This microfluidic technology aims to measure the functionality of human neutrophils directly from one droplet of blood on a platform that can be multiplexed. The central loading chamber serves as a whole blood source and also acts as a sink for the chemo attractant gradients that form from the 16 focal chemotactic chambers. The unique mechanical filtration of red blood cells enables probing the function of neutrophils in the natural context of blood, cellular and biochemical components.
The bifurcation between a channel leading to the focal chemotactic chamber and a channel exiting the device allows quantification and modulation of the directionality of migrating neutrophils. Thus, results can demonstrate how actively migrating neutrophils easily migrate past trapped red blood cells and accumulate in numbers over time. One unique feature of our microfluidic devices is they're very easy to manufacture and set up.
They consist of a layer of PDMS casted on a micro fabricated wafer that we bond on a glass bottom, single or multi-well plate. The main advantage of this technique is that it requires only a droplet of whole blood. The devices can be used with standard pipettes, have no moving parts, require no tubing or external syringe pumps.
The implications of this technique extend into clinical applications compared to ours required with traditional techniques. Our approach takes minutes from the time of blood collection to the neutrophil migration assays. The novel red blood cell filtering combs prevents RBCs from blocking the active migration of neutrophils and sets this methods apart from other microfluidic methods.
That requires removing the blood before the chemo assays perform. The mechanical jamming of red blood cells and channels distinguishes our technology and enables the testing of neutrophils in our physiologic environment of whole blood, including serum and platelets. So technology is precise and easy to use, and it can significantly advance our understanding of the role of neutrophils and C innate immune system in health and disease.
Fabricate the master mold wafer to define the migration channels according to the instructions from the manufacturer First pattern. The first three micron epoxy based negative photoresist layer then pattern the second 50 micron layer to define the cell loading and chemokine chambers. Now to the cast Polymethyl soane devices vigorously mix 20 grams of PDMS with two grams of initiator for five minutes.
Carefully pour the PDMS onto the patterned wafer and Degas in a vacuum desiccate for one hour. Then bake and cure the PDMS microfluidic devices in a 65 degree Celsius oven for at least three hours. To ensure that the PDMS is baked at the correct temperature, be sure to monitor the internal oven temperature with a thermometer.
Punch out the central whole blood loading chambers of 1.5 millimeter diameter. Also punch out whole donut shaped devices of 5.0 millimeters diameter. Remove the particles from the donut devices using adhesive tape.
Next, rinse a multi-well plate with deionized water and dry with nitrogen after placing the plate in a 60 degree Celsius oven for five minutes. Oxygen plasma treat for 35 seconds. Then add the donut devices and oxygen plasma.
Treat again for another 35 seconds. Carefully transfer the devices into the wells of the plate and bake with bonded devices on an 80 degree Celsius hot plate for 10 minutes. After oxygen plasma treatment, the device is hydrophilic and capillary effects can promote the priming of the small channels in the device.
For the chemo attractant solution, mix five microliters of 10 micromolar FMLP with five microliters of one milligram per milliliter fibronectin, and 490 microliters of HBSS. Slowly pipette the chemo attractant solution into the whole blood loading chamber pipette and additional 20 microliters of the chemo attractant around the outside of the device. Place the plate in a desiccate for 15 minutes.
Apply a vacuum to the device to prime the device with chemo attractant solution. Remove the plate from the desiccate and confirm wetting of the device channel by gradual decrease in size of the air bubble. Then wash the whole blood loading chamber and the outside of the device thoroughly to remove excess chemo attractant solution.
Next to generate a gradient of chemo attractant from each of the focal chemotactic chambers to the device center. Gently inject 100 microliters of PBS into the hole so that a droplet of PBS forms on top of the device. Tilt the plate and pipette one milliliter of PBS around the device to collect at the bottom of the well aspirate the liquid and repeat the PBS wash three times.
Next, fill each well with media to submerge the devices and allow 15 minutes for the gradient to stabilize. Then slowly pipette two microliters of whole blood into each whole blood loading chamber. For finger prick blood collection, wash hands with soap and water.
Dry the skin as an anticoagulant stain solution. Add one milliliter of 0.2%HSA and 10 microliters of 32.4 micromolar hooks. Stain to a heparin blood collection tube prick the healthy donor's finger and wipe away the first drop of blood.
Collect 50 microliters of blood into the anticoagulant stain solution and gently mix. Set up the bio chamber to 37 degrees Celsius, 80%humidity and 5%CO2. Immediately start the time-lapse imaging at 10 x or higher magnification for meaningful statistical analysis manually track at least 50 neutrophils in each sample.
Count the cells entering the focal chemotaxis chambers over time. Then using image J, calculate neutrophil velocities in the channel between the WBLC and FCC. Then to quantify directionality of the neutrophils, count the number of cells that pass the bifurcation and calculate the ratio between the number of cells that turn toward the focal chemotaxis chambers and the cells that exit the device.
Cells that are not directional do not follow the chemotactic gradient and therefore migrate in equal numbers toward the FCC and the exit channel. In this experiment, the whole blood neutrophil chemotaxis assay was validated by measuring the accumulation of neutrophils toward an FMLP gradient. Results confirm that the red blood cells are trapped by the filtration comb while the neutrophils are able to actively migrate out of whole blood.
The stable linear chemo attractant gradient formed by the whole blood microfluidic device was confirmed using FSE labeled dextran and the fluorescence levels were measured over time. Results obtained using the novel WB Chemotaxis platform reveal that neutrophils from all blood sources migrate with consistent velocity and with similar total migratory cells. Importantly, this system allows quantitation of the neutrophils, therefore differentiates chemotaxis from chemo Kinesis Once mastered, this technique can be done in less than 10 minutes if it is performed properly.
The incorporation of our device in a 12 or 24 well plate using standard clean room techniques, facilities, the screening of multiple conditions simultaneously. Using this technique, we are able to measure neutrophil velocity and directionality at the single cell level. Unlike traditional methods that measure bulk endpoint cell migration, Remember to carefully prime the microfluidic device and load both samples slowly so that RBCs will not be pushed past the com of the device.
The Protocol described is particularly useful for measuring transient neutrophil chemotactic deficiencies, allowing repeated samplings over multiple time points, but could also be adapted to measuring migration of other cell types from different model organisms. This method can provide insight into temporary alterations of neutrophil functionality in patients with various conditions associated with higher incidence of infections, including and trauma patients, patients after major surgery and cancer patients receiving chemotherapy.
