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Mar 2020

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Structural and Functional Mapping of Mesenchymal Bodies
间充质体的结构和功能图谱   

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Abstract

The formation of spheroids with mesenchymal stem/stromal cells (MSCs), mesenchymal bodies (MBs), is usually performed using bioreactors or conventional well plates. While these methods promote the formation of a large number of spheroids, they provide limited control over their structure or over the regulation of their environment. It has therefore been hard to elucidate the mechanisms orchestrating the structural organization and the induction of the trophic functions of MBs until now. We have recently demonstrated an integrated droplet-based microfluidic platform for the high-density formation and culture of MBs, as well as for the quantitative characterization of the structural and functional organization of cells within them. The protocol starts with a suspension of a few hundred MSCs encapsulated within microfluidic droplets held in capillary traps. After droplet immobilization, MSCs start clustering and form densely packed spherical aggregates that display a tight size distribution. Quantitative imaging is used to provide a robust demonstration that human MSCs self-organize in a hierarchical manner, by taking advantage of the good fit between the microfluidic chip and conventional microscopy techniques. Moreover, the structural organization within the MBs is found to correlate with the induction of osteo-endocrine functions (i.e., COX-2 and VEGF-A expression). Therefore, the present platform provides a unique method to link the structural organization in MBs to their functional properties.


Graphic abstract:


Droplet microfluidic platform for integrated formation, culture, and characterization of mesenchymal bodies (MBs). The device is equipped with a droplet production area (flow focusing) and a culture chamber that enables the culture of 270 MBs in parallel. A layer-by-layer analysis revealed a hierarchical developmental organization within MBs.


Keywords: Mesenchymal stromal cells (间充质基质细胞), Mesenchymal bodies (间充质体), Microfluidics (微流体), Droplets (飞沫), Spheroids (球体), Quantitative imaging (定量成像)

Background

Mesenchymal stem/stromal cells (MSCs) comprise a heterogeneous population of mesenchymal progenitors that are capable of differentiation into osteoblastic, chondrogenic, and adipogenic lineages (Dominici et al., 2006). MSCs also bear important trophic functions that regulate immune cell activities, promote angiogenesis, reduce tissue inflammation, and activate tissue-resident progenitors, making this cell type particularly suited for many tissue engineering/regeneration applications (Caplan and Correa, 2011). The formation of spheroids with MSCs (i.e., mesenchymal bodies, MBs) was recently found to enhance their differentiation potential and their secretory activities (Sart et al., 2014). However, it remains poorly understood how heterogeneous hMSCs self-organize in 3D, as well as the mechanisms linking their structural organization to their functional activities (Cesarz and Tamama, 2016).


Several methods have been used for the formation of MBs, including bioreactors and conventional well plates (Sart et al., 2014 and 2016; Sart and Agathos, 2018). However, while these methods enable the formation of a large number of MBs, they provide limited control over single aggregate stimulation or characterization (Sart et al., 2016; Sart and Agathos, 2018). Because MSCs constitute a heterogeneous population, the analysis of single aggregates at single-cell resolution is required to understand the mechanisms by which 3D cultivation induces functional changes on the behavior of a minority of very responsive cells or a global population shift. The macro-scale culture vessels and global population analysis therefore do not allow the MB structure and the cellular functions to be related (Sart et al., 2017; Sart and Agathos, 2018).


Here, we use a microfluidic platform that allows high density and controlled-sized MB formation within nanoliter drops. The microfluidic format enables a robust quantitative demonstration, using quantitative imaging at a single-cell level, that human MSCs self-organize in a hierarchical manner: The most undifferentiated MSCs are located in the core, while partially committed cells are located at the boundaries of the MBs. Moreover, we found that such structural organization correlated with the induction of osteo-endocrine functions. The microfluidic method and protocols developed here can find applications to characterize other types of organoids, to link cell sorting processes to phenotypic commitment, in view of understanding the mechanisms leading to tissue patterning in 3D stem cell cultures. The current method can be applied to any kind of stem cells cultivated in 3D, although the volume of droplets should be adapted to a specific type of stem cells. The data-driven approach developed in this protocol allows us to obtain a robust quantitative characterization of the structural and functional organization within organoids, which would be difficult to obtain using conventional population-based approaches that require the alteration of the cellular microenvironment.

Materials and Reagents

  1. Cell culture reagents

    1. T-175 cm2 flasks (Greiner, Cellstar, catalog number: 660175)

    2. Human mesenchymal stromal cells derived from the Wharton’s jelly of the umbilical cord (hMSCs), purchased from American Type Culture Collection (ATCC) (ref #PCS-500-010)

    3. α-modified Eagle’s medium (α-MEM) (Life Technologies, catalog number: 32561-029)

    4. TrypLETM Express (Life Technologies, catalog number: 12604013)

    5. Phosphate buffer saline (PBS) (Sigma-Aldrich, catalog number: D8662)

    6. Fetal bovine serum (FBS) (Life Technologies, catalog number: 10500-064)

    7. Penicillin- streptomycin (pen-strep) (Life Technologies, catalog number: 10378-016)

    8. Triton X-100 (Sigma-Aldrich, catalog number: X100)

    9. Ultra-low-melting agarose (Sigma-Aldrich, catalog number: A5030)

    10. Paraformaldehyde (PFA), 16% (Alpha Aesar, catalog number: 43368)

    11. Mouse anti-human CD146-Alexa Fluor 647 (clone P1-H12) (BD Biosciences, catalog number: 563619)

    12. Rabbit anti-COX-2 polyclonal antibody (Abcam, catalog number: ab15191)

    13. Rabbit anti-human VEGF-A monoclonal antibody (Abcam, catalog number: ab52917)

    14. Mouse anti-human RUNX-2 monoclonal antibody (Abcam, catalog number: ab76956)

    15. Alexa Fluor 488-conjugated goat anti-mouse IgG2a secondary antibody (Life Technologies, catalog number: A-21131)

    16. Alexa Fluor 594-conjugated goat polyclonal anti-rabbit IgG secondary antibody (Life Technologies, catalog number: A-11012)

    17. DAPI (Sigma-Aldrich, catalog number: 10236276001)

    18. VybrantTM multicolor cell labeling kit (Life Technologies, catalog number: V22889)

    19. hMSC culture medium (see Recipes)

