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Aug 2020

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OrganoPlate Micro-fluidic Microvessel Culture and Analysis
OrganoPlate微流控微血管培养与分析   

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Abstract

The endothelial cells from the microvasculature are key drivers and targets of inflammatory and thrombotic processes in microvascular diseases. The study of bioactive lipids in inflammatory processes has been largely based on two-dimensional endothelial cell cultures. Three-dimensional microvessels-on-a-chip provides an opportunity to monitor the inflammatory phenotype of human microvessels in a more physiological-relevant environment. This protocol describes the culture of endothelial cells as three-dimensional microvessels in the OrganoPlate. The microvessels are treated with tumor necrosis factor alpha to induce inflammation. The collection of samples from the microvessels is optimized for measuring bioactive lipids with liquid chromatography-mass spectrometry, providing a more informative metabolic readout as compared with functional assays.

Keywords: Microvessels (微血管), 3D cell culture (三维细胞培养), Oxidative stress (氧化应激), Metabolomics (代谢组学), Microfluidics (微流体), Inflammation (炎症), Organs-on-chips (器官芯片), Endothelial cells (内皮细胞)

Background

In recent decades, microvascular diseases have become a leading cause of mortality worldwide. Chronic activation of endothelial cells by cardiovascular risk factors can inflict the loss of pericytes, which play a critical role in microvascular stabilization. To diagnose and treat microvascular diseases, we aim to explore the association of circulating plasma factors with microvascular integrity.


In vitro mechanistic studies of bioactive lipids have been largely based on two-dimensional (2D) endothelial cell cultures that, due to lack of laminar flow and growth of cells on non-compliant stiff substrates, often display a proinflammatory phenotype. This complicates the assessment of inflammatory processes, such as tumor necrosis factor alpha (TNFα)-induced effects. Novel microfluidics-based perfused three-dimensional (3D) microvessels-on-a-chip models provide a unique opportunity to generate microvessels in a more physiological environment. The microvessels-on-a-chip are phaseguide-derived membrane-free microvessels grown from seeded endothelial cells on a 3D soft scaffold in the OrganoPlate 2-lane (van Duinen et al., 2017). To monitor the inflammatory phenotype of the 3D microvessels, we used an optimized targeted ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) measurement of a panel of pro- and anti-inflammatory bioactive lipids and generated expression profiles of TNFα-treated microvessels under flow (Junaid et al., 2020a).


Using the described protocol, we assessed the concentrations of prostaglandins, isoprostanes, lysophosphatidic acid (LPA) classes, sphingolipids, and platelet activating factor (PAF) secreted by primary human umbilical vein endothelial cells (HUVECs) seeded in the microvessels-on-a-chip. Conditioned medium perfused through TNFα-treated and control (untreated) microvessels was sampled, pooled, and measured by UPLC-MS/MS metabolomics (Schoeman et al., 2018). A strong differential response was observed between untreated and TNFα-treated microvessels. Moreover, the relative concentrations of the bioactive lipids found in the microvessels-on-a-chip are similar to those found in normal human blood vessels.


Most assays performed with the OrganoPlate to study microvessels are related to immunostaining, permeability, and angiogenesis (van Duinen et al., 2019, Junaid et al., 2020b). These assays study morphological changes by imaging phenotypes, and although relevant, the metabolic readout of microvessels using metabolomics is more informative. In this protocol, coupling LC/MS-based metabolomics techniques with the OrganoPlate is novel and allows us to measure bioactive lipids, providing exceptional information regarding microvascular disease onset and progression and the discovery of new biomarkers of inflammation and oxidative stress in this context. The results are easily translatable to physiological values found in human microvessels. Moreover, metabolic readouts for inflammation and oxidative stress are highly valuable in the organ-on-a-chip field with respect to drug research. The following protocol using the OrganoPlate was developed for researchers to study microvessels with approaches that suit other microfluidic devices and organ models.


Materials and Reagents

  1. 1.5-ml Eppendorf tubes

  2. 15-ml centrifuge tubes

  3. 50-ml centrifuge tubes

  4. 2-ml pipettes (sterile)

  5. 5-ml pipettes (sterile)

  6. 10-ml pipettes (sterile)

  7. P20 micropipette

  8. P1000 micropipette

  9. P20 tips (sterile)

  10. P1000 tips (sterile)

  11. 0.2-ml Biopur repeater pipette tips (VWR, catalog number: 89232-968)

  12. 1.0-ml Biopur repeater pipette tips (VWR, catalog number: 89232-972)

  13. 2.5-ml Biopur repeater pipette tips (VWR, catalog number: 89232-974)

  14. Cryovial of primary human umbilical vein endothelial cells (HUVECs; Lonza, catalog number: C2519A; stored in liquid nitrogen)

  15. Cell counting slides (Bio-Rad, catalog number: 1450011)

  16. T75 flasks

  17. Ice

  18. Dry ice

  19. Trypan Blue

  20. 1% gelatin

  21. Endothelial Cell Growth Medium 2 (EGM2; PromoCell, catalog number: C-39216; 4°C)

    1. Basal Medium 2 (Promocell, catalog number: C-22211; 4°C)

    2. Growth Medium 2 Supplement Mix (Promocell, catalog number: C-39216; -20°C)

  22. Penicillin/Streptomycin (Sigma-Aldrich, catalog number: P4333; -20°C)

  23. Phosphate-buffered saline (PBS)

  24. TrypLE (Gibco, catalog number: 12604013)

  25. OrganoPlate 2-lane (MIMETAS, catalog number: 9603-400-B)

  26. Rat tail collagen type 1 (Trevigen, catalog number: 3440-005-01; 4°C)

  27. 37 g/L Na2CO3 (Sigma-Aldrich, catalog number: S5761; 4°C)

