WSS. The static sample does not receive recirculating medium, unless a low flow is desired or needed to maintain the health of the cells.
    4. Prefill the 3-stop tubing with media, up to the elbow connector end by adjusting the analog rate on the peristaltic pump between 25-35. Turn off the peristaltic pump when media nears the end of the elbow connector orifice. Set and press the elbow connector to the first reservoir port of the slide. The elbow connector with tubing that runs on the opposite side, connects to the reservoir port for the corresponding channel. The end of this tubing goes into the centrifuge tube. Repeat for the subsequent slide reservoir ports. Fully assembled, the slide should resemble that in Video 1 and Figure 3.


      Figure 3. WSS setup of the IBIDI slide: Schematic and the actual tubing/pump breakdown for one single channel of the IBIDI slide

    5. Set the REGLO peristaltic pump setting to 85, which is equivalent to 8.5 ml/min.
      Note: Dyne is a unit of measure of force required to accelerate one gram of mass at a rate of 1 cm/sec2. In microfluidics this force is called shear stress, or at a distance of zero from the wall of the channel, wall shear stress (WSS). This flow rate on the IBIDI VI0.4 channel generates ~10.8 dyne/cm2 of WSS on the MSC cell monolayer, assuming η = 0.0075, where dynamical viscosity(η) is a function of the viscosity and temperature of the media. (See IBIDI link below)
      http://ibidi.com/img/cms/support/AN/AN11_Shear_stress.pdf
    6. Place the whole assembly into the incubator and start the pump after the one hour incubation period.
    7. Secure the incubator door and set timer for 3 h.
      Note: There is an alternative method of WSS utilizing syringe pumps, which can minimize flow pulsatility (Li et al., 2014).

  3. Harvesting splenocytes
    1. At 1.25 h prior to the end of the WSS, proceed with harvesting the spleen from a male C57BL/6 mouse. Recommended age is between 2-4 months old. After removing the spleen, immerse the tissue in 10 ml of chilled PBS. Transport to the lab.
    2. Pour out PBS. Prepare a 70-μm strainer (blue rimmed) in a 50 ml conical tube. Drop the spleen on the strainer. Using a 1 cc tuberculin syringe plunger, completely pulverize the tissue through the strainer.
    3. Using a serological pipet, flush the top of the strainer, using chilled 2% FBS in PBS. Flush at most 10 to 15 ml of buffer. From the bottom of the strainer, using a 1,000 μl pipette tip on pipettor, withdraw any residual suspension of cell-buffer mix and add to the cell suspension.
    4. Spin down for 5 min at 640 x g at 4 °C. After centrifugation, note that the supernatant is turbid, but there is a ‘red’ pellet at the bottom. Pour out the supernatant, without disturbing the pellet.
    5. Add 6 ml of RBC lysis buffer and break up the pellet with the buffer using a 5 ml serological pipet. Then let the mixture sit on an ice bath for EXACTLY 7 min of incubation time.
      Note: If splenocyte cell mixture is left too long in the RBC lysis buffer, it may affect the cell performance.
    6. Setup the 40 μm strainer with a fresh 50 ml conical. To the cell suspension add 20 to 30 ml of 2% FBS in PBS buffer to help quench the RBC lysis buffer. Draw up the suspension using a 10 ml serological pipet and run it through the 40 μm strainer.
    7. Change serological pipet and withdraw 10 ml of FBS-PBS buffer. Rinse the original 50 ml conical tube for the RBC lysis then transfer the suspension through the same 40 μm strainer.
    8. Again using a 1,000 μl pipet tip, collect any residual cell suspension under the strainer and transfer to the cell suspension.
    9. Spin down at 650 x g for 10 min at 4 °C.
    10. Carefully pour out the supernatant and add 10 ml of the MSC media. Re-suspend the pellet and perform a cell count. There are potentially 40-60 million splenocytes extracted from one mouse spleen, so it is advisable to dilute an aliquot 1:10 in MSC media prior to taking a 10 μl sample for counting.
    11. Combine 10 μl of the 1:10 aliquot of suspension and 10 μl of trypan blue. Transfer 10 μl of the stained cells and place into a hemocytometer with cover glass.
    12. Count the four outer quadrants. As before, approximately 30 cells per quadrant will provide an accurate estimate of cell concentration (cell number per ml).
    13. Once the cells per ml is calculated, take the required aliquot of cell suspension needed to obtain 6 x 106 cells per ml. Adjust volume to 1 ml if needed.