    20. Staining buffer (see Recipes)

    21. CD146 staining solution (see Recipes)

    22. Fixative solution (see Recipes)

    23. Agarose solution (see Recipes)

    24. Permeablization buffer (see Recipes)

    25. Blocking buffer (see Recipes)

    26. Primary antibodies solution (see Recipes)

    27. Secondary antibodies solution (see Recipes)


  2. Materials for microfabrication and microfluidics

    1. Brass plates (5 × 5 cm)

    2. Dry-film photoresists: Eternal Laminar E8020, Eternal Laminar E8013 (Eternal Materials), and Alpho NIT215 (Nichigo-Morton)

    3. K2CO3 (Sigma-Aldrich, catalog number: P5833)

    4. Poly(dimethylsiloxane) (PDMS) (Dow Corning, catalog number: SYLGARD 184)

    5. 3MTM NovecTM 1720 Electronic Grade Coating (3M)

    6. 3MTM FluorinertTM Electronic Liquid FC-40 (3M)

    7. PEG-di-Krytox (RAN Biotechnologies)

Equipment

  1. Equipment for cell culture

    1. CO2 incubator (Binder, CB 170)

    2. Cell sorter (flow cytometer) (BD Biosciences, FACSAria III)


  2. Equipment for microfabrication and microfluidics

    1. Office laminator (PEAK, pro PS320)

    2. Ultraviolet lamp (Hamamatsu, Lightningcure)

    3. Micromilling machine (Minitech Machinery, CNCMini-Mill/GX)

    4. Plasma cleaner (Harric, PDC-32G)

    5. 100 μl and 1 ml glass syringes (SGE, Analytical Science, Gas tight luer lock syringes)

    6. 1 ml plastic syringe (Terumo, SS+01T1)

    7. Syringe pumps (neMESYS Low-Pressure Syringe Pump, Cetoni GmbH)


  3. Microscopy

    1. Motorized wide-field microscope (Ti, Eclipse, Nikon), equipped with a CMOS (complementary metal-oxide semiconductor) camera (ORCA-Flash4.0, Hamamatsu), a fluorescence light-emitting diode source (Spectra X, Lumencor), and a 10× objective with a 4-mm working distance (extra-long working distance) and a 0.45 numerical aperture (NA) (Plan Apo λ, Nikon)

    2. Motorized (Ti2, Nikon) confocal spinning disc microscope equipped with lasers (W1, Yokogawa) and the same camera and objective as above

Software

  1. MATLAB (r2016a, MathWorks, Natick, MA)

Procedure

  1. Microfabrication of the microfluidic chips

    1. A detailed protocol on the fabrication of the chips can be found in Amselem et al. (2018) . The chips consist of two parts: 1) The top part comprises a flow focusing junction, a serpentine, diverging rails, and a culture chamber. The molds are fabricated using dry resins that are etched to produce the shape of the droplet generators and guiding channels by standard soft lithography. 2) The bottom of the chips consists of an array of 270 hexagonal capillary traps. The molds of the bottoms part of the chips are fabricated by micromilling the brass plates. The geometry and dimensions of the features can be found in Sart et al. (2020) (Figure 1).



      Figure 1. Chip design and depth of the different regions. 1. Inlet for the injection of the aqueous phase containing cells and culture medium; 2. Inlet for oil injection at the junction; 3. Inlet for oil injection to push the drops within the trapping chamber.


    2. Fabricate the tops and the floors of the chips by casting poly(dimethylsiloxane) (PDMS) (a mix of 90% of base and 10% of curing agents) into the molds.

    3. Place the two molds in an oven set up at 80°C for 2 h to promote the polymerization of the PDMS.

    4. Extract the two parts of the chips from the molds and cut them with a scalpel.

    5. Assemble the two parts of the chips by bonding the two surfaces for 40 s using a plasma cleaner.

    6. Flush the chips with Novec and heat them at 110°C for three cycles to render the inner walls of the chip fluorophilic.


  2. Cell culture, sorting, and loading in microfluidic droplets

    1. Cultivate hMSCs using hMSC culture medium, from passage 2 to 7 in regular T-175 cm2 flasks, and place them into a culture incubator, set up at 37°C and 5% CO2.

    2. Seed the cells into the flask at a density of 5 × 103 cells/cm2, subcultivate them every week using TrypLE, and change the medium every 2 days.

      Optionally, sort hMSCs based on their level of expression of CD146 by flow cytometry. CD146 is a marker of undifferentiated status, whose level of expression decreases upon differentiation (Sacchetti et al., 2007). To separate the CD146dim and CD146bright hMSCs, isolate the cells (passage 5) from the flasks, and incubate them with CD146 staining solution for 30 min. Wash the cells with straining buffer. Identify and select the hMSC population by plotting FSC-A and SSC-A signals using the flow cytometer. Eliminate cell doublets by gating the main population obtained by plotting FSC-H versus FSC-A signals. From this selected population, plot the FITC-A (or any fluorochrome for which the cells are not labeled) versus Alexa-647-A signal distribution; analyze the spread of expression of CD146 with the cytometer and sort 25% of the brightest and 25% of the dimmest cells.

    3. Connect two 1 ml syringes containing a 1% RAN in FC-40 solution to inlets #3 and #2, block inlet #1, then flush the oil of each syringe at 50 μl/min to remove the air from the chip (Figure 2, Step 1).

    4. Load a suspension of 6 × 106 bulk- or a mix of 50:50 CD146dim and CD146bright sorted- hMSCs/ml in culture medium supplemented with the agarose solution into a 100 μl glass syringe.

    5. Connect this syringe into inlet #1 (Figure 2, Step 2).

    6. Apply a flow rate of 8-8.5 μl/min to syringe 1 and a flow rate of 11 μl/min to syringe 2. The solution containing the cells is pinched at the junction. This yields to the formation of monodispersed droplets containing a suspension of about 380 cells each (Figure 2, Step 2).

    7. Apply a flow rate of 50 μl/min on syringe 3: the droplets are pushed on the rails that guide them evenly within the culture chamber. The drops are then captured within the capillary traps (Figure 2, Step 2).

    8. Stop the flow of syringe #1 and apply a flow rate of 100 μl/min to syringes #2 and #3 to remove the excess of non-anchored droplets in the culture chamber (Figure 2, step 3).

