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  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

Streszczenie

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Wprowadzenie

Neovascularization happens in many normal and pathological processes1,2,3,4, which include two major processes in adults, angiogenesis and arteriogenesis5. Besides the best-known growth factors, such as vascular endothelial growth factor (VEGF)6, mechanical stimulations, in particular the blood flow induced shear stress, is important in the regulation of neovascularization7. As we know, the magnitude and forms of shear stress vary dramatically and dynamically in different parts of the vasculature, resulting in important effects on vascular cells8,9,10,11,12. Previous studies have shown that shear stress may affect various aspects of ECs, including cell phenotypic changes, signal transduction, gene expression, and the communication with mural cells13,14,15,16,17,18,19,20; hence, regulate neovascularization21,22,23,24.

Therefore, to better understand neovascularization, it is important to reconstruct the process in natural cellular microenvironment in vitro. Recently, many models have been established to create micro-vessels and provide precise control of microenvironment25,26,27, taking advantage of advances in microfabrication and microfluidic technology. In these models, micro-vessels can be generated by hydrogel28,29, polydimethylsiloxane (PDMS) microfluidic chips30,31,32 or 3D bioprinting33,34. Some aspects of the microenvironment, such as luminal shear stress22,23,35,36, transendothelial flow37,38,39,40, biochemical gradient of angiogenic factors41,42, strain/stretch43,44,45, and co-cultured with other types of cells32,46 have been mimicked and controlled. Usually, a large reservoir or syringe pump was used to provide perfused medium. Transendothelial flow in these models was created by pressure drop between the reservoir and micro-tube22,23,38,40. However, the mechanical microenvironment was hard to maintain constantly in this way. Transendothelial flow would increase and then exceed the physiological level if a high flow rate with high shear stress was used for perfusion. Previous study showed that at the initial period of neovascularization, the velocity of transendothelial flow is very low due to the intact ECs and basement membrane, usually under 0.05 µm/s8. Meanwhile, though luminal shear stress in vascular system varies greatly, it is relatively high with mean values of 5-20 dyn/cm2,11,47. For now, the velocity of transendothelial flow in previous works have been generally kept between 0.5-15 µm/s22,38,39,40, and the luminal shear stress was usually under 10 dyn/cm2 23. It remains a difficult subject to constantly expose ECs to high luminal shear stress and physiological level of transendothelial flow simultaneously.

In the present study, we describe an in vitro 3D model to mimic the initial event of neovascularization (MIEN). We developed a microfluidic chip and an automatic control, highly efficient circulation system to form perfusion micro-tubes and simulate the process of sprouting48. With the MIEN model, the microenvironment of ECs stimulated at the initial period of neovascularization are firstly recapitulated. ECs can be stimulated by high luminal shear stress, physiological level of transendothelial flow and various VEGF distribution simultaneously. We describe the steps of establishing the MIEN model in detail and the key points to be paid attention to, hoping to provide a reference for other researchers.

Protokół

1. Wafer preparation

NOTE: This protocol is specific for the SU-8 2075 negative photoresist used during this research.

  1. Clean the silicon wafer 3 to 5 times with methanol and isopropanol on a spin coater as follows: first spin for 15 s at 500 rpm, and then spin for 60 s at 3,000 rpm.
  2. Transfer the silicon wafer to a hotplate, which is preheated to 180 °C and bake the wafer for 10 min.
  3. Remove the silicon wafer from the hotplate and cool it to room temperature. Clean the wafer again with compressed air before proceeding with the spin coating. Apply 4 mL of the SU-8 2075 photoresist to the center of the wafer.
  4. Obtain a feature height of 70 µm on the spin coater as follows: first spin for 12 s at 500 rpm, and then spin for 50 s at 2,100 rpm.
  5. Soft bake the wafer on a hotplate as follows: first bake for 8 min at 65 °C, then bake for 20 min at 95 °C. Next, remove the silicon wafer from the hotplate and cool it to room temperature before lithography.
  6. Place a photomask onto the photoresist film. Expose the wafer with a lithography equipment to achieve a total exposure of 200 mJ/cm2.
    NOTE: The photomask contains nine sets of patterns of the chip, so it can fabricate nine microfluidic sprouting chips each time.
  7. Post expose the wafer on a hotplate as follows: first bake for 5 min at 65 °C, and then bake for 20 min at 95 °C. Next, remove the silicon wafer from the hotplate and cool it to room temperature before developing.
  8. Transfer the wafer to a glass Petri dish filled with SU-8 developer (PGMEA) to start developing.
    CAUTION: The developer is irritating to the eyes and respiratory tract. Perform developing in a fume hood. Wear splash goggles, nitrile gloves, and airline mask during the operation.
  9. Shake the dish gently along the direction of the flow channel and change the developer after 10 min.
  10. Repeat step 1.9 3-5 times until the patterns can be clearly observed.
    NOTE: Make sure there is no photoresist residue left on the wafer, otherwise rinse the wafer in the developer again.
  11. Transfer the wafer to a preheated hotplate set to 120 °C and bake it for 30 min.
  12. Pipet 35 µL of silane onto a coverslip, then put the coverslip along with the wafer into a desiccator and pull vacuum. Seal the desiccator and leave the wafer under vacuum for 4 h to silanize the wafer to prevent the adhesion of PDMS during the soft-lithography processes.
    CAUTION: The silane is toxic. To prevent poisoning, perform silanization in the fume hood and wear nitrile gloves while handling.
  13. Release the vacuum from the desiccator. Remove the silanized wafer onto a preheated hotplate and bake it at 65 °C for 2 h.
  14. Store the wafer in a clean Petri dish until required.

