Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs)

1. Preparation of Solutions and Reagents

1. Prepare complete Human Endothelial-SFM medium (EC-SFM FULL) by adding platelet-poor plasma-derived serum (1%), basic fibroblast growth factor (bFGF, 20 ng/mL) and vascular endothelial growth factor (VEGF, 50 ng/mL) to Human Endothelial-SFM (see Table of Materials). Filter the medium through a 0.22 µm pore filter and store it at 4 °C for up to 2 weeks.
2. Prepare complete RPMI medium by adding fetal bovine serum (10%), 2-mercaptoethanol (0.05 nM), L-glutamine (1%), Penicillin-Streptomycin (25 U/mL) to RPMI medium (see Table of Materials). Store at 4 °C for up to 3 weeks.

2. Coating of Microfluidic Chips

1. Place a sterile biochip into a 10 cm sterile Petri dish.
2. Prepare fibronectin working solution by reconstituting the stock solution in phosphate-buffered saline (PBS) without Ca²⁺/Mg²⁺ to a final concentration of 50 µg/mL. Estimate 80 µL of working solution per biochip.
3. Inject 10 µL of fibronectin working solution into each channel of the biochip. Use a pipette with small 10 µL tips and make sure to form a tight contact between the channel inlet and the pipette tip (Figure 1C, left).
4. Close the lid of the Petri dish containing the biochip and place it in a humidified chamber (Figure 2B). Store the chamber at 4 °C overnight. To humidify the chamber, place a clean tissue paper at the bottom of the humidified chamber and add 5 mL of sterile H₂O.
5. Next day, before the assay, pre-warm the biochip at 37 °C in the incubator.

3. Preparation of hiPSC-ECs

NOTE: In the protocol described here, hiPSC-ECs were differentiated, purified, cryopreserved and thawed as previously described⁸.

1. Thaw and plate hiPSC-ECs in complete EC-SFM FULL into one 0.1 % gelatin-coated T75 flask, three to four days prior the assay.
2. The night prior to the assay, stimulate hiPSC-ECs with TNFα by adding EC-SFM FULL supplemented with 10 ng/mL TNFα and incubate overnight (~12 h) at 37 °C, as previously described ⁷.

4. hiPSC-ECs Dissociation and Seeding into Microfluidic Chip

1. On the day of the assay, take the flask of hiPSC-ECs from the incubator and examine the cells under the microscope. Ensure that hiPSC-ECs are 80–100% confluent and have a typical EC-like morphology.
2. Transfer the flask into the cell culture hood.
3. Aspirate the medium from the flask of hiPSC-ECs.
4. Briefly wash the cell culture by adding 10 mL of PBS without Ca²⁺/Mg²⁺. Aspirate the PBS.
5. Add 4 mL per flask of 1x dissociation enzyme. Incubate for 5 min at room temperature (RT) (Figure 1B).
6. Stop the dissociation reaction by adding 8 mL of Human Endothelial-SFM medium per flask. Gently detach and collect the hiPSC-ECs suspension in a 15 mL conical tube using a 5 mL pipette.
7. Count the total number of the cells in the suspension.
8. Centrifuge the cells at 300 x g for 3 min at RT.
9. Aspirate the supernatant and resuspend the cell pellet in EC-SFM FULL to a final concentration of 1.5 x 10⁷ cells/mL.
10. Take out the Petri dish with the biochip from the incubator and transfer it to the cell culture hood.
11. Aspirate the fibronectin solution from all the channels of the microfluidic chip.
12. Inject 6 µL of the cell suspension into each channel using a pipette with a small 10 µL tip (Figure 1C, middle). Make sure that the cell suspension is mixed well before the injection and avoid cell precipitation.
13. Examine the biochip under the microscope and ensure that the cells are uniformly distributed, and the cell density is high (Figure 2C).
14. Incubate the biochip for 15 min at 37 °C.
15. Add 40 µL of EC-SFM FULL into the medium reservoirs from both sides of each channel.
16. Incubate the biochip for 1 h at 37 °C (Figure 1C, right).
17. Add another 50 µL of EC-SFM FULL into the reservoirs from both sides of each channel.

5. Preparation of Human Leukocytes

NOTE: In the protocol described here, a commercially available monocytic cell line (THP-1) was used. Alternatively, peripheral blood mononuclear cells or neutrophils can be used. Isolation of peripheral blood monocytes and neutrophils can be performed using standard procedure¹¹. Perform all steps described in this paragraph in a cell culture hood.