    9. Stop the oil flows; the cells settle down at the bottom of the drops and start clustering.



      Figure 2. Protocol for cell loading and droplet formation


  3. Spheroid formation and culture in microfluidic droplets

    1. Place the chips into the CO2 incubator overnight to let the cells form spheroids (Figure 3 and Video 1).



      Figure 3. Kinetics of MB formation in microfluidic droplets. After stopping the oil flow, the cells are allowed to settle down to the bottom of the drops, where they start clustering. Cells are monitored continuously by live imaging, while forming MBs. Scale bar = 100 μm.


      Video 1. Morphology of MB in 3D. The MBs in liquid drops are subjected to recirculation by applying an oil flow around them. The protocol allows the MBs to rotate on their axis. The movie demonstrates the 3D structural organization of MBs.

    2. On the second day, place the chips at 4°C for 30 min to gel the agarose.

    3. Flush the chips with 1 ml of pure FC-40 oil, at 80 μl/min, to dilute the surfactant.

    4. Replace the oil phase with culture medium by slowly flowing the aqueous solution into the device. At this stage, the 3D aggregates are mechanically retained into the traps by the hydrogel. They can be regularly perfused with culture medium; thus, they are now ready for long-term culture while remaining fully viable (Sart et al., 2020).


  4. Spheroid labeling and imaging within microfluidic droplets

    To interrogate the structural organization within MBs:

    1. Label CD146dim- and CD146bright- hMSCs with Vybrant DiO (green) or Vybrant DiD (red) dyes (5 μl for 1 ml culture medium) for 30 min, prior to loading them into the microfluidic drops.

      Note: CD146 protein and the Vybrant dyes are soluble in Triton-X 100 solution; thus, this protocol does not allow to combine immunostaining with the detection of the different CD146 subpopulations.

    2. After spheroid formation and culture, image MBs using a fluorescent microscope (e.g., wide-field or confocal microscope equipped with large working distance objectives) (Figure 4).



      Figure 4. Spatial organization of CD146dim and CD146bright hMSCs within MBs. CD146dim and CD146bright are isolated by cell sorting, then labeled with Vybrant DiO (green) or Vybrant DiD (red) dyes prior to their loading into drops. Scale bar = 100 μm.


    To interrogate the functional organization within MBs (e.g., the regional expression of VEGF-A, COX-2, or RUNX-2):

    1. Fix the MBs by perfusing a solution of 200 µl of fixative solution, by first filling a 1 ml plastic syringe with PFA, then flowing at a flow rate of 80 μl/min.

    2. Incubate the aggregates of hMSCs with the PFA solution for 30 min at room temperature.

    3. Wash the culture chamber with PBS at the same flow rate (200 μl at 80 μl/min).

    4. Permeabilize the MBs by perfusing at 80 μl/min with a 200 μl of permeabilization buffer, and incubate for 5 min.

    5. Wash the culture chamber with PBS (200 μl at 80 μl/min).

    6. Block MBs by perfusion of a blocking buffer (200 μl at 80 μl/min) and incubate for 30 min.

    7. After blocking, perfuse the chamber with a solution of primary antibody (e.g., anti-COX-2 or anti-VEGF-A antibody or anti-RUNX-2; 200 μl at 80 μl/min), and incubate for 4 h.

    8. Wash the primary antibody solution by perfusing PBS (200 μl at 80 μl/min).

    9. Perfuse the solution of secondary antibodies (200 μl at 80 μl/min) and incubate for 1 h 30 min.

    10. Wash with PBS (200 μl at 80 μl/min).

    11. Image the MBs in the traps using a fluorescent microscope (Figure 5).

    12. To validate the specificity of the primary antibodies, incubate the sample with the secondary antibody only, then wash with PBS. Absence of fluorescent signal validates that the primary antibody is specific to its target (i.e., VEGF-A) and that excess of antibodies is effectively washed with PBS rinsing (Sart et al., 2020).

    13. To validate the absence of diffusion limitation, omit the blocking step and incubate with the secondary antibody only. Absence of diffusion limitation is demonstrated by homogeneous fluorescent signal distribution within the aggregate (Sart et al., 2020).



      Figure 5. Functional organization hMSCs within MBs: VEGF-A expression detected by immunostaining. Similar results were obtained for RUNX-2 and COX-2 immunostaining (Sart et al., 2020). Scale bar = 100 μm.

Data analysis

The objective of the data analysis is to provide a quantitative characterization of the structural and functional organization within mesenchymal bodies using image analysis. In the following experiments, the data are generated from about 270 replicates that have been reproduced in at least three different chips.

  1. Quantification of the structural organization within MBs using Matlab®

    Detailed information on data analysis can be found in Sart et al. (2017) .

    1. Identify the centroid of each spheroid.

    2. Measure the area (A) of each spheroid.

    3. Calculate R, the radius of each spheroid, defined as the square root of A/π.

    4. Measure the fluorescent intensity of each pixel of Vybrant DiO (green, corresponding to CD146dim hMSCs) or Vybrant DiD (red, corresponding to CD146bright hMSCs) stained cells, as well as their normalized distance (r/R) to the centroid (Figures 6A and 6B).

    5. Bin the values of fluorescence at specific (r/R).

    6. Normalize the two fluorescent signals against the DAPI signal to take into account the spherical shape of the aggregates.

    7. The relative intensity of the two different fluorescence signals indicates the relative abundance of the CD146dim vs. CD146bright hMSCs at different radii within the MBs (Figure 6C) (Sart et al., 2020).



      Figure 6. Data analysis methodology to quantify the structural organization in MBs. (A) Cell sorting yielded to the separation of CD146dim and CD146bright populations. (B) Position of the fluorescent signal of CD146dim and CD146bright labeled with Vybrant DiO (green) or Vybrant DiD (red) identified by their radial coordinates (r/R). Scale bar = 100 µm. (C) Quantification of the distribution of the different CD146 populations within MBs, calculated based on radial coordinates. Part C is extracted from Sart et al. (2020) .


  2. Quantification of the functional organization withing MB using Matlab®

    Detailed information on data analysis can be found in Sart et al. (2020) :

    1. Identify each local maximum of DAPI-stained nuclei (Figure 7A).

    2. Construct Voronoi diagrams (Chang et al., 2007) by drawing the perpendicular bisectors of the segments between each neighboring local DAPI maxima, which approximate the cell shapes inside the MBs (Figure 7A).