2. Microfluidic sprouting chip fabrication

  1. Combine 20 g of base agent and 2 g of curing agent (10:1 ratio) in a plastic beaker and mix them thoroughly with a mixing rod.
  2. Place the beaker into a desiccator and pull vacuum for 1 h to remove air bubbles in the PDMS mixture.
  3. Pour the PDMS mixture onto the wafer in the Petri dish and place the Petri dish back into the desiccator, degassing for another 30 min.
    NOTE: It is helpful to use double-sided adhesive tape to glue the wafer onto the bottom of the Petri dish to ensure the wafer is kept horizontal during degassing and curing.
  4. Remove the Petri dish from the desiccator and place it into an 80 °C dry oven for 3 h to cure.
  5. Carefully separate the PDMS layer from the wafer and cut the layer to nine chips with a scalpel according to the pattern.
    NOTE: Keep the feature side up after separation.
  6. Punch two hydrogel injection ports and four media injection ports out of each chip using a 1 mm and a 3.5 mm biopsy punch, respectively.
  7. Clean the punched chips with residue-free tape to remove PDMS residue. Place the chips and nine glass coverslips into a plasma cleaner and treat them with oxygen plasma for 30 s to form covalent bonding on the surface.
  8. Take out the chips and coverslips. Attach the feature side of the chips onto the coverslips.
  9. Place the attached chips into an 80 °C dry oven for 1 h to intensify the bonding.
  10. Autoclave the chips before use and keep them sterile for the rest of the procedure.

3. Surface modification and hydrogel injection

  1. For each chip, pipet 40 µL of 1 mg/mL poly-D-lysine (PDL) and inject it into channels in the chip from hydrogel injection port.
    NOTE: Make sure the PDL fills channels, especially in narrow channels near the ports.
  2. Incubate the chips at 37 °C for 4 h to modify the surface of the PDMS .
  3. Pipet 200 µL of sterile water and inject it into channels in the chip from hydrogel injection port to wash out PDL.
  4. Remove the chips into a 120 °C dry oven for 3 h to restore hydrophobicity.
  5. Place on ice a sterile tube and calculate the volume of Type I collagen to be used as the following equation.
    Final volume (50 µL) x Final collagen concentration (3 mg/mL in this research) / Concentration in bottle = volume collagen to be added
  6. Calculate the volume of NaOH to be used as the following equation.
    (volume collagen to be added) x 0.023 = volume 1 N NaOH
  7. Prepare 50 µL of hydrogel in the tube as the following protocol: add 5 µL of 10x PBS, 0.5 µL of phenol red, calculated volume of 1 N NaOH, 4 µL of 1 mg/mL Fibronectin, calculated volume of Type I collagen in turn. Add the proper amount of dH2O so that the total volume reaches 50 µL. Mix all the contents in the tube thoroughly.
    NOTE: Perform all the operations on ice. Use phenol red as an acid-based indicator to assist in visual determination of the pH of the hydrogel. The final hydrogel ends up orange when the pH is about 7.4 and the stiffness is about 15 kPa49.
  8. Pipet 2-3 µL of hydrogel for each chip and slowly inject it into the central hydrogel channel from hydrogel injection port.
  9. Incubate the chips at 37 °C for 30 min to allow gelation.
    ​NOTE: Seal the chips into a sealed box with 1 mL of sterile water if they are not used immediately. Sealed chips can be stored at 37 °C for at most 24 h.