  28. 1 M HEPES buffer (Gibco, catalog number: 15630-056; 4°C)

  29. Hank’s Balanced Salt Solution with calcium and magnesium (HBSS+; Life Technologies, catalog number: 24020117)

  30. Tumor necrosis factor alpha (TNFα; Sigma-Aldrich, catalog number: H8916; -20°C)

  31. Phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, catalog number: P8139; -80°C)

  32. 2-lane plate layout form (Supplementary Material 1)

  33. 70% ethanol

Equipment

  1. Pipetboy

  2. Eppendorf repeater M4 pipette (VWR, catalog number: 613-2890)

  3. OrganoFlow (MIMETAS, catalog number: MI-OFPR-L)

  4. Incubator (37°C, 5% CO2)

  5. Water bath (37°C)

  6. Eppendorf centrifuge 5810 (Sigma-Aldrich, catalog number: 022628188)

  7. Cell culture hood

  8. -80°C freezer

  9. Liquid chromatography-tandem mass spectrometer (Shimadzu, catalog number: LCMS-8060)

  10. Brightfield microscope

  11. TC20 automated cell counter (Bio-Rad, catalog number: 1450102)

Software

  1. Microsoft Excel

  2. GraphPad Prism (GraphPad Software)

  3. LabSolutions (Shimadzu)

  4. MetaboAnalyst (https://www.metaboanalyst.ca/)

Procedure

This protocol details every step of culturing microvessels in the OrganoPlate 2-lane up to measuring and analyzing bioactive lipids. All procedures handling cells and the OrganoPlate are sterile and carried out in the cell culture hood. A general outline of the workflow is described below:

Day 1: Initiate the culture process and maintenance.

Day 2: Refresh the medium in the T75 flask.

Day 5:

  1. Seed the collagen in the OrganoPlate 2-lane.

  2. Harvest the cells from the T75 flask.

  3. Seed the cells in the OrganoPlate 2-lane.

Day 8: Refresh the medium in the OrganoPlate 2-lane.

Day 9: Incubate the microvessels with TNFα and PMA overnight.

Day 10: Collect the medium from the microvessels, store the samples in a -80°C freezer and measure the bioactive lipids.


Day 1

  1. Initiate the culture process and maintenance

    1. Warm the 1% gelatin in a 37°C water bath.

    2. Prepare EGM2 by adding Growth Medium 2 Supplement Mix and 5 ml Penicillin/Streptomycin to the Basal Medium 2.

    3. Warm 50 ml EGM2 in a 37°C water bath.

    4. Pipette 5 ml 1% gelatin to a T75 flask and place in the incubator for 30 min.

    5. Pipette 11 ml 50 ml EGM2 into a 15-ml centrifuge tube.

    6. Quickly thaw a cryovial of HUVECs in a 37°C water bath, being careful not to submerge the entire vial.

      Important: Watch the cryovial closely; when the last sliver of ice melts, remove the cryovial.

    7. Dispense the cells into the 15-ml centrifuge tube containing 11 ml EGM2.

    8. Centrifuge the tube at 200 × g for 7 min to pellet the cells.

    9. While the cells are centrifuging, take the T75 flask containing 1% gelatin from the incubator. Aspirate the gelatin and pipette 12 ml EGM2 into the flask.

    10. Aspirate the supernatant from the centrifuged tube without disturbing the cell pellet.

    11. Resuspend the cells in 500 µl EGM2 by gently pipetting to break up any clumps.

    12. Pipette the cell suspension into the gelatin-coated T75 flask.

    13. Gently rock the flask to evenly distribute the cells and place in the incubator.


Day 2

  1. Refresh the medium in the T75 flask

    1. Warm 12 ml EGM2 in a 37°C water bath.

    2. Take out the T75 flask containing the HUVECs from the incubator (Figure 1).

    3. Remove the medium, replace it with the warmed, fresh medium, and return the flask to the incubator.



      Figure 1. Human umbilical vein endothelial cells (HUVECs) cultured in a T75 flask. Brightfield microscopy images were taken on days 1 and 2. Scale bars: 400 µm.


Day 5

  1. Seed the collagen in the OrganoPlate 2-lane

    1. Warm the 1% gelatin in a 37°C water bath.

    2. Place a 1.5-ml Eppendorf tube on ice.

    3. Pipette 60 µl 1 M HEPES buffer and 60 µl 37 g/L Na2CO3 in the 1.5-ml Eppendorf tube and mix. Place the tube on ice.

    4. Add 480 µl 5 mg/ml rat tail collagen type 1 to the HEPES/Na2CO3 mixture to a final concentration of 4 mg/ml collagen solution. Mix carefully to avoid the formation of bubbles. Place the tube back on ice.

    5. Lay and hold the OrganoPlate flat with your non-pipetting hand in the cell culture hood (Figure 2).



      Figure 2. The OrganoPlate 2-lane for culturing microvessels. The microfluidic platform is based on a 384-well plate interface on top and 96 microfluidic devices integrated at the bottom. 1 = gel inlet; 2 = medium inlet; 3 = observation window to view the perfusion and gel channels; and 4 = medium outlet.


    6. Align the repeater pipette perpendicular to the plate and gently place the repeater pipette at the center of the gel inlet (Figure 3). Make sure that there is contact, but not pressure, between the tip and the gel inlet. Pipette 2 µl 4 mg/ml collagen solution into the gel inlet of each microfluidic unit.



      Figure 3. Schematic representation of the OrganoPlate 2-lane with 96 tissue chips indicating the gel inlets. The wells in columns 1, 5, 9, 13, 17, and 21 are the gel inlets to load the collagen solution into the chips.


