In Vitro Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology

NOTE: All cell culture techniques must be performed under sterile conditions in a laminar flow hood and cells must be incubated at 37 °C in a humified atmosphere with 5% CO₂. Instructions for all cytokine preparation for both maintenance (rhbFGF) and for the differentiation protocol (rhBMP4, rhVEGF, rhbFGF, rhIL6, rhFLT3L, rhIGF1, rhIL11, rhSCF, rhEPO, rhTPO, rhIL3) are in Supplementary Table S1.

1. Culturing of hiPSCs – thawing, maintenance, and freezing of cells

1. Preparation of maintenance medium, growth factors, and other reagents
   1. Prepare the culture medium by adding the whole hESCs serum-free medium supplement, 36 mL of 25% of bovine serum albumin (BSA), and 1 mL of 55 mM of β-mercaptoethanol to Dulbecco's Modified Eagle Medium/F12 (DMEM/F-12) basal medium (see Table of Materials).
   2. Resuspend 1 mg of Rho Kinase inhibitor (iRock) in 1 mL of DMSO, make aliquots of 50 µL, and store them at -20 °C.
      NOTE: These aliquots can be kept at -20 °C for 1 year. Once thawed, they can be kept at 4 °C for 1 week.
   3. Thaw the vitronectin (VTN-N) solution on ice and aliquot 60 µL per vial prior to storing at -80 °C. Just before coating the plates, dilute the 60 µL stock in 6 mL of Dulbecco's phosphate-buffered saline (DPBS); the final concentration of 5 µg/mL.
2. hiPSC cell line thawing
   NOTE: The human pluripotent stem cell line SFCi55 was previously derived in-house and extensively used for differentiation into various cell types and different embryonic lineages^19,20,21,22.
   1. Coat one well of a 6-well plate with 1 mL of VTN-N solution for 1 h at 37 °C.
      ​NOTE: After incubation, coated plates can be stored at 4 °C for up to 1 week.
   2. Aspirate the VTN-N solution with an aspiration pipette and add 1 mL of prewarmed culture medium supplemented with 20 ng/mL of rhbFGF (Supplementary Table S1).
   3. Quickly defrost the vial containing the hiPSCs in a water bath and transfer the cell into 5 mL of prewarmed culture medium.
   4. Spin the cells down for 3 min at 300 × g at room temperature.
   5. Resuspend the cell pellet in 0.5 mL of culture medium supplemented with 20 ng/mL of rhbFGF.
   6. Transfer the cells to one coated well containing already 1 mL of medium.
   7. Add 5 µL of iRock into the wells containing the cells in a total of 1.5 mL of medium.
   8. Culture the cells in the incubator, change the medium daily during the week, and double-feed the cells, adding twice the normal volume of medium to the cells to ensure feeding over the weekend.
      NOTE: Cells are grown in the presence of iRock for 24 h only.