4. Cell seeding

  1. Pipet 20 µL of 125 µg/mL Fibronectin into one media injection port of the cell culture channel.
  2. Cut a pipette tip to fit the port of the cell culture channel with scissors.
  3. Insert the pipette tip into the other media injection port of the cell culture channel. Then, pipet out air from the cell culture channel to fill it with Fibronectin.
  4. Incubate the chips at 37 °C for 1 h.
  5. Before cell seeding, pipet 20 µL of ECM media into each media injection port and incubate the chips at 37 °C for 30 min.
  6. Then, pipet out all the media in all media injection ports.
  7. Next, pipet 5 µL of cell suspension into one media injection port of cell culture channel. Then, endothelial cells quickly spread over the entire channel under differential hydrostatic pressure.
    NOTE: Prepare the cell suspension by trypsinizing human umbilical vein endothelial cells (HUVECs) from the culture flask and centrifuging them at 400 x g. Then, resuspend the cells to 107 cells/mL in a tube.
  8. Add about 4-6 µL of ECM media to the other port to adjust the hydrostatic pressure and stop cell moving.
  9. Remove the chips to the cell incubator. Then, turn over the chips every 30 min until endothelial cells coat around the internal surface of the cell culture channel 2 h later (Figure 1).
    NOTE: To make the chip upside down, pipet a little water on the back of the coverslip. Then the chip can attach to the cover of the Petri dish.
  10. Use the pipette tip to remove the attached cells in the injection ports very carefully.
  11. Then, insert four barbed female Luer adaptors into the media injection ports and fill with ECM media.
    ​NOTE: The adaptors can function as fluid reservoirs to provide nutrients for the cells in the channel.
  12. Remove the chips to cell incubator. Change ECM media in Luer adaptors every 12 h.

5. Measurement of FITC-dextran diffusional permeability

NOTE: To assess barrier function of the micro-vessel, diffusional permeability of the EC culture channel with or without cell lining is assessed.

  1. Take out a microfluidic sprouting chip with hydrogel injected.
  2. Repeat steps 4.1-4.6.
  3. Remove all the Luer adaptors and pipet out all the media in four media injection ports.
  4. Place the chip onto the confocal laser scanning microscope.
  5. Pipet 5 µL of culture media containing 500 µg/mL 40 kDa FITC-dextran to one port of cell culture channel.
    NOTE: 40 kDa FITC-dextran has similar molecular size to VEGF-165 (39-45 kDa).
  6. Capture images every 3 s for 30 s. Thus, the diffusional permeability without cell lining is measured.
  7. Take out the microfluidic sprouting chip after HUVECs are confluent in the cell culture channel.
  8. Repeat steps 5.3-5.5.
  9. Capture images every 3 s for 30 s. Thus, the diffusional permeability with cell lining is measured.
  10. Calculate the diffusional permeability by quantifying changes of fluorescent intensity over time using the following modified equation50.
    Pd= (I2- I1) / ((I1- Ib)·Δt) ·S/d
    where, Pd is the diffusional permeability coefficient, I1 is the average intensity at an initial time point, I2 is the average intensity after delta time (Δt), Ib is the background intensity, S is the area of the channel in fluorescence images, and d is the total interval of micro-posts in fluorescence images. In the present work, Δt is set as 9 s.
    ​NOTE: The present equation is slightly different from the original50, due to the diffusion direction of the fluorescence in the chip is only from the EC channel to hydrogel channel, which is not like the circular tube diffusing in all the radial direction.

6. Microfluidic control system setup

NOTE: The microfluidic control system in the present study is consisted of a micro-syringe pump, an electromagnetic pinch valve, a bubble trap chip, a microfluidic chip, a micro-peristaltic pump, and a reservoir. Each part of the system can be replaced by alternatives able to perform the same function.