    7. Utilize a brightfield microscope to check the filling patterns in the observation windows of the loaded chips (Figure 4A). The results of one loaded chip should be similar to those shown in Figure 4B.

      Important: Meniscus formation as a shadow stretching along the entire observation window should be observed for a successfully loaded chip. If the collagen solution did not completely fill the channel, pipette an additional 2 µl.



      Figure 4. Schematic representation of the OrganoPlate 2-lane with 96 tissue chips indicating the observation windows. A. The wells in columns 3, 7, 11, 15, 19, and 23 are the observation windows. B. Correct loading of the collagen solution is confirmed under a brightfield microscope by observing a shadow stretching along the entire observation window. This is a successfully formed meniscus.


    8. Place the OrganoPlate in the incubator for 10 min to polymerize the collagen.

      Important: Avoid incubating longer than 10 min to prevent the gel from collapsing or shrinking.

    9. Pipette 25 µl HBSS+ into the gel inlet to prevent gel dehydration.

    10. Pipette 50 µl HBSS+ into the observation window for optical clarity.

    11. To coat the microchannels with gelatin, pipette 25 µl 1% gelatin into the medium inlet (Figure 5). Dispense the gelatin against the wall of the well to prevent air bubble formation.



      Figure 5. Schematic representation of the OrganoPlate 2-lane with 96 tissue chips indicating medium in- and outlets. The wells in columns 2, 6, 10, 14, 18, and 22 are medium inlets; the wells in columns 4, 8, 12, 16, 20, and 24 are medium outlets.


    12. Place the OrganoPlate in the incubator for at least 30 min.


  2. Harvest the cells from the T75 flask and seed in the OrganoPlate 2-lane

    1. Warm 50 ml EGM2 in a 37°C water bath.

    2. Observe the HUVECs in the T75 flask under the brightfield microscope. They should be 80-100% confluent. If not, refresh the medium and leave them for an extra day in the incubator.

    3. Aspirate the medium from the T75 flask.

    4. Rinse the cells with 10 ml room-temperature PBS.

    5. Aspirate the PBS from the flask.

    6. Carry out Steps D4 and D5 again.

    7. Cover the cells with 2 ml TrypLE solution.

    8. Place the cells into the incubator for 3-5 min. Periodically examine the cell layer microscopically to check for cell detachment.

    9. Allow the cell detachment to continue until approximately 90% of the cells are rounded up. At this point, tap the flask against the palm of your hand to release the majority of the cells from the cell culture surface.

    10. After the cells are released, neutralize the TrypLE in the flask with 5 ml EGM2.

    11. Quickly transfer the detached cells to a sterile 15-ml centrifuge tube.

    12. Rinse the flask with 5 ml EGM2 to collect residual cells and add this rinse to the centrifuge tube.

    13. Count the number of cells using the available cell counter device in your lab.

    14. Centrifuge the harvested cells at 200 × g for 7 min to pellet.

    15. While centrifuging the cells, transfer the OrganoPlate from the incubator to the cell culture hood.

    16. Aspirate the gelatin from the medium inlet using an aspirator. Place the tip of the aspirator in the corner of the well and allow it to touch the bottom of the plate.

    17. Pipette 25 µl EGM2 into the medium inlet.

    18. Place the OrganoPlate back in the incubator.

    19. After centrifuging, aspirate most of the supernatant.

    20. Dilute the cells to a final concentration of 15 × 106 cells/ml with EGM2.

    21. Transfer the cell suspension to a 1.5-ml Eppendorf tube.

    22. Transfer the OrganoPlate from the incubator to the cell culture hood. Lay and hold the OrganoPlate flat with your non-pipetting hand.

    23. Align the repeater pipette perpendicular to the plate and gently place the repeater pipette at the center of the medium outlet (Figure 5). Make sure that there is contact, but not pressure, between the tip and the medium outlet. Pipette 2 µl cell suspension into the medium outlet of each microfluidic unit. Regularly resuspend the cell suspension during seeding to ensure a homogenous cell density.

    24. Utilize a brightfield microscope to observe the cells in the observation window. The cells should be homogeneous in the chip (Figure 6). If cells are not present, pipette an additional 2 µl.



      Figure 6. Brightfield microscopy image of the OrganoPlate 2-lane chip after seeding the cells. Cells are in suspension next to the ECM gel.


    25. Place the OrganoPlate in the incubator for 1 h to allow the cells to attach.

    26. While the cells are attaching, clean and sterilize the OrganoFlow by spraying and wiping with 70% ethanol (Figure 7).



      Figure 7. The OrganoFlow for perfusing microvessels. MIMETAS OrganoFlow with the OrganoPlate 2-lane placed on top.


    27. Place the OrganoFlow on a level surface in the incubator. Plug in the power adapter and turn on the device by pressing the ON/OFF switch located at the back of the instrument.

    28. Press the CONFIRM button to calibrate the OrganoFlow.

    29. Use the UP and DOWN buttons to choose the interval mode. In this mode, the OrganoFlow will move the platform to the desired rocking angle and will remain in this position for the selected interval duration.

    30. Set the rocking angle to 7° and the interval to 8 min.

    31. Press the CONFIRM button to start rocking.

    32. After the cells have attached, add 50 µl EGM2 to the medium in- and outlets.

    33. Place the plate parallel on the OrganoFlow in the incubator as shown in Figure 7 for the perfusion of medium from the medium inlets to the medium outlets and vice versa.