1. Collect the leukocytes in a 50 mL conical tube.
2. Wash the leukocytes with 10 mL of PBS without Ca²⁺/Mg²⁺. Centrifuge the cells at 300 x g for 3 min at RT (Figure 1D).
3. Aspirate the supernatant and resuspend the cell pellet in 1 mL of PBS with DiOC6 dye (λ_ex = 485 nm, λ_em = 501 nm) (1:5,000). Incubate the cells in the dark for 10 min at RT.
4. Wash the leukocytes with 10 mL of PBS without Ca²⁺/Mg²⁺. Centrifuge the cells at 300 x g for 3 min at RT. Repeat the washing step one more time.
5. Count the total number of the cells in the suspension.
6. Aspirate the supernatant and resuspend the cell pellet in complete RPMI medium to a final concentration of 2.5 x 10⁶ cells/mL.

6. Preparation of the Microfluidic Pump

1. Assemble the microfluidic pump with the 8-channel manifold.
2. Connect the pump to a PC with the installed operating software.
3. Start the microfluidic software and load the protocol by clicking Open Protocol and navigate on the PC to select the microfluidic protocol.
4. Connect the software and the pump by selecting the Setup folder and clicking Run Assay Step.
5. Define the geometry of the channels for the correct flow rate control: select Update Geometry in the Geometry Setup folder and click Run Assay Step.
   NOTE: Make sure that the geometry details match the syringe and channels to be used (Geometry ID = 1, Number of channels = 8, Channel width = 800.0 µm, Channel depth = 120.0 µm, Syringe volume = 250 µL) and the viscosity of the sample (Liquid viscosity = 1.0).
6. Wash the pump by placing the inlet cable (orange) into the 50 mL conical tube containing 80% ethanol. Place the outlet cable (green) in the 50 mL conical empty tube (waste container).
7. Select the Washout/Pump Washout folder and click Run Assay Step (Wash volume = 2000 µL, Wash volume step = 250 µL, Washout direction = Output). Repeat the washing step once more.
8. Repeat the pump washing steps (Steps 6.6–6.7) with cell culture grade water, and Human Endothelial-SFM culture medium.
9. Continue with the washing step of the manifold.
10. Manually open the valve between the syringe and the manifold by placing the 3-way adapter into the correct position (indicator OFF is turned to the left).
11. Select Set Current Channel/Open step in the Washout/Manifold Wash folder and click Run Assay Step to open all valves of the manifold.
12. Wash the manifold manually by pushing 5 mL of 80% ethanol from the syringe, followed by 5 mL of cell culture grade water.
13. Wash the manifold by pushing 5 mL of Human Endothelial-SFM culture medium from the syringe.
14. Remove all large air bubbles that are trapped in the manifold. To do this, place the 8-well pin adapter into the 10 cm Petri dish with the medium and perform pushing/pulling with the syringe until all large air bubbles are gone (small bubbles are not a problem).
15. Select Set Current Channel/Close step in the Washout/Manifold Wash folder and click Run Assay Step to close all valves of the manifold.
16. Connect the green outlet cable from the pump to the manifold adapter.
17. Switch the 3-way valve back to the OFF position to close the syringe.
18. Select Washout/Cable Washout folder and click Run Assay Step to wash each port with RPMI medium individually, ensuring that all bubbles are removed from each cable (Dispense volume = 100 µL).
    NOTE: Each valve of the manifold is opened one-by-one, 1 to 8, and 100 μL of the medium is dispensed through each individual channel while keeping the other channels closed. Make sure that the liquid runs from each pin. If there is no liquid coming out, it is an indication of an air bubble or a clog.