  4. Preparing the co-culture
    1. The recommended ratio of MSCs to splenocytes is 1:30. After WSS exposure time is finished, disassemble the REGLO pump and channel reservoirs. Detach the elbow connectors from the IBIDI slides.
    2. Using a P200 pipettor and tip, remove the media from one end of the slide reservoir, down to the point that only the channel contains media (both reservoirs should be empty). Add 125 μl of fresh media at the opposite end of the slide reservoir, to rinse the channel. Repeat one more time.
    3. Again remove the media from the slide reservoir, but not the volume of the channel. Using a light vacuum and a glass Pasteur pipette, with a P200 pipet tip fixed at the end, carefully aspirate out all media in the channel, leaving only the MSCs. Work quickly.
    4. Using a P200 pipettor, gently mix the splenocyte suspension. Take 30 μl of this and inject into the IBIDI channel, directly into the channel as opposed to simply filling the reservoir. Repeat for all channels: static, WSS, other treatments, etc.
      Note: Always be sure to mix the splenocyte suspension prior to drawing for consistent cell numbers per channel.
    5. Place the slide with the co-culture into the 15 cm Petri dish used as a humidified chamber. Set the slides into the incubator for 30 min. This time is important for MSC-splenocyte interactions.
    6. You will need a splenocyte only culture used as a control for LPS treatment and non-treatment. Use a fresh IBIDI VI0.4 slide and fill four channels with 30 μl of splenocytes.
    7. Allow to incubate for 30 min with the co-cultured slides.
    8. After 30 min, fill the channel reservoirs by gently dispensing 45 μl of MSC media.
    9. Prepare a 2x concentration of lipopolysaccharide (LPS) at 2 μg/ml. The stock solution is 1 mg/ml and the final concentration in the channel will be 1 μg/ml.
    10. There will be 75 μl of media volume in the slide channels. Add 75 μl of the 2x LPS, alternating ~2 drops between each channel reservoir port until the 75 μl is dispensed for the one channel. After all channels on the slide are filled, gently pick up the slide with thumb and index finger and carefully rock it up to 10 ten times from reservoir port side to side to allow the media to flow and mix. Do not allow the media to overflow above the reservoir port rim, which causes media loss. This rocking motion allows for proper mixing of the LPS with the co-cultured cells and duplicate splenocyte only treatment group. The other duplicate splenocyte set remains untreated, with only MSC media. Place the slide cover on top of the slide and place in the humidified chamber.
    11. Place in the incubator for up to 18 h.

  5. mTNFα ELISA assay
    1. The mTNFα ELISA kit (R&D Systems) must be taken out of the refrigerator and allowed to equilibrate to room temperature for at least 30 min. The TNFα control and the TNFα standard should be each reconstituted with 1 ml of double deionized water. The 25x wash buffer should also be thawed.
    2. After incubation of the co-cultured cells and the splenocyte only control conditions (LPS treated and non-treated), collect the media from duplicate channels, combining together the two channels for each treatment group into individually marked 1.5 ml microcentrifuge tubes. A volume of two duplicate channels will be approximately 300 μl. Place the microcentrifuge tubes on ice.
    3. Transfer the tubes into a refrigerated table top centrifuge and spin down at 650 x g for 5 min at 4 °C. This step is required to remove any cell debris and splenocytes.
    4. After centrifugation, transfer the media supernatant into a fresh pre-labeled tube. Discard the first tube with the debris pellet.
    5. Follow the link to pages 5 and 6 of the TNFα standard preparation and assay procedure. Use the appropriate diluents for each and proceed. https://resources.rndsystems.com/pdfs/datasheets/mta00b.pdf.
      Note: The R&D ELISA assay procedure on page 6, steps 3 and 4, suggest to dilute you media sample 1:1 with the RD1-63 diluent into the pretreated well, in duplicates. This should be done for the TNF-α standards and control. For the actual sample, our experience has shown that undiluted sample into the well, allows for consistent pg/ml TNF-α values compared with an RD1-63 diluted sample, which can at times be inconsistent, possibly due to interfering contents in the media. We use straight 100 μl of media sample in duplicate wells for each condition, including the splenocyte only samples, both treated (with LPS) and untreated. After obtaining the values in pg/ml, final sample values calculated are reduced by multiplying to a factor of 0.5, to normalize to the diluted standard and control values, which initially are diluted.
    6. Continuing on the R&D assay procedure link from page 6, step 9, after adding the 100 μl of ‘Stop Solution’, wipe the bottom well plate surface using a paper towel to absorb any moisture from the washes and immediately take optical density readings using the Spectra MAX M2 microplate reader set at 450 nm wavelength. Wavelength correction, although recommended is not required due to the very minute difference in optical density differences related to bottom well surface imperfections.
    7. The concentration (pg/ml) for the standard and control mTNFα are factored by 1, since they were diluted by half with the RD1-63 diluent. For calculation of the actual media sample concentrations, you must include a multiplication factor of 0.5 to normalize to the standard and control.

Data analysis

  1. Based on the concentration (pg/ml) of TNFα detected by the microplate reader on the samples, plot an initial vertical bar graph, plotting the LPS ‘treated’ and ‘untreated’ splenocytes and the co-cultured static and WSS samples. Also, plot the TNFα positive control.
    Note: The TNFα positive control stock vial has a printed range of pg/ml concentration for media samples, which allows determination of whether the assay itself has worked. If the control numbers fall within the printed range of the stock vial for media, the assay is performing properly.
  2. If the assay worked well, the LPS ‘treated’ splenocyte only control will have the highest reading/concentration of mTNFα. The ‘untreated’ splenocyte control will register near zero, due to little or no production of TNFα in unactivated splenocytes (Figure 4).
    Note: MSCs in an un-sheared state have the ability to reduce inflammatory response or, in this case, splenocyte secretion of mTNFα. In WSS induced MSCs, the anti-inflammatory response is enhanced.