    3. Identify the position of each layer of cells within the aggregate (Figure 7B).

    4. Quantify the cellular cytoplasmic fluorescent signal (COX-2, VEGF-A, and RUNX-2) for each cell of the MBs (Figure 7C).

    5. Correlate the fluorescent signal of each cell to their cell layer position within the MBs (Figure 7D). To account for the variability of the cytoplasmic signal across the entire cell (nucleus included), the fluorescence signal of a single cell was defined as the mean signal of the 10% highest pixels of the corresponding Voronoi cell (Sart et al., 2020).

    6. Bin the values of fluorescence at specific cell layers.



      Figure 7. Data analysis methodology to quantify the functional organization in MBs: VEGF-A spatial distribution. (A) Detecting the DAPI signal enables drawing a Voronoi diagram, which allows estimating the area occupied by each cell in the aggregate. (B) The position of each cell is assigned to a layer number. (C) The fluorescent intensity in each cell defined from the Voronoi diagram is measured. (D) The fluorescent intensity of the protein of interest in each cell is correlated to the cell position (layer number) within the MBs, which allows mapping the cell function within the MBs. Similar results were obtained for RUNX-2 and COX-2 immunostaining (Sart et al., 2020).


    The results show that hMSCs self-organize in a hierarchical manner: most undifferentiated MSCs are located in the core, while partially committed cells are located at the boundaries of the MBs (Figure 6). Moreover, we found that such structural organization correlated with the induction of osteo-endocrine functions (i.e., COX-2 and VEGF-A expression) (Figure 7 and Sart et al., 2020 for RUNX-2 and COX-2).

Recipes

  1. hMSC culture medium

    Mix 50 ml of FBS and 5 ml of the pen-strep (100×) with 500 ml of α-MEM basal medium, which results in a medium containing 10% (v/v) FBS and 1× pen-strep.

  2. Staining buffer

    Mix 200 μl of FBS with 9.8 ml PBS, which results in a 2% FBS (v/v) solution in PBS.

  3. CD146 staining solution

    Mix 10 μl of Alexa Fluor 647-conjugated anti-CD146 antibody stock solution with 990 μl of staining buffer, which results 1:100 diluted CD146 antibody solution.

  4. Fixative solution

    Mix 1 ml of PFA stock solution (16%, v/v) with 3 ml of PBS, which results in a 4% PFA (v/v) solution.

  5. Agarose solution

    Weight 30 mg of agarose powder and mix it with 1 ml PBS. Place this solution in a oven set at 80°C for at least one hour, in order to melt the agarose. It results in a liquid agarose solution at concentration of 3% (w/v). Prior to cell loading into drops, mix 30 μl of the the 3% (w/v) agarose solution with 70 μl of medium containing 6 × 106 cells. This results in a solution containing cells at a concentration of 6 × 106 cells/ml and agarose at 0.9% (w/v).

  6. Permeablization buffer

    Mix 500 μl of Triton-X100 with 10 ml PBS, which results in a permeablization buffer containing 0.5% Triton-X100 (v/v).

  7. Blocking buffer

    Mix 500 μl of FBS with 9.5 ml PBS, which results in a bolocking buffer buffer containing a 5% FBS (v/v) solution.

  8. Primary antibodies solution

    Mix 10 μl of primary antibody stock solution (anti-COX2, anti-VEGF-A, anti-RUNX-2) with 990 μl of staining buffer, which results 1:100 diluted primary antibody solution.

  9. Secondary antibodies solution

    Mix 10 μl of Alexa Fluor 594-conjugated secondary antibody stock solution and 20 μl DAPI solution at 14 μM (prepared from 5 mg DAPI resuspended in 10 ml PBS, which stock solution (1.4 mM) is then diluted at 1:100, i.e., 10 μl into 990 μl PBS) with 970 μl of staining buffer, which results in a 1:100 diluted solution of secondary antibodies containging 0.3 μM DAPI.

Acknowledgments

C. Frot is gratefully acknowledged for the help with the microfabrication, and F. Soares da Silva is gratefully acknowledged for the help in flow cytometry. We acknowledge the support of the group of Biomaterials and Microfluidics (BMCF) of the Center for Innovation and Technological Research, as well as the Center for Translational Science (CRT)–Cytometry and Biomarkers Unit of Technology and Service (CB UTechS). The original paper (Sart et al., 2020) has been published in Science Advances (doi: 10.1126/sciadv.aaw7853).

Competing interests

The authors declare that they have no competing interests.

References

  1. Amselem, G., Sart, S. and Baroud, C. N. (2018). Universal anchored-droplet device for cellular bioassays. Methods Cell Biol 148: 177-199.
  2. Caplan, A. I. and Correa, D. (2011). The MSC: an injury drugstore. Cell Stem Cell 9(1): 11-15.
  3. Cesarz, Z. and Tamama, K. (2016). Spheroid Culture of Mesenchymal Stem Cells. Stem Cells Int 2016: 9176357.
  4. Chang, H., Yang, Q. and Parvin, B. (2007). Segmentation of heterogeneous blob objects through voting and level set formulation. Pattern Recognit Lett 28(13): 1781-1787.
  5. 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.
  6. Sacchetti, B., Funari, A., Michienzi, S., Di Cesare, S., Piersanti, S., Saggio, I., Tagliafico, E., Ferrari, S., Robey, P. G., Riminucci, M. et al. (2007). Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 131(2): 324-336.
  7. Sart, S. and Agathos, S. N. (2018). Towards Three-Dimensional Dynamic Regulation and In Situ Characterization of Single Stem Cell Phenotype Using Microfluidics. Mol Biotechnol 60(11): 843-861.
  8. Sart, S., Agathos, S. N., Li, Y. and Ma, T. (2016). Regulation of mesenchymal stem cell 3D microenvironment: From macro to microfluidic bioreactors. Biotechnol J 11(1): 43-57.
  9. Sart, S., Tomasi, R. F., Amselem, G. and Baroud, C. N. (2017). Multiscale cytometry and regulation of 3D cell cultures on a chip. Nat Commun 8(1): 469.
  10. Sart, S., Tomasi, R. F., Barizien, A., Amselem, G., Cumano, A. and Baroud, C. N. (2020). Mapping the structure and biological functions within mesenchymal bodies using microfluidics. Sci Adv 6(10): eaaw7853.
  11. Sart, S., Tsai, A. C., Li, Y. and Ma, T. (2014). Three-dimensional aggregates of mesenchymal stem cells: cellular mechanisms, biological properties, and applications. Tissue Eng Part B Rev 20(5): 365-380.

