To begin, centrifuge the isolated CD34-positive cells and resuspend the cell pellet in 300 microliters of pre-warmed SFM-34 medium that is supplemented with mixed 4 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.