7. Preparation of the Microfluidic Chip for Live Imaging

1. Ensure that the microfluidic setups are ready, and the live imaging chamber is pre-equilibrated to 37 °C and the level of CO₂ reaches 5% (Figure 3A, B).
2. Take the biochip from the incubator and position it in the microscope chip holder. Mount the chip holder on the microscope imaging stage (Figure 3C).
3. In order to connect the biochip to the pump via the manifold, place the 8-pin adapter close to the outlet reservoirs of the chip and deepen all pins in the medium (but do not connect completely).
4. Select the Washout | Connect to Chip folder and click Run Assay Step to dispense RPMI medium prior to connecting the chip (Dispense volume = 30 mL). Once the medium is dispensed, insert the 8-pin adapter into the outlet of the biochip.
   NOTE: During this step, all channels are set to On (open) and 30 µL of the medium is dispensed through each channel and following this the channels are set to Off (closed). This ensures that no air bubbles will be introduced into the channels of the microfluidic chip.
5. Manually aspirate the medium from all inlet reservoirs of the biochip with a pipette.
6. Select the Washout | Chip Washout folder and click Run Assay Step to perfuse 40 μL of EC-SFM FULL medium through each of the channels one-by-one to get rid of the dead cells/debris from the channel (Washout volume = 40 µL, Maximum washout shear = 40 dynes/cm²).

8. Flow Adhesion Assay and Image Acquisition

1. Manually aspirate the medium from the inlet reservoir of the channel of interest with a pipette.
2. Add 100 µL of leukocytes from Step 5.6 to the inlet reservoir of the channel of interest.
   NOTE: Gently mix the cells to make a single-cell suspension.
3. In the Cell Assay | Step Description folder, select Set Current Channel/Step Description and, in the list of channels, select only the current channel of interest and set to On (open) and set all other seven channels to Off (closed).
4. Select the Cell Assay | Step Description folder and click Run Assay Step.
   NOTE: Make sure that Variable Flow Rate Dispensing applies constant negative pressure to pull the leukocyte suspension from the inlet reservoir through the channel for 5 min (Shear set = Constant, Shear stress = -0.5 dyne/cm², Duration = 00:05:00, Scan delay = 00:00:00). The negative value of shear stress ensures the correct direction of the flow.
5. Aspirate the remaining leukocytes from the inlet reservoir.
6. Add 100 µL of complete RPMI medium to the inlet reservoir. Select Cell Assay/Step description folder and click Run Assay Step to perfuse the channel for 5 min with RPMI medium in order to wash out all non-adherent leukocytes (Shear set = Constant, Shear stress = -0.5 dyne/cm², Duration = 00:05:00, Scan delay = 00:00:00).
7. Acquire images from at least three to four different positions of the channel to image the adhered leukocytes (to ensure that representative areas are captured).
8. Repeat Steps 8.1–8.7 for functional assessment of all the rest of the channels.
   NOTE: It is possible to run multiple channels at once. To do so, open all the channels of interest during the Step 8.3. and perform all aforementioned Steps 8.4 –8.7 for multiple channels of interest.

9. Completing the Assay and Cleaning the Microfluidic Pump

1. After completing the experiment, export and save all images in a RAW format.
2. While saving the results, run the microfluidic pump and the manifold cleaning protocol.
3. For the microfluidic pump, run the Washout/Pump Washout folder first by perfusing 4 mL of cell culture grade water, followed by 4 mL of 80% ethanol as described in Steps 6.6–6.7 Dry the pump by running air for several minutes.
4. In order to clean the manifold, run Washout/Manifold Wash step with the syringe. Follow the steps to open the syringe and the manifold valves as described in Steps 6.10–6.11 Wash first with 80% ethanol and next with cell culture grade water. Dry the manifold by pushing air via the syringe. Close the syringe valve and the manifold valves as described in Steps 6.10 and 6.15.

10. Counting of Adherent Leukocytes

1. Download and install the image processing software¹⁴ from http://cellprofiler.org.
   NOTE: Image processing in this study was performed using software version 2.1.1. If using a newer version, the pipeline might require minor adaptation.
2. Download the pipeline Count_leukocytes.cpipe. Click File | Import | Pipeline from the file and navigate on PC to choose the pipeline Count_leukocytes.cpipe.
3. Drag-and-drop a folder with acquired RAW images of adherent leukocytes to the File list window in the Input modules | Images section for analysis.
4. Specify the typical size of leukocytes via Analysis modules | IdentifyPrimaryObjects. Specify the minimal and maximal diameter of adherent leukocytes in pixels.
5. Select the filtering criteria via Analysis modules | FilterObjects. Specify the minimally accepted area of objects in pixels.
6. Start the analysis by pressing the Analyze images button.
   NOTE: All the RAW images will be processed, and the results will be saved in a default output folder. The Image.txt output file contains the numbers of adherent leukocytes in each processed image (each number corresponds to one image, i.e., each acquired field of view).