简介

[摘要]具有间充质干/基质细胞 (MSC)、间充质体 (MB) 的球体的形成通常使用生物反应器或常规孔板进行。虽然这些方法促进了大量球体的形成,但它们对其结构或对其环境的调节提供了有限的控制。因此,到目前为止,很难阐明协调结构组织和诱导 MB 营养功能的机制。我们最近展示了一个基于液滴的集成微流体平台,用于 MB 的高密度形成和培养,以及对其中细胞结构和功能组织的定量表征。该协议首先将数百个 MSCs的悬浮液封装在毛细管陷阱中的微流体液滴中。液滴固定后,MSCs 开始聚集并形成密集的球形聚集体,显示出紧密的尺寸分布。通过利用微流控芯片和传统显微镜技术之间的良好拟合,定量成像用于提供人类MSC 以分层方式自组织的可靠证明。此外,发现MB内的结构组织与骨内分泌功能(即,COX-2和VEGF-A表达)的诱导相关。因此,本平台提供了一种独特的方法来将 MB 中的结构组织与其功能特性联系起来。

图文摘要:


液滴的形成一体化,文化微流体平台,以及间质组织(MBS)的特征。该设备配备了一个液滴生产区(流动聚焦)和一个培养室,可以并行培养 270 MB。逐层分析揭示了MB 内的分层发展组织。


[背景]间充质干/基质细胞(MSC)包括的异源群体的间充质祖细胞,其能够分化成成骨细胞的,chondrogen IC ,一个第二生脂谱系(Dominici等人,2006) 。MSC 还具有调节免疫细胞活性、促进血管生成、减少组织炎症和激活组织驻留祖细胞的重要营养功能,使这种细胞类型特别适合许多组织工程/再生应用(Caplan 和 Correa,2011)。将利用MSC(球体的形成,即,间充质组织,MBS)最近发现,以提高它们的分化潜能及其分泌活动(萨尔特等,2014) 。然而,人们对异质 hMSC 如何在 3D 中自组织以及将其结构组织与其功能活动联系起来的机制知之甚少(Cesarz 和 Tamama,2016 年)。
几种方法已用于形成 MB,包括生物反应器和常规孔板(Sart等人,2014 年和2016 年;Sart 和 Agathos ,2018 年)。然而,虽然这些方法能够形成大量 MB,但它们对单个聚合刺激或表征提供有限的控制(Sart等人,2016 年;Sart 和 Agathos,2018 年)。因为构成的MSC的异质群体,在单个单聚集体的分析-需要细胞分辨率理解,通过该三维培养诱导的非常敏感的少数细胞或全球人口移的行为功能的改变的机制。因此,宏观培养容器和全球种群分析不允许 MB 结构和细胞功能相关(Sart等,201 7;Sart 和 Agathos ,2018)。
在这里,我们使用了一个微流体平台,允许在纳升液滴内形成高密度和可控大小的 MB。微流体格式使得健壮定量示范,使用定量成像在一个单-细胞水平,即人MSC以分级的方式自组织:最未分化的MSC位于核心,而部分定型细胞位于边界MB。此外,我们发现这种结构组织与骨内分泌功能的诱导相关。鉴于了解导致 3D 干细胞培养中组织模式化的机制,此处开发的微流体方法和协议可以应用于表征其他类型的类器官,将细胞分选过程与表型承诺联系起来。 当前的方法可以应用到任何类型的在三维培养的干细胞,尽管液滴的体积应适合于一特定类型的干细胞的。该协议中开发的数据驱动方法使我们能够获得类器官内结构和功能组织的稳健定量表征,使用需要改变细胞微环境的传统基于人群的方法很难获得。

关键字:间充质基质细胞, 间充质体, 微流体, 飞沫, 球体, 定量成像


材料和试剂

 
细胞培养试剂
1. T-175 cm 2烧瓶(Greiner,Cellstar ,目录号:660175)      
2.来自脐带沃顿氏胶 (hMSCs) 的人类间充质基质细胞,购自美国典型培养物保藏中心 (ATCC) (ref #PCS-500-010)      
3.       α -改良的 Eagle 培养基(α -MEM)(Life Technologies,目录号:32561-029)
4. TrypLE TM Express (Life Technologies,目录号:12604013)      
5.磷酸盐缓冲盐水(PBS)(Sigma - Aldrich,目录号:D8662)      
6.胎牛血清(FBS)(Life Technologies,目录号:10500-064)      
7.青霉素-链霉素(p en-strep)(Life Technologies,目录号:10378-016)      
8. Triton X-100(Sigma-Aldrich,目录号:X100)      
9.超低熔点琼脂糖(Sigma-Aldrich,目录号:A5030)      
10.多聚甲醛(PFA),16%(Alpha Aesar,目录号:43368)   
11.小鼠抗人 CD146 - Alexa Fluor 647(克隆 P1-H12)(BD Biosciences,目录号:563619)   
12.兔抗- COX-2多克隆抗体(Abcam公司,目录号:ab15191)   
13.兔抗人VEGF-A单克隆抗体(Abcam,目录号:ab52917)   
14.小鼠抗人RUNX-2单克隆抗体(Abcam,目录号:ab76956)   
15. Alexa Fluor 488 偶联山羊抗小鼠 IgG2a 二抗(Life Technologies,目录号:A-21131)   
16. Alexa Fluor 594 -偶联山羊多克隆抗兔 IgG 二抗(Life Technologies,目录号:A-11012)   
17. DAPI(Sigma-Aldrich,目录号:10236276001)   
18. Vybrant TM多色细胞标记试剂盒(Life Technologies,目录号:V22889)   
19. hMSC 培养基(见配方)   
20.染色缓冲液(见配方)   
21. CD146 染色液(见配方)   
22.固定液(见配方)   
23.琼脂糖溶液(见食谱)   
24.通透缓冲液(见配方)   
25.阻塞缓冲区(见食谱)   
26. 一抗溶液(见配方)   
27.二抗溶液(见配方)   
 