    Figure 4. Initial plot with TNFα levels for each condition

  3. From the numbers generated for mTNFα concentration, normalize all values to percentage using LPS ‘treated’ splenocytes to represent maximal capacity for TNFα production (100%). The percent reduction of mTNFα between static, WSS, or other treatment groups relative to ‘treated’ splenocytes will provide a relative measure of MSC immunomodulatory performance (Figure 5).



    Figure 5. Percent reduction plot comparing WSS vs. static co-culture. WSS-exposed MSCs show enhanced immunomodulatory activity.

Note: Data presented in Figures 4 and 5 are from a single representative experiment demonstrating under ‘Data analysis’ the procedure for obtaining and charting TNFα ELISA assay numbers. The procedure described does not include technical replicates. Instead, we favor true biological replicates to establish reproducibility and these could include experiments set up on different days or using different cell lines. Please see our prior manuscript (Diaz et al., 2017) for a more in-depth study with statistically reproducible data for this immunomodulatory phenomena.

Recipes

  1. MSC media
    400 ml of MEMα media 100 ml of FBS
    5 ml of penicillin/streptomycin
    Run through the EMD-Millipore Stericup filtration unit
    Can be stored at 4 °C for up to 3 months

Acknowledgments

This work was supported by grants from the State of Texas Emerging Technology Fund, American Society of Hematology Scholar Award, National Institutes of Health K01DK092365, and Mission Connect: a Program of the TIRR Foundation (014-121, 016-118) to P.L.W.

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  14. Nitzsche, F., Müller, C., Lukomska, B., Jolkkonen, J., Deten, A. and Boltze, J. (2017). Concise review: MSC adhesion cascade-insights into homing and transendothelial migration. Stem Cells 35: 1446-1460.
  15. Plumas, J., Chaperot, L., Richard, M. J., Molens, J. P., Bensa, J. C. and Favrot, M. C. (2005). Mesenchymal stem cells induce apoptosis of activated T cells. Leukemia 19(9): 1597-1604.
  16. Sacchetti, B., Funari, A., Michienzi, S., Di Cesare, S., Piersanti, S., Saggio, I., Tagliafico, E., Ferrari, S., Robey, P.G. Riminucci, M. and Bianco, P. (2007). Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 131: 324-336.
  17. Scallan, J., Huxley, V. H. and Korthuis, R. J. (2010). Fluid movement across the endothelial barrier. In: Scallan, J., Huxley, V. H. and Korthuis, R. J. (Eds.). Capillary Fluid Exchange: Regulation, Functions, and Pathology. Morgan & Claypool Life Sciences.
  18. Schrimpf, C., Teebken, O. E., Wilhelmi, M. and Duffield, J. S. (2014). The role of pericyte detachment in vascular rarefaction. J Vasc Res 51(4): 247-258.
  19. Sekiya, I., Larson, B. L., Smith, J. R., Pochampally, R., Cui, J. G. and Prockop, D. J. (2002). Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the yields of early progenitors and evaluate their quality. Stem Cells 20(6): 530-541.
  20. Shepro, D. and Morel, N. M. (1993). Pericyte physiology. FASEB J 7: 1031-1038.
  21. Singer, N. G. and Caplan, A. I. (2011). Mesenchymal stem cells: Mechanisms of inflammation. Annu Rev Pathol 6: 457-478.
  22. Zvaifler, N. J., Marinova-Mutafchieva, L., Adams, G., Edwards, C. J., Moss, J., Burger, J. A. and Maini, R. N. (2000). Mesenchymal precursor cells in the blood of normal individuals. Arthritis Res 2: 477-88.