  1. Bubble trap chip fabrication
    NOTE: The bubble trap chip is used to remove air bubbles in circulation. The chip consists of three PDMS layers. The top layer is constructed using soft lithography to form grid structure as the liquid chamber. Each channel of the grid structure is 100 µm wide. The bottom layer is a PDMS chunk with a hole. Between the two layers, a 100 µm thin PDMS film is laid.
    1. Repeat step 1 to prepare the wafer for bubble trap chip.
    2. Repeat steps 2.1-2.5 to fabricate the top layer and bottom layer of the bubble trap chip.
      NOTE: The photomask of the top layer contains three sets of patterns, so cut the PDMS layer to three chips according to the pattern.
    3. Punch six holes on the end of inlet and outlet channels of the top layer using a 3.5 mm biopsy punch.
    4. Punch two holes on the corresponding position of bottom layer using a 6 mm biopsy punch according to the pattern on the top layer.
    5. Repeat steps 2.1-2.2 to prepare 10 g of PDMS mixture and pour it on the center of a cleaned silicon wafer.
    6. Fabricate a 100 µm PDMS film by applying the following spin protocol: spin for 15 s at 500 rpm, increase the spin speed to 1,300 rpm and hold here for 45 s.
    7. Transfer the wafer to a preheated hotplate set to 180 °C and bake it for 30 min.
    8. Clean the punched layers with residue-free tape to remove PDMS residue. Place the top layers and wafer into a plasma cleaner and treat them with oxygen plasma for 30 s to form covalent bonding on the surface.
    9. Take out top layers and wafer. Attach the feature side of top layers onto the PDMS film.
    10. Cut the film carefully along the edge of the top layers with a needle.
    11. Slowly separate the film from the wafer and turn over the chips to make the film side up after separation.
    12. Place the bottom layers and attached chips into a plasma cleaner and treat them with oxygen plasma for 30 s again.
    13. Take out bottom layers and attached chips. Attach the bottom layers onto the PDMS film and the bubble trap chips are done.
      NOTE: Align the holes on the bottom layer with the patterns on the top layer when attaching.
    14. Place the chips into an 80 °C dry oven for 1 h to intensify the bonding.
  2. Assemble the microfluidic control system
    NOTE: All the parts of the system, such as bubble trap chip, reservoir, tubes, and connectors are used after autoclave sterilization, except electronic equipment. Assemble the microfluidic control system on a clean bench.
    1. To assemble the microfluidic control system, prepare two polytetrafluoroethylene tubes, two short silicone tubes, three long silicone tubes, one barbed female Luer adaptor, one Y type connector, and three L type connectors.
      NOTE: The advantage of polytetrafluoroethylene tube is low elasticity, therefore, less media is needed in pipeline. Silicone tube, in contrast, has high elasticity, therefore, it is suitable for the pinch valve and peristaltic pump.
    2. Fill the syringe with 10 mL of preheated (37 °C) ECM medium.
      NOTE: Preheating helps the medium release dissolved gas.
    3. Connect a polytetrafluoroethylene tube to the syringe by a barbed female Luer adaptor. Then, connect the other end of polytetrafluoroethylene tube to a Y type connector.
    4. Next, connect two long silicone tubes to the other two ends of Y type connector with one tube connecting to the reservoir and the other tube connecting to the bubble trap chip.
    5. Connect another long silicone tube to the reservoir.
    6. Next, use two short silicone tubes to connect all inlet and outlet holes on the top layer of the bubble trap chip. Connect a polytetrafluoroethylene tube to the backend of the chip.
    7. Next, fix the syringe onto the micro-syringe pump.
    8. Clip two long silicone tubes into the electromagnetic pinch valve.
    9. Next, switch the electromagnetic pinch valve to open the pipeline between the syringe and the reservoir. Inject the media to the reservoir using a micro-syringe pump to exhaust air in the tube.
    10. Then, switch the valve again to open the pipeline between the syringe and the bubble trap chip. Inject the media to fill the liquid chamber and the backend tube of the bubble trap chip.

7. Endothelial sprouting assay

NOTE: A stage top incubator assembled with phase contrast microscope is used in the present study to observe the process of sprouting in real time. The stage top incubator can maintain the temperature, humidity, and CO2 control on microscope stages, being good for live cell imaging. But the equipment is not necessary for the assay. The protocols provided here can also be worked in a basic cell incubator.

  1. Take out endothelial sprouting chips from the cell incubator.
  2. Then, remove Luer adaptors on the cell culture side. Insert two pipe plugs into the hydrogel injection ports of the microfluidic sprouting chip.
    NOTE: The plugs can transform from needles and their main function is to prevent media from spilling.
  3. Connect the backend tube of bubble trap chip to one port of cell culture channel.
    NOTE: Make sure there are no bubbles in the tube before connection.
  4. Insert a T type connector to the other port and connect it to long silicone tube connected with the reservoir.
  5. Clip the long silicone tube into the micro-peristaltic pump.
  6. Then, insert an air filter to the reservoir.
  7. Next, assemble the microfluidic sprouting chip to the stage top incubator.
  8. Next, connect the vacuum pump to the holes in the bottom layer of bubble trap chip with a TPU tube.
  9. Set up the circulation volume and flow rate in the custom program, which controls the micro-syringe pump and electromagnetic pinch valve simultaneously (Figure 2).
    NOTE: The flow rate across the endothelial cell culture channel is calculated according to the classic equation.
    τ = 6µQ / Wh2
    where, τ is shear stress (dyn/cm2), µ is viscosity of the medium (8.8 x 10-4 Pa•s), Q is the flow rate across the endothelial cell culture channel (ml/s), h is channel height (70 µm), and W is channel width (1,000 µm). The viscosity of the medium is measured using a coaxial cylinder type rotational viscometer. The height and width of the channel are predetermined and manually confirm using a phase contrast microscope at 4x magnification. The circulation volume is 5 mL and flow rate is 85 µL/min (average 0.2 m/s in cell culture channel) for 15 dyn/cm2 shear stress according to calculation.
  10. Next, set up the flow rate of micro-peristaltic pump.
    NOTE: The flow rate of micro-peristaltic pump is slightly higher than micro-syringe pump, in order to prevent the media from spilling.
  11. Turn on the micro-syringe pump. Then, the circulation control system is established.

