Our research explores how mechanical cues instruct the fate of endothelial cells during development for which we combine human induced pluripotent stem cell differentiation with fluidic mediated mechanical stimulation. Endothelial cells are constantly exposed to the movement of blood, so important signals are missed out in study culture, which makes this the main challenge in studying endothelial cell biology using traditional culture systems. Mechanical cues in fact activate many crucial signaling pathways that might be overlooked in cell's culture in study conditions.
Our protocol provides an easy step-by-step guide on how to differentiate and mechanically stimulate induced pluripotent stem cell derived endothelial cells. This will help many laboratories in adopting culture to easily address questions about endothelial cell behavior while underflow. To begin, on day zero prewarm serum free differentiation medium or SFD medium containing mix one cytokine to 37 degrees Celsius.
Then under a sterile tissue culture hood, add the mix one cytokine supplemented medium to each well of a cell repellent six well plate. To prepare the cells, aspirate the culture medium from a six well plate containing human induced pluripotent stem cells or human iPS cells. Wash them with DPBS followed by aspirating the DPBS.
Then add the dissociation reagent to the cells and incubate them at room temperature for a minute. After aspirating the dissociation reagent, incubate the cells for another three minutes at room temperature. Then tap the plate containing the cells 10 times on each side to detach the cell clusters.
Add one milliliter of pre warmed mix one cytokine supplemented SFD medium to the cells per well. Using a Pasteur pipette, transfer the cell clusters from each well to one well of the cell repellent plate containing the medium with mix one cytokine. After introducing the plate into the incubator, move it back and forth as well as side to side to disperse the content evenly and incubate it for embryoid bodies or EB formation.
The next day, swirl the cell repellent plate containing the EBs to collect them into the center of the wells. Using a Pasteur pipette, transfer the EB suspension from the wells to a 15 milliliter centrifuge tube. Wait five to 10 minutes for the EBs to settle at the bottom of the tube.
Meanwhile, wash the cell repellent plates with sterile water or DPBS to remove single cells or debris. Add two milliliters of mix one cytokine supplemented SFD medium to each well of the plate. Now gently aspirate the supernatant from the centrifuge tube without dislodging the EBs.
Resuspend the EBs in the calculated volume of SFD medium containing mix one cytokine. Add one milliliter of EB suspension to each well of the washed cell repellent plate already containing two milliliters of medium. Incubate the plate after dispersing the EBs by gently moving the plate as mentioned before.
After gathering the EBs at the center of the wells, add CHIR99021 on the side of each well, avoiding direct contact with the cells. Incubate the wells as demonstrated previously. On days three and six of differentiation, collect the EBs and perform media changes as demonstrated previously for day one.
Use SFD medium supplemented with mix three cytokines on day three and use SFD medium supplemented with mix four cytokines on day six. After collecting and settling the Ebs from the cell repellent plate into a 15 milliliter centrifuge tube as demonstrated previously, aspirate the supernatant, then add one milliliter of cell dissociation reagent per well of EB collected in the tube to resuspend the EBs. Next, transfer one milliliter of this EB suspension to each well of the cell repellent plate washed with water.
After incubating the plate for 10 minutes in the incubator, use a P1000 pipette to dissociate the EBs in each well by gently pipetting up and down no more than 10 times. Then add five milliliters of washing buffer per well of dissociated EBs. Collect the cells into a 50 milliliter centrifuge tube by passing them through a 40 micron strainer.
After counting the cells in the filtered suspension, spin the cells down for 10 minutes at 300 G.Resuspend the cells in 300 microliters of washing buffer by gently pipetting a few times, ensuring no clumps are present. Proceed to isolate the CD34 positive cells using a CD34 microbead kit per the manufacturer's instructions. EBs of good quality showed a defined edge by day two of the differentiation and appeared clear and bright when observed under a microscope.
The presence of darker areas indicated cell death within the EBs. Following CHIR99021 treatment with varying concentrations on day two, quantifying the number of CD34 positive cells at day eight of differentiation revealed the response of the cell line to the treatment. Flow cytometry showed that the CD34 positive enrichment using the magnetic beads provided about 85%CD34 positive cells.
To begin, centrifuge the isolated CD34 positive cells and resuspend the cell pellet in 300 microliters of prewarmed SFM-34 medium that is supplemented with mix four cytokines and count the cells using 10 microliters of the cell suspension. After counting and resuspending the cells in a final volume of 150 microliters cytokine supplemented SFM-34, add 0.5 microliters of ROCK inhibitor to it. Slowly aspirate the laminin from fluidic chips by placing the tip of a P200 pipette inside the reservoir on the edge of the channel.
Then add the cell suspension into the channel consistently, ensuring no bubbles are formed. Incubate the chip overnight at 37 degrees Celsius and 5%carbon dioxide to attach the cells to the channel completely. When the endothelial cells are fully attached, aspirate the medium as described previously.
Add 200 microliters of cytokine supplemented SFM-34 into the chip. Replace the medium daily until the cells reach 90 to 100%confluency. Install the perfusion set into the unit per the manufacturer's protocol.
Inside a hood, attach an empty fluidic chip to it and add cytokine supplemented SFM-34 medium to fill both reservoirs under sterile conditions. Transfer the fluidic unit to the incubator, connecting it to the pump for bubble removal and calibration. Carefully remove the fluidic unit with the connected set from the incubator and transfer it into the hood along with the chips containing the cells.
Clamp the tubing on both sides of the test chip. Remove the clamped tubing from the test chip and connect the chip containing the cells to the tubing. After removing or opening the clamps, transfer the system to the incubator and connect the air pump to the fluidic unit.
Remove the fluidic unit from the pump and move it into the hood. Clamp the tubing on both sides of the chip and remove the tubing from the reservoirs on the chip. After gently removing the medium from the chip, wash the cells with Dulbecco's phosphate-buffered saline, or DPBS.
Add 150 microliters of dissociation buffer and incubate for three minutes at 37 degrees Celsius. When the cells are detached, collect the dissociation buffer from one reservoir and wash the channel once with DPBS. Wash the reservoir with the washing buffer to collect the remaining cells from the chip.
Finally, add one milliliter of washing buffer to the collected cells before using 10 microliters from it to count the cells. Before the stimulation, cells showed random orientation, but with stimulation they reoriented parallel to the direction of the flow. CD34 expression profile of cells cultured under flow for five days and percentage of CD34 positive cells retrieved from the fluidic channel showed the effectiveness of the protocol.