3. Maintenance and passaging of hiPSCs
   1. Change the medium daily with fresh prewarmed culture medium supplemented with 20 ng/mL of rhbFGF.
   2. Passage the cells when they reach approximately 80% confluency, generally twice a week.
      1. To passage the cells, coat a plate with VTN-N as described before in steps 1.2.1 and 1.2.2.
      2. Aspirate the medium from the wells with cells and wash them with DPBS.
      3. Aspirate the DPBS and add 1 mL of dissociation reagent (see Table of Materials) and incubate for 1 min.
      4. Aspirate the dissociation reagent and incubate for a further 3 min. Firmly tap the plate 10 times on each side.
         NOTE: The dissociation step might need cell-type specific optimization in the incubation time and the tapping procedure.
      5. Add 1 mL of culture medium to the cells and with a Pasteur pipette, wash once the with the medium to ensure that most of the colonies are collected.
      6. Add 150 µL of the cell suspension to each well to provide a passage ratio of 1 well into 6.
         ​NOTE: Immediately after thawing a new vial, it is better to passage the cells at a lower ratio such as 1:1 or 1:2 for one or two passages to allow them to reach a steady growing phase before passaging at a ratio of 1:6.
      7. Culture the cells in the incubator, change the medium daily during the week, and double-feed the cells once over the weekend.
4. hiPSCs cell line freezing
   NOTE: Freeze cells within their first two passages after thawing to ensure to maintain a constant low passage batch of frozen vials to start the culture. Freeze the cells when they reach a confluency of approximately 80%.
   1. Change the medium to fresh prewarmed culture medium supplemented with 20 ng/mL of rhbFGF and 5 µL of iRock and incubate for at least 1 h.
   2. Detach the cells as described in steps 1.3.2.2-1.3.2.5.
   3. Collect the detached cells in a 15 mL centrifuge tube containing 5 mL of culture medium.
   4. Centrifuge for 3 min at 300 × g at room temperature.
   5. Aspirate the supernatant and add 1 mL of cryopreservation solution (see Table of Materials).
   6. Using a Pasteur pipette, gently pipette the cells up and down to mix them in the cryopreservation solution.
      ​NOTE: Avoid excessive pipetting, which might result in dissociating the cell clusters.
   7. Divide the cell suspension into two cryopreservation vials with 0.5 mL each.
   8. Transfer the cryopreservation vials to a cryopreservation container precooled at 4 °C.
   9. Transfer the container with the cells to a -80 °C freezer for 24 h before transferring the vials to liquid nitrogen for long-term storage.