用于微细加工和微流体的材料
黄铜板 (5 × 5 cm)
干膜光刻胶:Eternal Laminar E8020、Eternal Laminar E8013(Eternal Materials)和 Alpho NIT215(Nichigo-Morton)
K 2 CO 3 (Sigma-Aldrich,目录号:P5833)
聚(二甲基硅氧烷)(PDMS)(道康宁,目录号:SYLGARD 184)
3M TM Novec TM 1720 电子级涂层 (3M)
3M TM Fluorinert TM电子液体 FC-40 (3M)
PEG-di-Krytox(RAN 生物技术)
 
设备
 
细胞培养设备
CO 2培养箱(粘合剂,CB 170)
细胞分选仪(流式细胞仪)(BD Biosciences, FACSAria III )
 
微加工和微流体设备
办公室覆膜机(PEAK、pro PS320)
紫外线灯(滨松、Lightningcure)
微型铣床(Minitech Machinery、CNCMini-Mill/GX)
等离子清洁器(Harric,PDC-32G )
100 μ l 和 1 ml玻璃注射器(SGE、分析科学、气密鲁尔锁注射器)
1 ml l塑料注射器 (Terumo, SS+01T1)
注射泵(neMESYS 低压注射泵,Cetoni GmbH)
 
显微镜
电动宽视场显微镜(Ti,Eclipse,Nikon),配备 CMOS(互补金属氧化物半导体)相机(ORCA-Flash4.0,Hamamatsu),荧光发光二极管源(Spectra X,Lumencor)和具有 4 毫米工作距离(超长工作距离)和 0.45 数值孔径 (NA)的 10 ×物镜(Plan Apo λ ,尼康)
电动(Ti2,尼康)共聚焦旋转圆盘显微镜,配备激光器(W1,横河)以及与上述相同的相机和物镜
 
软件
 
MATLAB (r2016a, MathWorks, Natick, MA)
程序
 
微流控芯片的微加工
1.可以在Amselem等人中找到有关芯片制造的详细协议。(2018 年)。该芯片包括的两个部分:1)顶部部分包括流动聚焦结,蛇纹石,发散轨道,和一个培养腔室中。模具是使用干树脂制造的,这些树脂经过蚀刻以通过标准软光刻法产生液滴发生器和引导通道的形状。2) 芯片底部由 270 个六边形毛细管阱阵列组成。芯片底部的模具是通过微铣削黄铜板制成的。特征的几何形状和尺寸可以在Sart等人中找到。(2020 年)(图 1 )。      
 
 
图 1. 不同区域的芯片设计和深度。1 、含有细胞和培养基的水相进样口;2 、交界处注油口;3 、注油口,推动捕集室内的液滴。
 
2.通过将聚二甲基硅氧烷 (PDMS)(90% 的基础和 10% 的固化剂的混合物)浇铸到模具中来制造芯片的顶部和底部。      
3.将两个模具放在 80 °C的烘箱中 2小时,以促进 PDMS 的聚合。      
4.提取从模具芯片的两个部分,并用手术刀切割它们。      
5.通过使用等离子清洁器将两个表面粘合40秒来组装芯片的两个部分。      
6.用 Novec 冲洗芯片并在 110 °C 下加热三个循环,使芯片内壁具有亲氟性。      
 
细胞培养,分拣,和装载在微流体液滴
培育的hMSCs使用hMSC的培养米ë dium ,从通道2至7在常规T-175厘米2个烧瓶,并将其放置在一个培养箱中,在37建立℃下和5 %CO 2 。
将细胞以 5 × 10 3细胞/cm 2的密度接种到烧瓶中,每周使用 TrypLE 进行传代,每 2 天更换一次培养基。
或者,通过流式细胞术根据 CD146 的表达水平对 hMSC 进行分类。CD146 是未分化状态的标志物,其表达水平在分化时降低(Sacchetti等,2007)。为了分离 CD146暗淡和 CD146明亮的hMSC,将细胞(第 5 代)从烧瓶中分离出来,并用CD146 染色溶液将它们孵育30 分钟。用过滤缓冲液清洗细胞。识别并通过使用绘图FSC-A和SSC-A信号选择的hMSC人口的流式细胞仪。通过对通过绘制 FSC- H与 FSC- A信号获得的主要群体进行门控来消除细胞双峰。从这个选定的群体中,绘制 FITC-A(或任何未标记细胞的荧光染料)与 Alexa-647-A 信号分布的关系图;用细胞仪分析 CD146 表达的扩散,并分选 25% 的最亮的细胞和 25% 的最暗的细胞。
将两个1毫升注射器containi毫微克1%在FC-40的溶液,以入口RAN#3和#2,数据块入口#1,然后在50冲洗每个注射器的油μ升/分钟,以除去所述空气从所述芯片(图 2,步骤 1 ) 。
加载悬架6的× 10 6块体或50:50的混合CD146暗淡和CD146亮sorted-的hMSCs /毫升补充有在培养基琼脂糖溶液为100 μ升玻璃注射器。
将此注射器连接到入口 #1(图 2,步骤 2 )。
适用的8-8.5流速微升/ min至注射器1和11的流速μ升/ min至注射器2含有细胞的溶液的交界处被夹持。这导致形成单分散液滴,每个液滴包含约 380 个细胞的悬浮液(图 2,步骤 2 )。
在注射器 3 上应用 50 μl /min 的流速:液滴被推到导轨上,在培养室中均匀地引导它们。然后液滴被毛细管捕集器捕获(图 2,步骤 2 )。
停止注射器 #1 的流动,并将 100 μl /min 的流速应用于注射器 #2 和 #3,以去除培养室中多余的非锚定液滴(图 2,步骤 3 )。
停止油流;细胞在水滴底部安定下来并开始聚集。
 
图 2. 细胞加载和液滴形成的协议
 
微流体液滴中的球体形成和培养
将芯片放入 CO 2培养箱过夜,让细胞形成球体(图 3 和视频1 )。
 
 
图 3.微流体液滴中 MB 形成的动力学。停止油流后,细胞被允许沉降到液滴的底部,在那里它们开始聚集。细胞通过实时成像连续监测,同时形成 MB。小号Cale的巴= 100 μ米。
 