    Day 8

    1. Refresh the medium in the OrganoPlate 2-lane

      1. Warm 15 ml EGM2 in a 37°C water bath.

      2. Take out the OrganoPlate from the incubator.

      3. Aspirate the medium from the medium in- and outlets. Place the tip of the aspirator in the corner of the wells and allow it to touch the bottom of the plate.

        Important: Placing the tip of the aspirator directly on top of the medium in- and outlets may disturb the microvessels or cause the formation and trapping of air bubbles.

      4. Pipette 50 µl warmed, fresh EGM2 into the medium in- and outlets.

      5. Return the OrganoPlate to the OrganoFlow in the incubator.


    Day 9

    1. Incubate the microvessels with TNFα and PMA overnight

      1. Take out the OrganoPlate from the incubator.

      2. Observe the cells under a brightfield microscope. They should have formed fully confluent microvessels. An example of a microvessel that is perfectly arranged against collagen in the OrganoPlate 2-lane is shown in Figure 8.



        Figure 8. Microvessel in the OrganoPlate 2-lane. The microvessel was cultured against collagen gel, forming a perfusable vessel.


      3. Note in the 2-lane plate layout form, the microvessels that are perfectly arranged (Supplementary Material 1).

      4. Partition the microvessels into groups, each containing four microvessels.

      5. Select which groups will be the control, treated with 15 ng/ml TNFα, or treated with 20 ng/ml PMA. In Steps F9-F11, 50 µl medium will be pipetted into the medium in- and outlets of each microvessel.

      6. Dilute TNFα in EGM2 to a final concentration of 15 ng/ml.

      7. Dilute PMA in EGM2 to a final concentration of 20 ng/ml.

      8. Aspirate the medium from the medium in- and outlets of the selected microvessels. Place the tip of the aspirator in the corner of the wells and allow it to touch the bottom of the plate.

        Important: Placing the tip of the aspirator directly on top of the medium in- and outlets may disturb the microvessels or cause the formation and trapping of air bubbles.

      9. Pipette 50 µl 15 ng/ml TNFα into the medium in- and outlets of the microvessels selected in Step F5.

      10. Pipette 50 µl 20 ng/ml PMA into the medium in- and outlets of the microvessels selected in Step F5.

      11. Pipette 50 µl EGM2 into the medium in- and outlets of the microvessels selected in Step F5. These are the controls.

      12. Return the OrganoPlate to the OrganoFlow in the incubator and incubate for 18 h.


    Day 10

    1. Collect the medium from the microvessels and store samples in a -80°C freezer

      1. Label 1.5-ml Eppendorf tubes with the experimental conditions.

      2. Take out the OrganoPlate from the incubator.

      3. Use a P200 micropipette to collect the medium from the medium in- and outlets.

        Important: For each condition, the medium from four microvessels should be pooled to form one sample to allow analysis of metabolites at low concentrations due to low cell numbers.

      4. Store the samples in a -80°C freezer.

      5. Carry out sample preparation according to the LC-MS platform available in your lab. For our work, we used the method described in the Materials and Methods section of Schoeman et al. (2018) to measure prostaglandins, isoprostanes, LPA classes, sphingolipids, and PAF. We used the serum extraction method. The samples were measured by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to obtain results such as those shown in Figure 9.



        Figure 9. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) chromatogram of prostaglandin E2. Microvessels were untreated (control) or treated with 15 ng/ml TNFα and 20 ng/ml PMA for 18 h.

    Data analysis

    1. Use LabSolutions for peak determination and integration.

      Note: Other software that are compatible with the available LC-MS platform in your lab can also be used for peak determination and integration.

    2. Use MetaboAnalyst to determine the fold change by dividing the concentrations of bioactive lipids in TNFα- and PMA-treated samples with the concentrations of bioactive lipids in the control samples. Go to https://www.metaboanalyst.ca/ > Click Here to Start > Statistical Analysis > Upload your Data > Fold Change Analysis.

    3. Export the data to Microsoft Excel and calculate the log2 of the fold change.

    4. Copy and paste the calculated log2 from Microsoft Excel to GraphPad Prism and create a bar graph.

    Acknowledgments

    Our work was supported by the RECONNECT CVON Groot consortium, which is funded by the Dutch Heart Foundation. We acknowledge support from the TKI METABOCHIP project, which is co-funded by the PPP Allowance made available by Health-Holland, Top Sector Life Sciences & Health, to stimulate public–private partnerships. The measurement of bioactive lipids was derived from work by Schoeman et al. (2018). This protocol was derived from Junaid et al. (2020a) (doi: 10.7554/eLife.54754).

    Competing interests

    TH is the co-founder of the company that makes the OrganoPlates and OrganoFlow used in the protocol.

    References

    1. Junaid, A., Schoeman, J., Yang, W., Stam, W., Mashaghi, A., van Zonneveld, A. J. and Hankemeier, T. (2020a). Metabolic response of blood vessels to TNFalpha. Elife 9: e54754.
    2. Junaid, A., Tang, H., van Reeuwijk, A., Abouleila, Y., Wuelfroth, P., van Duinen, V., Stam, W., van Zonneveld, A. J., Hankemeier, T. and Mashaghi, A. (2020b). Ebola Hemorrhagic Shock Syndrome-on-a-Chip. iScience 23(1): 100765.
    3. Schoeman, J. C., Harms, A. C., van Weeghel, M., Berger, R., Vreeken, R. J. and Hankemeier, T. (2018). Development and application of a UHPLC-MS/MS metabolomics based comprehensive systemic and tissue-specific screening method for inflammatory, oxidative and nitrosative stress. Anal Bioanal Chem 410(10): 2551-2568.
    4. van Duinen, V., van den Heuvel, A., Trietsch, S. J., Lanz, H. L., van Gils, J. M., van Zonneveld, A. J., Vulto, P. and Hankemeier, T. (2017). 96 perfusable blood vessels to study vascular permeability in vitro. Sci Rep 7(1): 18071.
    5. van Duinen, V., Zhu, D., Ramakers, C., van Zonneveld, A. J., Vulto, P. and Hankemeier, T. (2019). Perfused 3D angiogenic sprouting in a high-throughput in vitro platform. Angiogenesis 22(1): 157-165.