2. Differentiation of hiPSCs into endothelial cells

1. Preparation of differentiation medium, cytokines, and growth factors
   1. Prepare serum-free differentiation medium (SFD) according to Table 1. Use this medium from Day 0 to Day 5 of differentiation.
   2. Prepare serum-free medium for CD34⁺ cells (SFM-34) by adding 34 nutrient supplement and 5 mL of L-glutamine supplement to the 34 SFM Basal Medium (see Table of Materials). Use this medium from Day 6 of differentiation onwards.
   3. Resuspend 1 mg of CHIR99021 in 716 µL of DMSO to obtain a 3 mM solution. Incubate at room temperature until fully resuspended; if needed, warm up quickly at 37 °C. Make 20 µL aliquots and store them at -20 °C for up to 6 months. Use immediately after thawing and do not freeze again or store.
   4. Resuspend the cytokines according to the instruction in Supplementary Table S1. Store all cytokines' aliquots at -80 °C.
2. Endothelial cells differentiation
   NOTE: For each day of the differentiation, prepare 18 mL (3 mL medium/well) of prewarmed SFD medium, according to the cytokines' mixes described in Table 2.
   1. Day 0 – formation of embryoid bodies (EBs)
      1. Prepare 18 mL of SFD medium with Mix 1 cytokine according to Table 2, for each 6-well plate (3 mL/well).
      2. Add 2 mL of prewarmed SFD medium with Mix 1 cytokine in each well of a cell-repellent 6-well plate (see Table of Materials).
      3. To form EBs, follow the steps described in steps 1.3.2.2-1.3.2.4.
         NOTE: Ensure that hiPSCs are 70-80% confluent to start the differentiation.
      4. Add 1 mL of prewarmed SFD medium with Mix 1 cytokines to each well of detached cell clusters.
      5. Use a Pasteur pipette to gently transfer the cell clusters into a single well of cell-repellent well for EB formation at a ratio of 1:1.
      6. After placing the plate in the incubator, move it forward and back, right and left, to disperse the EBs evenly in the well.
   2. Day 1 – medium change to the EBs
      ​NOTE: This step is only necessary if, by day 1 of differentiation, there are many single cells in suspension alongside the EBs.
      1. Prepare 18 mL of SFD medium with Mix 1 cytokine according to Table 2, for each 6-well plate (3 mL/well).
      2. Swirl the plate with the EBs to move them into the center and collect them using a Pasteur pipette into a 15 mL centrifuge tube.
         NOTE: If the EBs look clumped together as in strings, separate them by pipetting them up and down with a P1000 before collecting them into the 15 mL centrifuge tube.
      3. Wait 5-10 min for the EBs to settle at the bottom of the tube.
         NOTE: If the EBs are too small, centrifuge them for 5 min at 100 × g to help them settle.
      4. Wash the cell-repellent plates with sterile water or DPBS to remove any single cells or debris.
      5. Carefully and slowly aspirate the supernatant from the EBs without dislodging them.
      6. Add 2 mL of SFD with Mix 1 cytokines to each well of the cell-repellent plates.
      7. Resuspend the EBs using 1 mL of SFD medium with Mix 1 cytokines for each starting well – for a 6-well plate, add 6 mL of medium.
      8. Transfer the EBs to the cell-repellent plates in 1 mL volume per well, which already contains 2 mL of SFD medium.
      9. After placing the plate in the incubator, move it forward and back, right and left, to disperse the EBs evenly in the well.
   3. Day 2 – addition of CHIR99021
      1. Swirl the EBs into the center of the plate and add CHIR99021 according to Table 2 on the side of the well to avoid direct contact with the cells.
         NOTE: If the medium was not changed at Day 1, replace the whole medium instead of adding CHIR alone. Prepare 18 mL of SFD medium with Mix 2 according to Table 2, for each 6-well plate (3 mL/well).
      2. After placing the plate in the incubator, move it forward and back, right and left, to disperse the EBs evenly in the well.
   4. Day 3 – medium change to the EBs and addition of Mix 3 cytokines
      1. Prepare 18 mL of SFD medium with Mix 3 cytokines according to Table 2, for each 6-well plate (3 mL/well).
      2. Collect the EBs as described in steps 2.2.2.2-2.2.2.4.
      3. Add prewarmed 2 mL of SFD medium with Mix 3 cytokines to the cell-repellent plates.
      4. Carefully aspirate the supernatant from the EBs. Add 1 mL/well of SFD with Mix 3 cytokines.
      5. Distribute the EBs between the wells as described in steps 2.2.2.8-2.2.2.9.
   5. Day 6 – medium change for SFM-34 and addition of Mix 4 cytokines
      1. Prepare 18 mL of SFD medium with Mix 4 cytokines according to Table 2, for each 6-well plate (3 mL/well).
      2. Collect the EBs as described in steps 2.2.2.2-2.2.2.4.
      3. Add 2 mL of prewarmed SFM-34 medium with Mix 4 cytokines to the cell-repellent plates.
      4. Carefully aspirate the supernatant from the EBs. Add 1 mL/well of SFM-34 with Mix 4 cytokines.
      5. Distribute the EBs between the wells as described in steps 2.2.2.8-2.2.2.9.