 
视频 1. MB 的 3D 形态。在液滴的MB是由经受再循环施加的它们周围的油流。该协议允许 MB 绕其轴旋转。这部电影展示了 MB 的 3D 结构组织。
 
上吨他第二天,将芯片在4 ℃下30分钟,以胶凝琼脂糖。
用 1 ml 纯 FC-40 油以 80 μl /min 的速度冲洗芯片,以稀释表面活性剂。
通过将水溶液缓慢流入设备中, 用培养基替换油相。在这个阶段,3D 聚集体被水凝胶机械地保留在陷阱中。它们可以定期用培养基灌注;因此,他们现在已经准备好长-长期培养,而其余的完全可行的(萨尔特等,2020) 。
 
微流体液滴内的球体标记和成像
要询问 MB 内的结构组织:
标记 CD146暗淡- 和 CD146明亮- hMSCs 与 Vybrant DiO(绿色)或 Vybrant DiD(红色)染料(5 μl用于 1 ml培养基)30 分钟,然后将它们加载到微流体滴中。
注:CD146 蛋白和 Vybrant 染料可溶于 Triton-X 100 溶液;因此,该协议不允许将免疫染色与检测不同的 CD146 亚群结合起来。
球状体形成和培养后,图像使用荧光显微镜的MB(例如,宽-场或者配备有大的工作距离的物镜共焦显微镜)(图4 )。
 
 
图 4. MB 内CD146暗淡和 CD146明亮hMSC 的空间组织。CD146暗淡和CD146亮通过细胞分选,然后用标记的分离Vybrant细胞DIO(绿色)或Vybrant细胞DiD的(红色)之前将其装载到滴染料。小号Cale的棒= 100微米。
 
询问的MB内的功能单位(例如。,VEGF-A的表达区域,COX-2 ,或RUNX-2):
通过灌注200的溶液固定的MB μ升的固定溶液,通过第一填充1米升塑料注射器与PFA,然后在80的流速流动微升/分钟。
在室温下用 PFA 溶液孵育 hMSCs 的聚集体 30 分钟。
在相同的流速(200用PBS洗培养室μ升在80 μ升/分钟)。
在80灌注透化的MB μ升/分钟与200 μ升的透化缓冲液中,并孵育5分钟。
(200用PBS洗培养室μ升在80 μ升/分钟)。
块的MB通过灌注的一个阻挡buffe - [R (200 μ升在80 μ升/分钟)孵育30分钟。
(阻塞,灌注腔室与初级抗体溶液后例如,抗- COX-2或抗VEGF-A抗体或抗RUNX-2 ; 200 μ升在80 μ升/分钟),并孵育˚F或4小时。
由(200灌注PBS洗一次抗体溶液μ升在80 μ升/分钟)。
灌注二抗溶液(200微升,80微升/分钟)并孵育 1小时30分钟。
洗涤用PBS(200 μ升在80 μ升/分钟)。
使用荧光显微镜对陷阱中的 MB 进行成像(图 5 )。
为了验证一抗的特异性,只用二抗孵育样品,然后用 PBS 清洗。荧光信号的缺失证实一抗对其靶标(即,VEGF-A)具有特异性,并且用PBS漂洗有效地洗涤了过量的抗体(Sart等人,2020)。
为了验证不存在扩散限制,请省略封闭步骤并仅与二抗一起孵育。聚集体内均匀的荧光信号分布证明没有扩散限制(Sart等人,2020 年).
 
 
图 5. MB 内的功能组织 hMSC:通过免疫染色检测 VEGF-A 表达。RUNX-2 和 COX-2 免疫染色获得了类似的结果(Sart等,2020)。比例尺 = 100 μ m。
 
数据分析
 
数据分析的目的是使用图像分析提供间充质体内结构和功能组织的定量表征。在以下实验中,数据是从在至少三个不同芯片中复制的大约270 次重复中生成的。
A.使用 Matlab ®量化 MB 内的结构组织      
关于数据分析的详细信息可以在Sart等人中找到。(2017 年)。
确定每个球体的质心。
测量每个球体的面积 (A) 。
计算每个球体的半径 R,定义为 A/ π 的平方根。
测量Vybrant DiO(绿色,对应 CD146暗淡hMSC)或 Vybrant DiD(红色,对应 CD146亮hMSC)染色细胞的每个像素的荧光强度,以及它们到质心的归一化距离 (r/R) (图小号6A和6乙)。
将特定 (r/R) 处的荧光值分箱。
将两个荧光信号针对 DAPI 信号标准化,以考虑到聚集体的球形形状。
两种不同荧光信号的相对强度表明了MB 内不同半径的 CD146暗与 CD146亮hMSC的相对丰度(图6C )(Sart等人,2020)。
 
 
图 6.量化 MB 结构组织的数据分析方法。(A)细胞分选产生了 CD146暗淡和 CD146明亮种群的分离。(B)P CD146的荧光信号的osition暗淡和CD146亮标记Vybrant细胞DIO(绿色)或Vybrant细胞DiD的(红色)通过它们的径向坐标(r / R)来标识。小号Cale的棒=100μm的。(C)的定量的不同CD146人口分布小号的MB内,基础计算的径向坐标。C 部分摘自 Sart等人。(2020 年)。
 
B.使用 Matlab ®用 MB 量化功能组织      
关于数据分析的详细信息可以在Sart等人中找到。(2020 年):
识别 DAPI 染色核的每个局部最大值(图 7A )。
通过绘制每个相邻局部 DAPI 最大值之间的线段的垂直平分线来构建 Voronoi 图(Chang等人,2007),其近似于MB 内的单元形状(图 7A )。
确定聚合内每层单元格的位置(图 7 B )。
量化蜂窝细胞质荧光信号(COX-2,VEGF-A ,为的MB(中的每个小区和RUNX-2)图7 Ç )。
将每个细胞的荧光信号与其在 MB 中的细胞层位置相关联(图 7 D )。为了说明整个细胞(包括细胞核)的细胞质信号的可变性,单个细胞的荧光信号被定义为相应 Voronoi 细胞的 10% 最高像素的平均信号(Sart等人,2020 年).
Bin 特定细胞层s的荧光值。
 
 
图 7. 量化 MB 功能组织的数据分析方法:VEGF-A 空间分布。(A)d etecti纳克的DAPI信号使绘制荷兰国际集团Voronoi图,它允许estimat荷兰国际集团的所占面积通过在聚集体的每个小区。(B)每个单元格的位置都分配给一个层号。(C)测量从 Voronoi 图定义的每个单元格中的荧光强度。(D)每个细胞中感兴趣的蛋白质的荧光强度与 MBs 内的细胞位置 (层数) 相关, 这允许映射MBs 内的细胞功能。对于RUNX-2和COX-2免疫染色,获得类似的结果(Sart的等人,2020) 。
 