简介

[摘要]来自微血管系统的内皮细胞是微血管疾病炎症和血栓形成过程的关键驱动因素和靶点。炎症过程中生物活性脂质的研究主要基于二维内皮细胞培养物。三维微血管-ON-A芯片提供了一个机会来监测人体的炎症表型的微血管更生理-相关的环境。这个协议描述内皮细胞的培养物作为三维微血管在OrganoPlate 。该微血管用肿瘤坏死因子 α 治疗以诱导炎症。从样品的收集微血管是用于测量生物活性脂质用液相色谱优化-质谱法,provid荷兰国际集团更翔实的代谢读出作为比较用的功能测定。

[背景]在最近的几十年里,微血管疾病已成为世界范围内死亡的主要原因。由心血管危险因素血管内皮细胞的慢性激活可以造成周细胞的损失,其发挥稳定微血管了至关重要的作用。为了诊断和治疗微血管疾病,我们旨在探索循环血浆因子与微血管完整性的关联。

生物活性脂质的体外机理研究主要基于二维 (2D) 内皮细胞培养物,由于缺乏层流和细胞在不合规的硬质基质上生长,通常表现出促炎表型。这个复杂的炎症过程的评估,如肿瘤坏死因子α(TNFα)-诱导的效应。新型基于微流体的灌注三维 (3D)微血管芯片模型提供了在更生理环境中生成微血管的独特机会。芯片上的微血管是相导衍生的无膜微血管,从有机板2 通道中的 3D 软支架上接种的内皮细胞生长(van Duinen等,2017)。为了监测 3D微血管的炎症表型,我们使用优化的靶向超高效液相色谱-串联质谱 (UPLC-MS/MS) 测量一组促炎和抗炎生物活性脂质,并生成TNF 的表达谱。α -治疗微血管下流动(朱奈德等人,2020A) 。

使用所描述的协议,我们评估了前列腺素,浓度异前列烷,溶血磷脂酸(LPA)类,鞘脂,和血小板活化因子(PAF)由初级人分泌的脐静脉内皮细胞(HUVEC)在接种微血管-on-α-芯片。条件培养基通过TNFα灌注-处理组和对照(未处理)微血管进行取样,汇集,并测量由UPLC-MS / MS代谢物组学(斯库曼等人,2018) 。A S未处理和TNFα之间观察到仲差动响应-治疗微血管。此外,在发现了生物活性脂质的相对浓度微血管-on的单芯片是相似于那些在正常人体血管中发现。

大多数测定与所执行的OrganoPlate到研究微血管都涉及免疫染色,渗透性,和血管生成的(van Duinen的等人,2019年,朱奈德等人,2020B) 。这些试验研究形态变化的成像phenotyp ES和一个lthough相关,代谢读出微血管利用代谢组学的更多的信息。在这个协议中,耦合LC / MS -基于与所述代谢技术OrganoPlate是新颖的并且使我们能够测量生物活性脂质,provid荷兰国际集团特殊的信息有关的微血管疾病发病和进展和所述的炎症和氧化应激的新的生物标记物的发现在这方面。结果很容易转化为在人体微血管中发现的生理值。此外,代谢读出ş炎症和氧化应激是在器官上的单芯片领域极具价值相对于药物研究。以下使用OrganoPlate 的协议是为研究人员开发的,用于使用适合其他微流体设备和器官模型的方法研究微血管。

关键字:微血管, 三维细胞培养, 氧化应激, 代谢组学, 微流体, 炎症, 器官芯片, 内皮细胞



材料和试剂


1.5 -毫升Eppendorf管
15 -毫升离心管
50 -毫升离心管
2 - ml 移液器(无菌)
5 - ml 移液器(无菌)
10 - ml 移液器(无菌)
P20 微量移液器
P1000 微量移液器
P20 吸头(无菌)
P1000 吸头(无菌)
0.2 - ml B iopur中继器移液器吸头(VWR,目录号:89232-968)
1.0 - ml B iopur 中继器移液器吸头(VWR,目录号:89232-972)
2.5 - ml B iopur 中继器移液器吸头(VWR,目录号:89232-974)
原代人脐静脉内皮细胞的冷冻管(HUVECs;Lonza,目录号:C2519A ;储存在液氮中)
细胞计数载玻片(Bio-Rad,目录号:1450011)
T75 ˚F拉斯克小号

干冰
台盼乙略
1% 明胶
内皮细胞生长培养基 2(EGM2;PromoCell ,目录号:C-39216 ;4°C )
基础培养基 2(Promocell ,目录号:C-22211 ;4°C )
生长培养基 2 补充混合物(Promocell ,目录号:C-39216 ;-20°C )
青霉素/链霉素(Sigma-Aldrich,目录号:P4333 ;-20°C )
磷酸盐-缓冲盐水 (PBS)
TrypLE (Gibco ,目录号:12604013)
OrganoPlate 2-lane(MIMETAS,目录号:9603-400-B)
鼠尾胶原1型(Trevigen公司,目录号:3440-005-01 ; 4℃ )
37 g/L Na 2 CO 3 (Sigma-Aldrich,目录号:S5761 ;4°C )
1 M HEPES 缓冲液(Gibco,目录号:15630-056 ;4°C )
含钙和镁的 Hank 平衡盐溶液(HBSS+;Life Technologies,目录号:24020117)
肿瘤坏死因子α(TNFα;Sigma-Aldrich,目录号:H8916 ;-20°C )
Phorbol 12-肉豆蔻酸酯 13-乙酸酯(PMA;Sigma-Aldrich,目录号:P8139 ;-80°C )
2车道板布局形式(补充材料1)
70%乙醇
设备