3. CD34⁺ cells isolation and seeding into the chip

NOTE: CD34⁺ cells are isolated via a positive isolation approach with a CD34 microbead kit (see Table of Materials), that contains CD34 microbeads conjugated to monoclonal mouse antibodies anti-human CD34 antibodies and FcR Blocking reagent (Human IgG). It is important to validate the efficiency of the column isolation by staining cells before and after the isolation for flow cytometry analysis, Below it is indicated when cells need to be taken for this analysis.

1. Prepare materials and reagents.
   1. Prepare washing buffer by adding 5 mL of 5% BSA solution and 200 µL of EDTA 0.5 M to 45 mL of DPBS to obtain PBS + 0.5% BSA + 2 mM EDTA. Prepare fresh for each isolation, filter-sterilize, and keep refrigerated until use.
   2. Coat fluidic chips with laminin solution prepared by diluting the rhLaminin 521 1:50 in DPBS Ca²⁺ Mg²⁺. Coat each chip with the appropriate volume for the chip in use and incubate in the incubator for 2 h ahead of seeding.
      NOTE: Other matrices can be employed for the coating and should be tested for the specific cell type/experiment.
   3. Prepare Mix 4 SFM-34 medium by supplementing 18 mL of SFM-34 medium with Mix 4 cytokines according to Table 2 and place the mixture in a 50 mL tube in the incubator with the lid slightly unscrewed to facilitate gas exchange.
   4. Place the selected perfusion sets and any other tubing to be used in the incubator to degas.
2. Day 8 – dissociation of EBs and CD34⁺ isolation
   1. Collect the EBs as described in steps 2.2.2.2-2.2.2.5.
   2. Add 1 mL of cell dissociation reagent per starting well of EBs collected (if 6 wells were collected, add 6 mL).
   3. Transfer back 1 mL of the EB suspension in the cell dissociation reagent to each well of the cell-repellent plate.
   4. Incubate for 10 min in the incubator.
   5. Gently pipette the EBs up and down against the well with a P1000, no more than 10 times.
   6. Repeat steps 3.2.4-3.2.5.for a total of 3x.
      NOTE: If the EBs are difficult to dissociate, repeat the above steps 4x in total.
   7. Add 5 mL of washing buffer for each well of dissociated EBs.
   8. Collect the cells into a 50 mL centrifuge tube by passing them through a 40 µm strainer. Take 10 µL of the cell suspension to count the cells.
      NOTE: To test for the efficiency of the isolation, transfer 10⁵ cells/tube into two different tubes (unstained control and presorted test sample) that will be used later for flow cytometry (as described in steps 4.3.9-4.3.13). For a 6-well plate, ~10⁶ cells should be collected after filtration.
   9. Spin the cells down for 10 min at 300 × g.
   10. Resuspend the cells in 300 µL of washing buffer, gently pipetting a few times to make sure that no clumps are present. Continue following the manufacturer's protocol (see Table of Materials).
3. CD34⁺ cell seeding into fluidic chips
   NOTE: The fluidic chip used in the protocol has a channel height of 0.6 mm and length of 50 mm, for a total growth area of 2.5 cm² (Supplementary Figure S1). This type of chip is seeded with a total volume of cell suspension of 150 µL. Different chips can be used, and the volume of seeding and the cell density should be adapted according to the growth area. Additional optimization might be needed depending on the cell line used and its growth.
   1. Resuspend the isolated CD34⁺ cells in 300 µL of prewarmed SFM-34 medium with Mix 4 cytokines.
   2. Take 10 µL of the cell suspension and count the cells.
   3. Resuspend 2.5 × 10⁵ cells in a final volume of 150 µL supplemented SFM-34; add 0.5 µL of iRock.
      NOTE: To test for the efficiency of the isolation, transfer 10⁵ cells/tube into a tube (post-sorted test sample) that will be used later for flow cytometry (as described in steps 4.3.9-4.3.13).
   4. Slowly aspirate the laminin from fluidic chips by putting the tip of a P200 inside the reservoir on the edge of the channel.
      NOTE: If the liquid is difficult to collect, slowly lift one side of the chip to help the liquid move to the opposite reservoir.
   5. Add the cell suspension steadily into the channel to make sure no bubbles are formed.
      ​NOTE: Perform steps 3.3.4-3.3.5 quickly but gently to avoid the laminin drying out and the formation of bubbles in the channels of the chip. If bubbles are formed, lift one side of the chip and gently tap the slide to mobilize the bubbles; when they reach the reservoir, they will rise to the air interface and should not be able to renter the channel.
   6. Transfer the chip to the incubator and leave them overnight so that the cells are completely attached to the channel and look elongated.
   7. When the cells are fully attached, aspirate the medium as in step 3.3.4 and replace it with 200 µL of cytokine-supplemented SFM-34.
   8. From now on, replace the medium daily until the cells have reached 90%-100% confluency.