简介

认为基于间充质干细胞(MSC)的细胞疗法的有益作用主要是由能够抑制慢性或急性损伤,感染,自身免疫和移植物抗宿主病相关炎症的能力介导的。 为了专门解决由血流或壁剪应力(WSS)引起的摩擦力对人MSC免疫调节功能的影响,我们利用微流体在动脉腔壁上建模WSS。 随后通过在共培养测定中通过活化的小鼠脾细胞测量TNF-α产生来量化MSC的抗炎效力。 TNF-α抑制测定作为MSC效力的功能评估的可重现平台,并且表现出作为MSC治疗功效的替代测定的预测价值。
【背景】间充质干细胞(MSC)的免疫调节活性由直接细胞相互作用和旁分泌因子介导(Singer和Caplan,2011;英语,2013)。 MSCs被认为是源于与骨髓和各种组织内脉管系统内皮细胞相关的周细胞(Sacchetti et al。,2007; Crisan et al。,2008)。这种独特的血管周围位置将它们置于血流中的炎症和其他可溶性因子附近,使其监测系统信号。事实上,将壁壁细胞募集到内皮是血管成熟的关键事件,周细胞在血管维持和完整性中起关键作用(Benjamin et al。,1998; Schrimpf等,2014)。周细胞可能通过流体从小动脉和毛细血管流出,通过基底膜中的间上皮裂缝或间隙来监测系统信号,这可以将容器外部的内皮细胞的基底外侧表面暴露于相当大的流体摩擦力或壁剪切应力(WSS)腔内力量(Scallan等,2010)。 MSC和其他类型的周细胞也可能通过突出到血管腔与血管内膜的细胞质投射(如巨核细胞)中观察到血管内皮细胞开放的腔内环境,尽管更典型的是它们以分支过程吞噬血管(Shepro和Morel,1993; Murphy et al。,2013)。在炎症或损伤的情况下,例如由于对中枢神经系统的创伤,周细胞已经显示出与血管周围水肿同时发生的微血管和与血管发芽相关的损伤组织(Dore-Duffy等人,2000 ;Göritzet al。,2011)。被描述为具有MSC的特征的细胞已被检测到在人类外周血中循环(Zvaifler等,2000),尽管围绕健康和甚至受伤个体的循环中MSC的证据存在一些争议(Hoogduijn等,2014) 。在这些情况下,内皮细胞周细胞相互作用的破坏可能会加剧可能影响MSC迁移或侵袭的血管高渗透性(Mills et al。,2013)。由于MSC是锚定依赖性细胞,所以可能的运动方式将包括附着于血管壁,导致直接暴露于管腔内WSS。在静脉内给予MSCs的治疗应用中,WSS在处理,输注和贩运期间将是不可避免的刺激(Nitzsche等,2017)。
   我们已经表明,WSS典型的动脉血流通过粘着斑激酶(FAK),NF-κB和COX2促进信号传导(Diaz等,2017; Lee等,2017)。增加的COX2导致前列腺素E2(PGE2)生物合成升高。 MSC分泌的PGE2在调节天然和适应性免疫细胞中起着核心作用。因此,暴露于WSS的MSC更有效地抑制存在炎性线索的免疫细胞活化(Diaz等,2017; Lee等,2017)。为了量化暴露于流体流动的细胞中的MSC免疫调节活性,我们共同培养MSC和脂多糖活化的小鼠脾细胞,以适应常用的混合淋巴细胞反应(Plumas等,2005)。通过物种特异性ELISA测量TNF-α以确定活化的小鼠脾细胞的细胞因子产生,从而限制对免疫细胞活性的分析,并且能够单独测定人类MSC的细胞因子产生。使用该测定作为MSC效能的替代测量,我们确定MSC在流体剪切应激下的瞬时暴露提高了其在炎性刺激存在下限制免疫细胞活化的能力。在培养物中少量3小时的WSS预处理MSC是提高治疗大鼠创伤性脑损伤模型的治疗效果的有效手段。这些数据表明,WSS增强了MSC的免疫调节和神经保护功能。结合补充研究,将PGE2作为MSC治疗功效的效力标记(Kota等,2017),我们的研究表明,可以利用机械转导来改善可用于神经损伤患者的细胞治疗。这种共培养测定可以容易地适应于经历各种处理的MSC的抗炎效力的分析,包括遗传工程化的MSC。