吸管小子
的Eppendorf ř epeater M4移液器(VWR,目录号:613-2890)
OrganoFlow (MIMETAS,目录号:MI-OFPR-L)
培养箱(37°C,5% CO 2 )
水浴 (37°C)
Eppendorf c entrifuge 5810(Sigma-Aldrich,目录号:022628188)
细胞培养罩
-80°C 冰箱
液相色谱串联质谱spectromet ë R(岛津制作所,产品目录号:LCMS-8060)
明场显微镜
TC20一个utomated Ç ELL Ç ounter(Bio-Rad公司,目录号:1450102)
软件


微软Excel
GraphPad Prism(GraphPad 软件)
LabSolutions(岛津)
MetaboAnalyst ( https://www.metaboanalyst.ca/ )
程序


该协议详细介绍了在OrganoPlate 2 泳道中培养微血管直至测量和分析生物活性脂质的每一步。所有处理细胞和有机板的程序都是无菌的,并在细胞培养罩中进行。下面描述了工作流程的一般概述:


第1天:Initiat e本培养过程和维护。


第 2 天:刷新T75 烧瓶中的培养基。


第 5 天:


种子的中的胶原OrganoPlate 2车道。
收获的来自T75瓶的细胞。
种子的细胞中OrganoPlate 2车道。
第 8 天:刷新OrganoPlate 2 泳道中的培养基。


9日:孵育的微血管与TNFα和PMA过夜。


第10天:收集的从介质微血管,存储所述样品中的一个-80℃的冰箱中,并测量所述生物活性脂质。


第一天


Initiat Ë的培养过程和维护
暖的在1%明胶一个37℃的水浴中。
通过向基础培养基 2添加生长培养基 2 补充混合物和 5 毫升青霉素/链霉素来制备 EGM2 。
温热50毫升EGM2在一个37℃的水浴中。
移液管5毫升1%明胶到一个T75烧瓶中,并在培养箱中30分钟的地方。
移液管11毫升50毫升EGM2在到一个15 -毫升离心管中。
迅速解冻一离心管的在HUVEC中一个37℃的水浴中,小心不要淹没整个小瓶中。
重要提示:密切观察冷冻管;当最后SL我版本冰融化,取出的冷冻管。


分配的细胞进入15 -毫升离心管中含有11毫升EGM2。
将管以 200 × g离心7 分钟以沉淀细胞。
尽管小区是离心机ING ,取T75瓶含有从培养箱中1%的明胶。吸出明胶和吸管12毫升EGM2在烧瓶中。
在不干扰细胞沉淀的情况下从离心管中吸出上清液。
通过轻轻移液打散任何团块,将细胞重悬在 500 µl EGM2 中。
移液管将细胞悬浮液在明胶-包被的T75烧瓶中。
轻轻摇动烧瓶,使细胞均匀分布并放入培养箱中。
第 2 天


刷新T75 烧瓶中的培养基
暖12毫升EGM2在一个37℃的水浴中。
从孵化器中取出含有HUVEC的 T75 烧瓶(图 1)。
取出培养基,用温热的新鲜培养基代替,然后将烧瓶放回培养箱。
                                                                                                                                            图1 。人脐静脉内皮细胞(HUVECs)中培养一个T75烧瓶中。在第1天和第2天拍摄明场显微镜图像。比例尺s :400 µm。


第 5 天


种子的胶原蛋白在OrganoPlate 2车道
暖的在1%明胶一个37℃的水浴中。
放置一个1.5 -在冰上毫升Eppendorf管中。
移液管加入60μl的1M HEPES缓冲液和60微升37克/ L的Na 2 CO 3在1.5 -毫升Eppendorf管中并混合。将管子放在冰上。
添加 480 µl 5 毫克/毫升r在尾胶原类型 1 到 HEPES/Na 2 CO 3混合物到4 毫克/毫升胶原溶液的最终浓度。小心混合以避免形成气泡。将试管放回冰上。
用非移液手将OrganoPlate平放在细胞培养罩中(图 2)。
                                                                                                                                            图2 。用于培养微血管的OrganoPlate 2 通道。微流体平台是基于一个384上-在顶部孔板接口和96个集成微流体装置在底部。1 = 凝胶入口;2 = 介质入口;3 = 观察窗以查看灌注和凝胶通道;和 4 = 中等出口。


将中继器移液器与板垂直对齐,然后将中继器移液器轻轻放置在凝胶入口的中心(图 3 )。确保吸头和凝胶入口之间有接触,但没有压力。移液管2微升的4mg / ml胶原溶液中,以各微流体单元的凝胶入口。
FIGUR ê 3 。带有 96 个组织芯片的OrganoPlate 2 泳道示意图,指示凝胶入口。在,,,17列1,5 9 13孔中,和图21是凝胶入口加载的胶原溶液进入芯片。


利用明场显微镜检查加载芯片的观察窗口中的填充图案 (图 4A)。结果小号一个装载芯片的应类似于那些在图4B中所示。
重要的是:中号eniscus形成为阴影沿拉伸的整体观察窗口应为一个成功加载芯片被观察到。如果胶原溶液并没有完全填充沟道,吸管一个附加的2微升。


