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.
