This method can help to answer key questions in the human pluripotent stem cell biology field about the functionality and inflammatory responses of human induced pluripotent stem cell-derived endothelial cells. The main advantage of this technique is that it provides a detailed description of the experimental setup and data analysis for the assessment of leukocyte adhesion to human induced pluripotent stem cell-derived endothelial cells. For microfluidic chip coating, place a sterile biochip in a 10-centimeter sterile Petri dish and use a 10-microliter pipette tip to inject 10 microliters of freshly prepared fibronectin working solution into each channel of the biochip.
Cover the Petri dish and place it in a humidified chamber overnight at four degrees Celsius. The next morning, pre-warm the biochip in a 37-degree Celsius incubator, and re-suspend human induced pluripotent stem cell-derived endothelial cells harvested from an 80 to 100%confluent cell culture at the desired concentration in a fresh endothelial cell serum-free medium. Next, aspirate the fibronectin solution from all of the channels of the microfluidic chip, and thoroughly mix the cells to obtain a homogeneous cell suspension before using a 10-microliter pipette tip to inject six microliters of cells into each channel.
Examine the biochip under the microscope to confirm that the cells are uniformly distributed with a high cell density. After 15 minutes at 37 degrees Celsius, add 40 microliters of endothelial cell serum-free medium FULL into the medium reservoirs on both sides of each channel. And return the biochip to the incubator for an hour.
Then, add another 50 microliters of endothelial cell serum-free medium FULL into the reservoirs on both sides of each channel. For live imaging of the seeded cells, confirm that the microfluidic setups are ready, that the live imaging chamber is equilibrated to 37 degrees Celsius, and that the CO2 level has reached 5%Place the biochip into the microscope chip holder and mount the chip holder onto the microscope imaging stage. To connect the biochip to the pump via the manifold, place the eight-pin adapter close to the outlet reservoirs of the chip and deepen all the pins in the medium without connecting the pins completely.
In the microfluidic pump software, select Washout/Connect to chip, and click Run Assay Step to dispense 30 microliters of RPMI medium through each pin. Then insert the eight-pin adapter into the outlet of the biochip. Use a pipette to manually aspirate the medium from all of the inlet reservoirs of the biochip, and select Washout/Chip Washout and Run Assay Step to perfuse one channel at a time with 40 microliters of endothelial cell serum-free medium Full to remove any dead cells and debris.
When all of the channels have been washed replace the medium from the inlet reservoir of the channel of interest with 100 microliters of human leukocytes diluted to a 2.5 times 10 to the six cells per milliliter concentration in RPMI medium. Click Cell Assay/Step description and Set Current Channel/Step description and set the channel of interest to On and the other seven channels to Off. Select Cell Assay Step description and click Run Assay Step.
Replace the remaining leukocytes in the inlet reservoir with 100 microliters of complete RPMI medium. And click Cell Assay Step description and Run Assay Step to perfuse the channel for five minutes with RPMI medium to remove any nonadherent leukocytes. Then acquire images from at least three to four different positions in the channel to visualize the adhered leukocytes.
To quantify the adherent leukocytes open the CellProfiler image processing software and import the Count Leukocytes Pipeline file. Under Input modules select Images and drag-and-drop the folder with the raw images of the adherent leukocytes to the File list window. Under Analysis modules select Identify Primary Objects and indicate the minimal and maximal diameters of the adherent leukocytes in pixels.
Under Analysis modules select FilterObjects. Then select the filtering criteria and enter the minimally accepted area of the objects in pixels and click Analyze Images to begin the analysis. Optimal human induced pluripotent stem cell-derived endothelial cell dissociation should result in a single cell suspension free of cell clumps.
Typically, 15 minutes after injection the endothelial cells will attach to the bottom of the channel and begin to spread. Within one hour of the injection the endothelial cells should form a well-spread monolayer across the channel at which point they are ready for use in the Flow assay. Using the free open-source image processing software as demonstrated allows the filtering of identified objects based on a minimal area, and the exclusion of falsely identified objects such as cell debris, autofluorescence, or any other nonspecific fluorescent signal.
While attempting this procedure, it is important to remember to optimize the stimulation with pro-inflammatory cytokines and to include primary human umbilical vein endothelial cells as a positive control. Don't forget that working with primary mammalian cell and cell lines can be extremely hazardous and that precautions such as wearing laboratory coat, gloves and eye protection should always be taken while performing this procedure.