FIGUR ê 4 。带有 96 个组织芯片的OrganoPlate 2-lane 的示意图表示观察窗口。一个。在,,,19列3 7 11,15的孔中,和图23是该观察窗。乙。的正确装载的胶原溶液是通过观察阴影沿拉伸明场显微镜下确认在整个观察窗。这是一个成功形成的弯液面。


将OrganoPlate放入培养箱中 10 分钟以聚合胶原蛋白。
重要的是:避免incubati纳克比10分钟更长的时间来防止凝胶塌陷或收缩。


移液管将25μlHBSS +中以凝胶入口,以防止凝胶脱水。
移液管将50μlHBSS +中以用于光学清晰度的观察窗。
为了涂覆有明胶的微通道,移液管将25μl的1%明胶在到所述介质入口(图5) 。将明胶分配在井壁上以防止气泡形成。
网络古尔5 。OrganoPlate 2-lane 的示意图,带有 96 个组织切片,指示培养基入口和出口。在18列2,6,10,14,各孔,和22是介质入口; Ť他孔中,立柱4 ,12,16,20 8 ,和24是介质出口。


将有机板放入培养箱中至少 30 分钟。
              收获的从所述T75烧瓶中并种子细胞OrganoPlate 2车道。
温热50毫升EGM2在一个37°C水浴。
在明场显微镜下观察 T75 烧瓶中的 HUVEC。它们应该是 80 - 100% 汇合。如果没有,刷新中,让他们对一个ñ额外一天的孵化器。
从 T75 烧瓶中吸出培养基。
冲洗用10毫升室温的细胞-温度PBS。
从烧瓶中吸出 PBS。
进行小号TEPS d 4和d 5试。
用 2 ml TrypLE溶液覆盖细胞。
放置细胞进入培养箱3 - 5分钟。定期在显微镜下检查细胞层以检查细胞脱离。
允许细胞脱离继续,直到大约 90% 的细胞被四舍五入。在这一点上,点击对你的手掌烧瓶释放大部分的从细胞的细胞培养表面。
后的细胞被释放,中和的TrypLE在用5ml EGM2烧瓶中。
快速分离的细胞转移到无菌的15 -毫升离心管中。
用 5 ml EGM2 冲洗烧瓶以收集残留细胞并将冲洗液加入离心管中。
使用计数细胞数量的可用细胞计数装置在实验室。
将收获的细胞以 200 × g离心7 分钟以沉淀。
在离心细胞的同时,将OrganoPlat e从培养箱转移到细胞培养罩中。
吸出明胶从所述介质中使用入口一个Ñ吸气。将抽吸器的尖端放在孔的角落,使其接触板的底部。
移液管将25μl在EGM2到所述介质入口。
将OrganoPlate放回培养箱中。
后的C entrifuging ,一大部分上清液spirate。
用 EGM2将细胞稀释至终浓度为 15 × 10 6细胞/ml。
细胞悬液转移至一个1.5 -毫升Eppendorf管中。
将有机板从培养箱转移到细胞培养罩。用您的非移液手平放并握住OrganoPlate 。
将中继器移液器与板垂直对齐,然后将中继器移液器轻轻放置在介质出口的中心(图 5)。确保尖端和介质出口之间有接触,但没有压力。吸取 2 µl 细胞悬浮液到每个微流体单元的介质出口。接种以确保期间定期重悬细胞悬浮液一同质的细胞密度。
利用明场显微镜观察观察窗中的细胞。芯片中的细胞应该是同质的(图 6)。如果c厄尔不存在,吸管的额外2微升。
Figu重新6 。接种细胞后的OrganoPlate 2 通道芯片的明场显微镜图像。细胞悬浮在 ECM 凝胶旁边。


放置OrganoPlate培养箱中1个小时,以允许所述细胞附着。
而该细胞被附着,清洁和消毒OrganoFlow通过喷雾,并用70%的乙醇(图7)擦拭。
FIGUR ê 7 。用于灌注微血管的OrganoFlow 。MIMETAS OrganoFlow与OrganoPlate 2 通道置于顶部。


将OrganoFlow放在培养箱中的水平表面上。插上电源适配器和转通过按压位于仪器背面的ON / OFF开关在设备上。
按确认按钮校准OrganoFlow 。
用UP和DOWN按钮选择的间隔模式。在此模式下,OrganoFlow会将平台移动到所需的摇摆角度,并在选定的间隔持续时间内保持在该位置。
设置摇动角度至7°和所述间隔到8分钟。
按确认按钮开始摇摆。
后的细胞已附着微升EGM2添加50到所述介质入口和出口。
将板平行放置在培养箱中的OrganoFlow上,如图 7 所示,用于从介质入口到介质出口的介质灌注,反之亦然。
第 8 天


              刷新OrganoPlate 2 通道中的培养基
在温热15毫升EGM2一个37℃的水浴中。
从培养箱中取出有机板。
从培养基入口和出口吸出培养基。将抽吸器的尖端放在孔的角落,使其接触板的底部。
重要提示:将抽吸器的尖端直接放在培养基入口和出口的顶部可能会干扰微血管或导致气泡的形成和捕获。


移液管将50μl温热,新鲜EGM2在向所述介质入口和出口。
将OrganoPlate放回培养箱中的OrganoFlow 。
第 9 天


              孵育的微血管与TNFα和PMA过夜
从培养箱中取出有机板。
观察下将细胞一明场显微镜。它们应该已经形成完全融合的微血管。图 8显示了在OrganoPlate 2 泳道中与胶原蛋白完美排列的微血管示例。
Figu重8 。OrganoPlate 2 泳道中的微血管。的微血管是针对胶原凝胶培养,形成了可灌注容器。