关键字:生物机械力, 炎症, 免疫调节, 间充质干细胞, 效能测定, TNF-α

材料和试剂

  1. Falcon培养处理的烧瓶,225cm 2(Corning,Falcon ®,目录号:353139)
  2. Falcon 15 ml锥形离心管(Corning,Falcon ®,目录号:352097)
  3. 5 ml血清移液管(MIDSCI,目录号:MWB-5)
  4. Fisherbrand高级微量离心管,1.5 ml(Fisher Scientific,目录号:05-408-129)
  5. IBIDIμ-Slide VI 0.4 ibiTreat,无菌幻灯片(IBIDI,目录号:80606)
  6. Fisherbrand P200低保留气溶胶屏障移液管吸头(Fisher Scientific,目录号:02-717-165)
  7. Falcon Petri Dish 150 x 15 mm(Corning,Falcon ®,目录号:351058)
  8. Greiner Petri Dish 35 x 10 mm(Greiner Bio One International,目录号:627161)
  9. 三通硅胶管,1.52 mm I.D. (Cole-Parmer,目录号:SK-07624-36)
  10. 弯头路厄连接器(IBIDI,目录号:10802)
  11. Falcon圆底聚丙烯管(Corning,Falcon ®,目录号:352006)
  12. EASYStrainer,70μm细胞筛,无菌(Phenix Research Products,目录号:TCG-542070)
  13. Falcon 50ml锥形离心管(Corning,Falcon ®,目录号:352098)
  14. 1 cc结核菌素注射器柱塞
  15. SHARP P1000精密阻隔移液管吸头(Denville Scientific,目录号:P1126)
  16. EASYStrainer,40μm细胞筛,无菌(Phenix Research Products,目录号:TCG-542040)
  17. 10ml血清移液管(MIDSCI,目录号:MWB-10)
  18. Fisherbrand硼硅酸盐玻璃巴斯德移液器(Fisher Scientific,目录号:13-678-20C)
  19. 纸巾
  20. EMD-Millipore Stericup真空过滤器,500ml(EMD Millipore,目录号:SCGPU05RE)
  21. Parafilm M TM (Bemis,目录号:PM996)
  22. 道康宁硅胶实验室管材1.57毫米x 3.18毫米O.D (道康宁,目录号:2415569)
  23. 人骨髓(BM)MSC(全骨髓抽吸物)(AllCells,目录号:ABM001-0)使用Ficoll梯度随后通过塑性粘附从全骨髓中分离MSC,然后在MSC培养基中培养(参见食谱) /> 注意:由于存在典型的MSC生长,外观和表面标志物表达,用于这项工作的MSC被预先筛选,并在其使用前扩展用于库存循环保存(Sekiya等人,2002; Dominici等人, 2006)。
  24. 雄性C57BL / 6小鼠(JACKSON LABORATORY,目录号:000664);推荐年龄在2-4个月以前
  25. Hyclone Dulbecco的磷酸盐缓冲盐水(DPBS)溶液,500ml,不含钙的镁(GE Healthcare,Hyclone ,目录号:SH30028.FS)
  26. Gibco-Tryp-LE Express酶,1x,500ml(Thermo Fisher Scientific,Gibco TM,目录号:12604021)
  27. 千分尺 - 台盼蓝溶液,0.4%(Thermo Fisher Scientific,Gibco TM,目录号:15250061)
  28. 亚特兰大生物胎牛血清(FBS),胚胎干细胞合格,500毫升(亚特兰大生物公司,目录号:S10250)
  29. 红细胞裂解缓冲液hybri-max(Sigma-Aldrich,目录号:R7767-100ML)
  30. 脂多糖,BioXtra(Sigma-Aldrich,目录号:L6529)
  31. R& D系统小鼠TNF-αQuantikine ELISA试剂盒(R& D Systems,目录号:MTA00B)
  32. 用谷氨酰胺和核苷介质进行的HycloneMEMα修饰(GE Healthcare,Hyclone TM,目录号:SH30265.FS)
  33. Gibco-Penicillin-streptomycin,10,000U / ml(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  34. MSC媒体(见食谱)

设备

  1. Hettich Rotofix 32A带有15毫升和50毫升锥形管的旋转桶(Hettich Lab Technology,型号:Rotofix 32A)
  2. 带真空抽吸的无菌罩(贝克公司,型号:SterilGARD ® III Advance )
  3. Hausser Scientific Bright-Line TM带有玻璃杯的计数室(Hausser Scientific,目录号:3110V)
  4. P2-20 XL3000i移液器(Denville Scientific,目录号:P3950-20A)
    注意:本产品已停产。
  5. P20-200 XL3000i移液器(Denville Scientific,目录号:P3950-200A)
    注意:本产品已停产。
  6. P100-1000 XL3000i移液器(Denville Scientific,目录号:P3950-1000A)
    注意:本产品已停产。
  7. 三洋CO 2培养箱(SANYO,型号:MCO-18AIC)
  8. Ismatec REGLO蠕动12辊泵(Cole-Parmer,目录号:ISM796B)
  9. Hettich Mikro 200R冷冻微量离心机(Hettich Lab Technology,型号:MIKRO 200R)
  10. 色谱酶标仪(Molecular Devices,型号:SpectraMax M2)
    注意:本产品已停产。
  11. 37°C水浴(Fisher Scientific,型号:Model 210,目录号:15-462-10Q)
    注意:本产品已停产。

程序

  1. 播种MSCs
    1. 将经冷冻的人类骨髓(BM)MSC冷冻储存液以每个冷冻小瓶2百万条解冻,并在37℃下具有5%CO 2 。理想通道介于1-5之间。 2〜3天后,实验使用80%贴壁细胞融合
    2. 完全取出培养基,加入5ml无菌DPBS室温冲洗细胞单层。完全吸出。
    3. 加入5ml Tryp-LE Express离解试剂。允许体积扩散到单层,将烧瓶置于组织培养箱内5-8分钟
    4. 通过加入5毫升MSC培养基收集细胞悬浮液(参见食谱)以减少Tryp-LE效应。将内容物加入干净的15 ml锥形离心管中
    5. 用2-3ml的MSC培养基重新冲洗烧瓶以收集残余细胞
    6. 将内容物加入同一个15 ml离心管中
    7. 将离心管放入旋转铲斗台式离心机(Hettich Rotofix 32A)中,并以617“x”(2000 rpm)旋转悬浮液5分钟。
    8. 使用真空吸力吸出媒体混合物,不会干扰细胞沉淀
    9. 用手指轻轻敲打15 ml离心管的圆锥形末端,以帮助球团脱落。通过与5ml血清移液管轻轻混合,将2 ml MSC培养基重新悬浮细胞沉淀
    10. 一旦混合,快速取10μl细胞悬浮液等分细胞进行细胞计数,并将其置于新鲜的1.5 ml微量离心管中。加入悬浮液,10μl台盼蓝。通过移液5-8次混合。
    11. 取最终10μl该溶液的等分试样,并放入带有玻璃杯的血细胞计数仪(Hausser Scientific)(图1)。