结果表明 hMSCs 以分层方式自组织:大多数未分化的 MSCs 位于核心,而部分定型的细胞位于 MBs 的边界(图 6 )。此外,我们发现与骨内分泌功能的诱导相关(这样的结构组织即,COX-2和VEGF-A表达)(图7和萨尔特等人,2020年RUNX-2和COX-2)。
 
食谱
 
要准备的hMSC的培养基:
将 50 ml FBS 和 5 ml p en-strep (100X)与500 ml α -MEM 基础培养基混合,从而产生含有10% (v/v) FBS 和 1 X p en-strep的培养基。
准备染色缓冲液:
将 200 μl的 FBS 与 9.8 ml 的PBS 混合,从而在 PBS 中生成 2% FBS (v/v)溶液。
制备CD146 染色溶液:
混合10微升的Alexa氟647-缀合的抗CD146抗体原液与990微升染色的缓冲,这将导致1:100稀释的CD146抗体溶液。
制备固定溶液:
将 1 ml PFA 库存溶液(16% ,v/v )与 3 ml PBS 混合,得到 4% PFA (v/v)溶液。
制备琼脂糖溶液:
称取 30 毫克琼脂糖粉并将其与 1 毫升 PBS 混合。地方个是在80℃下至少一小时的烘箱组解决方案,为了以熔化AG一个玫瑰。它导致一个液体琼脂糖溶液在浓度3 %(W / V) 。前小区负载在以滴,混合30微升的3%(W / V)琼脂糖溶液与70微升含有介质的6×10 6细胞。这导致溶液中含有浓度为 6 x 10 6细胞/ml 的细胞和 0.9% (w/v) 的琼脂糖。
准备渗透缓冲液:
混合5 00微升的的Triton-X100用10ml PBS ,该结果在一个透化缓冲液含0.5%的Triton-X100(V / V)。
准备 b锁定缓冲区:
混合500微升FBS与9. 5米升PBS,这导致bolocking缓冲器缓冲含5%FBS(V / V)溶液。
准备初级抗体溶液:
将 10 μl一抗原液(抗 COX2、抗 VEGF-A 、抗 RUNX-2)与 990 μl染色缓冲液混合,得到 1:100 稀释的一抗溶液。
制备二次抗体溶液:
混合10微升的Alexa氟594缀合的二级抗体原液和2 0微升DAPI溶液在1 4 μ中号(从制备5毫克DAPI再悬浮于1 0 ml的PBS,其股票溶液(1 。4毫摩尔),然后在稀释1:100 ,即1 0微升到990微升PBS ),用9 7 0 μ染色缓冲液升,这导致了1:100稀贡献的第二抗体的溶液containging 0 3 μ中号DAPI。
 
致谢
 
C. Frot 对微加工的帮助表示感谢,F. Soares da Silva 对流式细胞术的帮助表示感谢。我们感谢创新和技术研究中心的生物材料和微流体 (BMCF)小组以及转化科学中心 (CRT)-细胞计量学和生物标志物技术和服务部门 (CB UTechS) 的支持。原始论文(Sart et al. , 2020)已发表在 Science Advances (doi: 10.1126/sciadv.aaw7853 ) 上。
 
利益争夺
 
作者声明他们没有相互竞争的利益。
 
参考
 
1. Amselem, G.、Sart, S.和Baroud, C. N. (2018 年)。用于细胞生物测定的通用锚定液滴装置。方法细胞生物学杂志148 :177 - 199。      
2. Caplan, AI 和 Correa, D. (2011)。MSC:一家伤病药店。细胞 干细胞9(1):11-15。                    
3. Cesarz, Z. 和 Tamama, K. (2016)。间充质干细胞的球体培养。干细胞国际2016:9176357。                    
4. Chang, H.、Yang, Q. 和 Parvin, B. (2007)。通过投票和水平集公式分割异构 blob 对象。模式识别 Lett 28(13): 1781-1787。      
5. Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F., Krause, D., Deans, R., Keating, A., Prockop, D. 和霍维茨,E.(2006 年)。定义多能间充质基质细胞的最低标准。国际细胞治疗学会立场声明。细胞疗法8(4):315-317。                    
6. Sacchetti, B., Funari, A., Michienzi, S., Di Cesare, S., Piersanti, S., Saggio, I., Tagliafico, E., Ferrari, S., Robey, PG, Riminucci, M 。等人。(2007)。骨髓血窦中自我更新的骨祖细胞可以组织造血微环境。单元格131(2):324-336。      
7. Sart, S. 和 Agathos, SN (2018)。使用微流体技术实现单干细胞表型的三维动态调节和原位表征。Mol Biotechnol 60(11): 843-861。      
8. Sart, S., Agathos, SN, Li, Y. 和 Ma, T. (2016)。间充质干细胞 3D 微环境的调节:从宏观到微流体生物反应器。生物技术杂志11(1): 43-57。      
9. Sart, S., Tomasi, RF, Amselem, G. 和 Baroud, CN (2017)。多尺度细胞计数和芯片上 3D 细胞培养的调节。国家通讯社8(1): 469。      
10. Sart, S., Tomasi, RF, Barizien, A., Amselem, G., Cumano, A. 和 Baroud, CN (2020)。使用微流体绘制间充质体内的结构和生物学功能。Sci Adv 6(10):eaaw7853。   
11. Sart, S., Tsai, AC, Li, Y. 和 Ma, T. (2014)。间充质干细胞的三维聚集体:细胞机制、生物学特性和应用。组织工程 B 部分修订版20(5):365-380。                 
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Sart, S., Tomasi, R. F., Barizien, A., Amselem, G., Cumano, A. and Baroud, C. N. (2021). Structural and Functional Mapping of Mesenchymal Bodies. Bio-protocol 11(19): e4177. DOI: 10.21769/BioProtoc.4177.
  2. Sart, S., Tomasi, R. F., Barizien, A., Amselem, G., Cumano, A. and Baroud, C. N. (2020). Mapping the structure and biological functions within mesenchymal bodies using microfluidics. Sci Adv 6(10): eaaw7853.
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