注意在 2 通道板布局形式中,完美排列的微血管(补充材料 1)。
分区微血管中到组,E ACH包含荷兰国际集团4个微血管。
选择哪些组将作为对照,用 15 ng/ml TNFα处理,或用 20 ng/ml PMA 处理。在小号TEPS ˚F 9 -F 11 ,将50μl介质将中移取到各自的IN-的介质和出口微血管。
将 EGM2 中的 TNFα 稀释至最终浓度为 15 ng/ml。
将 EGM2 中的 PMA 稀释至终浓度为 20 ng/ml。
从所选微血管的介质入口和出口吸出介质。将抽吸器的尖端放在孔的角落,使其接触板的底部。
重要提示:将抽吸器的尖端直接放在培养基入口和出口的顶部可能会干扰微血管或导致气泡的形成和捕获。


移液管将50μl15纳克/ ml的TNFα在到的IN-介质和出口的微血管中选择的小号TEP ˚F 5 。
移液管将50μl20毫微克/毫升PMA中以的IN-介质和出口的微血管中选择的小号TEP ˚F 5 。
移液管将50μl在EGM2到的IN-介质和出口的微血管中选择的小号TEP F5 。这些是控件。
将OrganoPlate放回培养箱中的OrganoFlow并孵育18 小时。
第 10 天


              收集的从培养基中微血管和存储样本中一个-80℃冰箱
用实验条件标记 1.5 - ml Eppendorf 管。
从培养箱中取出有机板。
使用一个P200微量移液器收集媒体从媒体入口和出口。
重要的是:对于每个条件,所述介质从4个微血管应该合并,以形成一个样品以允许analys我由于低细胞数s以低浓度的代谢物。


存储的样品中一个-80℃冷冻机中。
根据您实验室中可用的 LC-MS 平台进行样品制备。对于我们的工作,我们使用了所描述的方法中号aterials和中号ethod小号区段的舒曼等人。(2018)来测量前列腺素,异前列烷,LPA类,神经鞘脂,和PAF。我们使用了血清提取方法。将样品测量由超高性能液C hromatography串联质谱法(UPLC-MS / MS),以获得结果,如示出的那些在图9中。
Figu重新9 。前列腺素 E2 的超高效液相色谱串联质谱 (UPLC-MS/MS) 色谱图。微血管未经处理(对照)或用 15 ng/ml TNFα 和 20 ng/ml PMA 处理 18 小时。


数据分析


使用 LabSolutions 进行峰测定和积分。
注意:与您实验室中可用的 LC-MS 平台兼容的其他软件也可用于峰测定和积分。


使用MetaboAnalyst通过将生物活性脂质的浓度在TNFα以确定倍数变化-和PMA -与生物活性脂质的浓度处理的样品的对照样品。转到https://www.metaboanalyst.ca/ >点击这里开始>统计分析>上传d ATA>倍数变化分析。
将数据导出到 Microsoft Excel 并计算折叠变化的 log2。
将计算出的 log2 从 Microsoft Excel 复制并粘贴到 GraphPad Prism 并创建条形图。
致谢


我们的工作得到了由荷兰心脏基金会资助的 RECONNECT CVON Groot 财团的支持。我们承认支持从该TKI METABOCHIP项目,这是共同资助的PPP津贴提供通过Healt ^ h - ^ h olland ,顶部区生命科学与健康,刺激公共-私营伙伴关系。生物活性脂质的测定是从工作衍生通过斯库曼等。(2018) 。该协议源自Junaid等人。(2020a) ( doi : 10.7554/eLife.54754 ) 。


利益争夺


TH 是在协议中使用OrganoPlates和OrganoFlow的公司的联合创始人。


参考


Junaid, A.、Schoeman, J.、Yang, W.、Stam, W.、Mashaghi , A.、van Zonneveld, AJ 和Hankemeier , T. (2020a)。血管对 TNFalpha 的代谢反应。Elife 9:e54754。
Junaid, A., Tang, H., van Reeuwijk, A., Abouleila, Y., Wuelfroth, P., van Duinen, V., Stam, W., van Zonneveld, AJ, Hankemeier, T. 和 Mashaghi, A . (2020b)。埃博拉出血性休克综合征芯片。iScience 23(1):100765。
舍曼,J 。Ç 。,危害,A 。Ç 。,范威格尔,M 。,伯杰,R 。,弗里肯,R 。Ĵ 。和Hankemeier , T. (2018)。基于 UHPLC-MS/MS 代谢组学的炎症、氧化和亚硝化应激的综合全身和组织特异性筛查方法的开发和应用。肛门Bioanal化学410(10):2551至2568年。
van Duinen, V., van den Heuvel, A., Trietsch, SJ, Lanz, HL, van Gils, JM, van Zonneveld, AJ, Vulto, P. 和 Hankemeier, T. (2017)。96 条可灌注血管用于体外研究血管通透性。科学报告7(1):18071。
van Duinen, V., Zhu, D., Ramakers, C., van Zonneveld, AJ, Vulto, P. 和 Hankemeier, T. (2019)。在高通量体外平台中灌注 3D 血管生成发芽。血管生成22(1):157-165。
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Copyright Junaid and Hankemeier. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Junaid, A. and Hankemeier, T. (2021). OrganoPlate Micro-fluidic Microvessel Culture and Analysis. Bio-protocol 11(13): e4070. DOI: 10.21769/BioProtoc.4070.
  2. Junaid, A., Schoeman, J., Yang, W., Stam, W., Mashaghi, A., van Zonneveld, A. J. and Hankemeier, T. (2020a). Metabolic response of blood vessels to TNFalpha. Elife 9: e54754.
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