      图1. MSC的细胞计数配置文件。 200μm中四个象限之一的示例。

    12. 计数四个外象限。细胞数应该接近并且不少于每个象限约30个细胞(图1)
    13. 计算细胞浓度(每ml细胞数)乘以4象限计数乘以稀释因子2乘以因子2,500的总和。这将等于每毫升细胞。
    14. 一旦计算出每毫升的细胞数,取所需的细胞悬液等分试样,以获得每毫升2×10 5个细胞。
    15. 使用的载玻片是通道体积为30μl的IBIDI VI 0.4 六通道载玻片。使用P200移液器和提示将32μl您的细胞悬浮液加入每个通道。将移液器吸头插入滑道容器,并将细胞直接注入通道(图2A)

      图2. IBIDI VI的种子方法 0.4 幻灯片。 A.将细胞悬浮液直接吸入幻灯片水槽内通道; B.所有渠道填补;加湿室设置。

    16. 如果在添加细胞后形成气泡,拿起IBIDI滑块,轻轻敲击边缘,以使气泡朝向通道的储存器移动。将幻灯片盖板放在IBIDI幻灯片上(图2B)。
    17. 将载玻片放入15厘米的陪替氏培养皿中,装有一个装有3毫升DPBS的35毫米培养皿(图2C)。将盖子放回15厘米的盘子上。
      注意:此设置为IBIDI幻灯片提供了一个“加湿室”,可防止组织培养箱内的介质蒸发。
    18. 将培养皿放入37℃的培养箱中,使细胞附着30分钟
    19. 30分钟后,取出滑块,并通过移液管分配每个通道储液罐2滴,直到完全分离125μl的MSC培养基来填充个体通道。
      注意:每个通道的这种交替逐滴分配媒体有助于最大限度地减少流量中断,从而允许非连接的MSC均匀地通过通道。
    20. 在37°C的时间过夜。

  2. 为WSS设置IBIDI幻灯片
    1. 在WSS之前,必须在IBIDI幻灯片的每个通道中补充媒体。需要每个治疗组(静态,WSS和其他条件)的重复通道,以便在随后的ELISA测定中获得足够的培养基进行分析。每个通道都有一个储存口。从一端抽出125μl旧的培养基,不要在通道内抽出30μl的体积(见视频1,开始时间为1:21分钟)。

      Video 1. Demonstration of steps 1-5 for setting up IBIDI slide for WSS

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


    2. 向对面的水库口,加入125μl新鲜的MSC培养基。重复一次以确保媒体的完整更改。
    3. 等待WSS开始前一个小时。在此期间,设置了REGLO蠕动泵以及三通管,滑动弯头连接器和圆底14ml离心管,预填充6 ml MSC培养基。这是流量储存器,将为IBIDI滑块的各个通道供应再循环。每个通道都有一个单独的6毫升WSS储存器。静态样品不接收循环介质,除非需要或需要低流量以维持细胞的健康
    4. 通过调节25-35之间的蠕动泵上的模拟速率,预先填充三通管路与介质,直到弯头连接器端。当介质靠近弯头连接器孔口的末端时,请关闭蠕动泵。将弯头连接器固定并滑动到滑块的第一个储存口。弯管连接器与管道相对运行,连接到相应通道的储存口。该管的末端进入离心管。重复以后的滑动储存器端口。完全组装,幻灯片应与视频1和图3中的相似。


      图3. IBIDI幻灯片的WSS设置:IBIDI幻灯片的单个通道的原理图和实际管道/泵故障

    5. 将REGLO蠕动泵设置设置为85,等于8.5 ml / min。
      注意:Dyne是以1厘米/秒的速度加速1克质量所需的力的单位。在微流体中,这种力称为剪切应力,或者与通道壁的距离为零,壁面剪切应力(WSS)。 IBIDI VI 通道上的此流量产生〜10.8达因/厘米 2 http://ibidi.com/img /cms/support/AN/AN11_Shear_stress.pdf
    6. 将整个组件放入培养箱中,并在一个小时的潜伏期后启动泵。
    7. 固定孵化器门并设置定时器3小时。
      注意:WSS采用注射泵的替代方法,可以最大程度地减少流动脉动(Li et al。,2014)。