4. Application of continuous flow to endothelial cells – Aorta-on-a-chip

1. Prepare materials and reagents.
   1. Prepare Mix 4 SFM-34 medium by supplementing 18 mL of SFM-34 medium with Mix 4 cytokines according to Table 2 and place it in a 50 mL tube in the incubator with the lid slightly unscrewed to facilitate gas exchange.
   2. Place the selected perfusion sets and any tubing to be used for the fluidic setup in the incubator to degas.
2. Fluidic system assembly
   1. Install the perfusion set into the unit according to the manufacturer's protocol.
      ​NOTE: Remember to use clamps in the system. If sliding clamps are used for this step, they need to be slid on the tubing before connecting to the chip.
   2. Attach a new fluidic chip and add the cytokine-supplemented SFM-34 medium, making sure to fill both reservoirs under sterile conditions in the hood.
   3. Perform the bubble removal program and the calibration step.
   4. Remove the fluidic unit with the connected set from the incubator and transfer it into the hood; take also the chips containing the cells from the incubator.
   5. Clamp the tubing on both sides of the test chip.
   6. Remove the tubing from the test chip.
      NOTE: Check that no bubbles are present in the Luer connector at the end of the tubing. If bubbles are visible, carefully aspirate them using a P200 pipette and if necessary, add more medium to make sure that the connector is filled with medium.
   7. Connect the chip containing the cells with the tubing.
   8. Remove or open the clamps.
   9. Transfer the system to the incubator and connect the air pump to the fluidic unit.
   10. Start the preselected program using the pump-dedicated software (Supplementary Figure S2) with the gradual increase of shear stress described in Table 3.
       NOTE: Depending on the specific experiment that is needed, the stimulation program might need optimization. Described here is a gradual shear stress increase leading to the final value of 5 dyn/cm², which mimics the shear stress calculated to be experienced by the wall of the dorsal aorta at the onset of fetal circulation ⁹. Independently of the final shear stress that will be employed, it is necessary to gradually increase over time to allow the cells to adapt to the force without causing the cells to detach from the chip. If the selected stimulation protocol is longer than 3 days, cytokines should be topped up into the system by adding 1 mL of SFM-34 containing the Mix 4 cytokines that would normally be added into 18 mL. To do this, the pump program is quickly paused and 500 µL of supplemented medium is added to each of the two syringes in the fluidic set.
3. Cell collection for analysis
   1. Prewarm the dissociation buffer in a water bath.
   2. Remove the fluidic unit from the incubator and move it into the hood.
   3. Clamp the tubing flanking the chip on both sides and remove the tubing from the reservoirs on the chip.
   4. Gently remove the medium from the chip and replace it with DPBS Ca²⁺ Mg²⁺ to wash the cells.
      NOTE: This step of washing with PBS can be skipped if the cells start to detach.
   5. Gently add 150 µL of dissociation buffer and incubate for 3 min at 37 °C.
      NOTE: Check under the microscope if the cells are single and detached; otherwise, incubate for an additional 2 min. It is essential that the cells are completely detached from the channel before aspirating the medium, as the chip does not allow to aid cell detachment by pipetting. Other solutions can be employed to detach the cells, such as trypsin or EDTA-based buffers.
   6. Collect the dissociation buffer containing the cells from one reservoir and transfer to a 15mL centrifuge tube and wash the channel once with DPBS to collect all the detached cells.
   7. Add 1 mL of washing buffer to the 15 mL tube with the cells and take 10 µL to count the cells.
   8. Divide the cell suspension in flow cytometry tubes to have 10⁵ cells per test tube.
   9. Spin the tubes for 5 min at 300 × g.
   10. Prepare the staining solution to have 50 µL for each test tube to stain. Add the CD34 PerCP-efluor710 or CD34-PE at 1:100 and 1:200 dilution, respectively.
   11. Resuspend the cells in 50 µL of staining solution and incubate for 30 min at 4 °C.
   12. Wash the cells by adding 2 mL of washing buffer and spin for 5 min at 300 × g.
   13. Resuspend the pellets in 100 µL of staining solution and acquire the data using a flow cytometer.
       NOTE: The cells can also be lysed directly in the chip for RNA extraction using 150 µL of RNA lysis buffer or fixed for imaging using 4% paraformaldehyde in DPBS for 10 min at room temperature.
4. Cell orientation analysis
   1. Analyze the images to quantify changes in cell orientation using FIJI²³ (Supplementary Figure S3).
      1. Open the Region of Interest (ROI) manager from the Analyze | Tools | ROI manager menu.
      2. Draw the cell contours manually using the polygon selection tool and add them to the ROI manager by clicking Add or using the CTRL+T shortcut.
      3. Measure the orientation of each ROI by choosing the Fit ellipse measure in the Analyze | Set Measurements menu.
      4. Apply the measurement to all ROIs through the More… Multi measure command in the ROI manager.
         NOTE: This step will fit an ellipse to each ROI and generate a table containing the length of the major and minor ellipse axes, as well as the angle.
      5. Export the table to a CSV file to be imported into other software to plot.
         NOTE: The script used for the plots is available at https://gist.github.com/nicolaromano/708b3231d730ee7f70763a7cf885
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