8. Data analysis

NOTE: To quantify the sprouts, the normalized area of sprouting, average sprout length, and longest sprout length were calculated. Results represent mean ± SEM obtained from three independent studies. Statistical significance (P < 0.05) is assessed by Student's t-test.

  1. Fix cells and sprouts in the microfluidic sprouting chip after experiments with 4% paraformaldehyde in PBS for 15 min. Stain nuclei with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; 1: 1000; Sigma-Aldrich) for 10 min and then stain cytoskeleton with TRITC phalloidin (P5285, 1: 100; Sigma-Aldrich) for 1 h. Wash cells with PBS three times at 5 min intervals between each step.
  2. Take confocal images of the chips in a tiling mode and stitch them using image editing software.
  3. Count the number of fluorescent pixels of Z-projection images using a custom code in programming software to quantify normalized area of sprouting (See Supplemental File).
  4. Manually identify and label each tip of sprouts in Z-projection images and calculate distances between sprouts tips to ECs basement membrane using another custom code to quantify average sprout length and longest sprout length (See Supplemental File).

Wyniki

The in vitro 3D model to mimic the initial event of neovascularization (MIEN) presented here consisted of a microfluidic sprouting chip and a microfluidic control system. The microfluidic sprouting chip was optimized from previous publications22,23,37,40,51,52,53. Briefly, it contai...

Dyskusje

For a long time, real-time observation of neovascularization has been a problem. Several approaches have been developed recently to create perfused vessels lining with ECs and adjacent to extracellular matrix for sprouting22,32,40,46,54, but the mechanical microenvironment is still hard to maintain constantly. It remains a difficult subject to mimic the initia...

Ujawnienia

The authors have nothing to disclose.

Podziękowania

This work was supported by the National Natural Science Research Foundation of China Grants-in-Aid (grant nos. 11827803, 31971244, 31570947, 11772036, 61533016, U20A20390 and 32071311), National key research and development program of China (grant nos. 2016YFC1101101 and 2016YFC1102202), the 111 Project (B13003), and the Beijing Natural Science Foundation (4194079).

Materiały

NameCompanyCatalog NumberComments
0.25% Trypsin-EDTAGenviewGP3108
Collagen I, rat tailCorning354236
DAPISigma-AldrichD9542
Electromagnetic pinch valveWokun TechnologyWK02-308-1/3
Endothelial cell medium (ECM)Sciencell1001
Fetal bovine serum (FBS)Every GreenNA
FibronectinCorning354008
FITC-dextranMiragen60842-46-8
Graphical programming environmentLab VIEWNA
Image editing softwarePhotoShopNA
Image processing programImageJNA
IsopropanolSigma-Aldrich91237
Lithography equipmentInstitute of optics and electronics, Chinese academy of sciencesURE-2000/35
MethanolSigma-Aldrich82762
Micro-peristaltic pumpLead FluidBT101L
Micro-syringe pumpLead FluidTYD01
Oxygen plasmaMING HENGPDC-MG
ParaformaldehydeSigma-AldrichP6148
PBS (10x)BeyotimeST448
Permanent epoxy negative photoresistMicrochemSU-8 2075
Phenol Red sodium saltSigma-AldrichP5530
Polydimethylsiloxane (PDMS)Dow CorningSylgard 184
Poly-D-lysine hydrobromide (PDL)Sigma-AldrichP7886
PolytetrafluoroethyleneTeflonNA
Program softwareMATLABNA
Recombinant Human VEGF-165StemImmune LLCHVG-VF5
Sodium hydroxide (NaOH)Sigma-Aldrich1.06498
Stage top incubatorTokai HitNA
SU-8 developerMicrochemNA
Trichloro(1H,1H,2H,2H-perfluorooctyl)silaneSigma-Aldrich448931
TRITC PhalloidinSigma-AldrichP5285

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