  3. 收获脾细胞
    1. 在WSS结束前1.25小时,继续从雄性C57BL / 6小鼠收获脾脏。推荐年龄在2-4个月之间。取出脾脏后,将组织浸入10ml冷冻的PBS中。运送到实验室。
    2. 倒出PBS。在50ml锥形管中准备一个70μm过滤器(蓝色边框)。把脾脏放在过滤器上。使用1cc结核菌素注射器柱塞,通过过滤器完全粉碎组织
    3. 使用血清移液管,用PBS中的冷冻2%FBS冲洗过滤器的顶部。冲洗最多10至15 ml缓冲液。过滤器的底部,使用移液器上的1,000μl移液管吸头,取出细胞缓冲液混合物的残留悬浮液,并加入细胞悬浮液中。
    4. 在4℃下以640×g旋转5分钟。离心后,注意上清液是浑浊的,但底部有一个“红”颗粒。倒出上清液,不要打扰沉淀。
    5. 加入6ml RBC裂解缓冲液,并用5ml血清移液管用缓冲液分离沉淀。然后让混合物在冰浴上放置7分钟的孵育时间。
      注意:如果脾细胞混合物在RBC裂解缓冲液中留下太长时间,可能会影响细胞表现。
    6. 使用新鲜的50ml圆锥形设置40μm过滤器。向细胞悬浮液中加入20至30ml的PBS缓冲液中的2%FBS以帮助猝灭RBC裂解缓冲液。用10ml血清移液器吸取悬浮液,并将其穿过40μm过滤器
    7. 更换血清移液管并取出10ml的FBS-PBS缓冲液。冲洗原来的50ml锥形管,用于RBC裂解,然后将悬浮液转移通过相同的40μm过滤器
    8. 再次使用1,000μl的吸头,收集过滤器下的任何残留细胞悬浮液,并转移到细胞悬浮液中
    9. 在4℃下以650×x×10分钟旋转10分钟
    10. 仔细倒出上清液,加入10 ml的MSC培养基。重新悬挂颗粒并进行细胞计数。从一只小鼠脾脏中可以提取四千万到六千万个脾细胞,因此建议在服用10μl样品进行计数之前,在MSC培养基中稀释等分试样1:10。
    11. 将10μl的1:10等分悬浮液和10μl台盼蓝混合。转移10μl染色细胞,放入带有玻璃杯的血细胞计数器
    12. 计数四个外象限。如前所述,每个象限约30个细胞将提供细胞浓度(每毫升细胞数)的准确估计
    13. 一旦计算每毫升细胞,取所需的细胞悬浮液等分试样,以获得每毫升6×10 6个细胞。如果需要,将体积调整至1 ml。

  4. 准备共同文化
    1. MSCs与脾细胞的推荐比例为1:30。在WSS曝光时间完成后,拆卸REGLO泵和通道储存器。从IBIDI幻灯片中分离肘连接器。
    2. 使用P200移液器和尖端,从墨盒的一端取下介质,直到只有通道包含介质(两个储存器应为空)。在幻灯片储存器的另一端添加125μl新鲜介质,以冲洗通道。再重复一次。
    3. 再次从墨盒中取出介质,而不是通道的音量。使用轻型真空和玻璃巴斯德吸管,将P200吸头固定在末端,小心吸出通道中的所有介质,仅留下MSC。快速工作
    4. 使用P200移液器,轻轻混合脾细胞悬液。取30μl,并注入IBIDI通道,直接进入通道,而不是简单地填充储存器。对所有渠道重复:静态,WSS,其他治疗,等等。
      注意:始终确保在绘制之前混合脾细胞悬浮液,以保证每个通道的细胞数量一致。
    5. 将载玻片与共培养物置于用作加湿室的15厘米培养皿中。将载玻片放入培养箱中30分钟。这一次对于MSC-脾细胞相互作用是重要的
    6. 您将需要一种仅用于脾细胞培养,作为LPS治疗和非治疗对照。使用新鲜的IBIDI VI 0.4 幻灯片,并用30μl脾细胞填充四个通道。
    7. 允许用共同培养的载玻片孵育30分钟。
    8. 30分钟后,通过轻轻分配45μlMSC培养基填充通道储存器。
    9. 以2μg/ ml制备2x浓度的脂多糖(LPS)。原液为1 mg / ml,通道最终浓度为1μg/ ml
    10. 在幻灯片通道中将有75μl的媒体音量。加入75μl2x LPS,在每个通道储存器端口间交替〜2滴,直到为一个通道分配75μl。在幻灯片上的所有通道充满后,用拇指和食指轻轻拿起幻灯片,并从储存口侧面小心摇晃10次,以使介质流动并混合。不要让介质溢出到油箱端口边缘上方,导致介质损坏。这种摇摆运动允许LPS与共培养细胞和仅复制脾细胞治疗组的适当混合。其他复制脾细胞集合仍然未经处理,只有MSC培养基。将滑盖放在滑块顶部,放在加湿室中。
    11. 放在孵化器长达18小时。

  5. mTNFαELISA测定
    1. 必须将mTNFαELISA试剂盒(R& D Systems)从冰箱中取出并使其平衡至室温至少30分钟。 TNFα对照和TNFα标准物均应用1ml双去离子水重构。 25x洗涤缓冲液也应该解冻。
    2. 在共培养细胞和仅脾细胞控制条件(LPS处理和未处理)孵育后,从重复通道收集培养基,将每个处理组的两个通道组合在一起,分别标记为1.5ml微量离心管。两个重复通道的体积约为300μl。将微量离心管放在冰上。
    3. 将试管转移到冷藏台式离心机中,并在4℃下以650×g离心5分钟。这个步骤需要去除任何细胞碎片和脾细胞。
    4. 离心后,将培养基上清液转移到新鲜的预先标记的管中。用碎屑丸丢弃第一根管。
    5. 请点击链接到第5和第6页的TNFα标准品制备和测定程序。为每个使用适当的稀释剂并继续。 https://resources.rndsystems.com/pdfs/datasheets/ mta00b.pdf
      注意:第6页,第3步和第4步的R& D ELISA测定程序建议将RD1-63稀释剂的介质样品1:1稀释到预处理的孔中,重复。应该对TNF-α标准和对照进行。对于实际样品,我们的经验表明,未稀释的样品进入孔,与RD1-63稀释样品相比,允许与RD1-63稀释样品相比持续的pg / ml TNF-α值,其可能有时不一致,可能是由于介质中的干扰物质。我们在每个条件的双重孔中直接使用100μl的培养基样品,包括仅使用脾细胞样品(LPS)和未处理的样品。在获得pg / ml值后,通过乘以0.5的因子减少最终样品值,将其标准化为稀释的标准品和最初稀释的对照值。
    6. 继续从第6页第9步的R& D测定程序链接,加入100μl'Stop Solution'后,用纸巾擦拭底部孔板表面,以吸收洗涤液中的任何水分,并立即获取光密度读数使用设置在450 nm波长的Spectra MAX M2微孔板读数器。波长校正虽然由于与底部井表面缺陷相关的光密度差异非常小的差异而不需要推荐。
    7. 标准和对照mTNFα的浓度(pg / ml)由1,因为RD1-63稀释剂稀释了一半。要计算实际的介质样品浓度,必须包括0.5的倍增因子才能标准化和控制。

数据分析

  1. 根据酶标仪在样品上检测到的TNFα的浓度(pg / ml),绘制初始垂直条形图,绘制LPS处理的'和'未处理'脾细胞和共培养的静态和WSS样品。另外,绘制TNFα阳性对照。
    注意:TNFα阳性对照储备小瓶的印刷范围为培养基样品的pg / ml浓度,这可以确定测定本身是否有效。如果控制数字落在介质的储存小瓶的打印范围内,则该测定正确执行。
  2. 如果测定效果很好,LPS处理的“脾细胞控制”将具有最高的mTNFα读数/浓度。未经治疗的脾细胞控制将注册在零附近,因为在未激活的脾细胞中TNFα的产生很少或不产生(图4)。
    注意:未剪切状态的MSC具有减少炎症反应或在这种情况下是mTNFα的脾细胞分泌的能力。在WSS诱导的MSC中,抗炎反应增强。


    图4.每个条件的TNFα水平的初始图

  3. 从mTNFα浓度产生的数字,使用LPS处理的脾细胞将所有值归一化为百分比,代表TNFα产生的最大容量(100%)。静态,WSS或其他治疗组相对于“处理”脾细胞的mTNFα的减少百分比将提供MSC免疫调节性能的相对量度(图5)。



    图5.比较WSS与静态共同文化的减少百分比图。 WSS暴露的MSC显示增强的免疫调节活性

注意:图4和图5中显示的数据来自一个代表性的实验,在“数据分析”下证明了获得和绘制TNFαELISA测定数量的程序。所描述的过程不包括技术重复。相反,我们赞成真正的生物重复来建立重复性,这些可能包括在不同的日子里建立的实验或使用不同的细胞系。请参阅我们以前的手稿(Diaz et al。,2017),对这种免疫调节现象进行统计学重现的数据进行更深入的研究。

食谱

  1. MSC媒体
    400毫升MEMα培养基100毫升FBS
    5 ml青霉素/链霉素
    穿过EMD-Millipore Stericup过滤装置
    可在4°C下储存多达3个月

致谢

这项工作得到了德克萨斯州新兴技术基金,美国血液学学者奖,美国国立卫生研究院K01DK092365和Mission Connect:TIRR基金会计划(014-121,016-118)给P.L.W.的资助。

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Diaz, M. F., Evans, S. M., Olson, S. D., Cox, C. S. and Wenzel, P. L. (2017). A Co-culture Assay to Determine Efficacy of TNF-α Suppression by Biomechanically Induced Human Bone Marrow Mesenchymal Stem Cells. Bio-protocol 7(16): e2513. DOI: 10.21769/BioProtoc.2513.
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