Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. Inflammation dysregulation often causes altered vascular endothelium barrier function and excessive leukocyte trafficking, which can result in organ damage. The biomimetic microfluidic assay, what we call bMFA, reproduces topography and flow conditions of in-vivo microvascular networks and allows for realtime assessment of rolling, firm adhesion, spreading, and migration of neutrophils into the tissue compartment.
A strength of the biomimetic microfluidic assay is the ability to use primary human cells and clinically relevant patient samples to increase clinical translation and rapidly screen potential therapeutics. Demonstrating the procedures will be Mr.Qingliang Yang, a graduate research assistant from my laboratory. Start inserting the tube of around one inch in length within the ports using fine forceps, except for one inlet port.
Using jaw clamps, clamp the two outlet ports and the tissue compartment together. Dilute the fibronectin stock solution to 100 micrograms per milliliter with PBS. Load a one-milliliter syringe connected to a 24-gauge blunt needle with the diluted fibronectin solution and connect the syringe to a four-inches-long tubing.
Insert the tubing into the open inlet port, then push the plunger until human fibronectin is released from another inlet port and clamp it. Repeat the process for the remaining ports until all the channels, tissue compartment, and tubing are filled with the human fibronectin solution. Remove the needle but keep the four-inch-long tubing inserted and unclamped.
To perform degassing, connect the unclamped tubing to the pneumatic primer connected to a compressed nitrogen tank with a pressure of five pounds per square inch for 15 minutes. Under the microscope, check that there are no air bubbles trapped within the channel or tissue compartment and reconnect the device if the air bubbles are present. Remove the device from the pneumatic primer and incubate it at 37 degrees Celsius for one hour.
Using pre-warmed human lung microvascular endothelial cell culture media, flush all the channels and tissue compartment. After harvesting the endothelial cells as described in the text manuscript, pellet the cells by centrifugation for five minutes at 150 times G.In the programmable syringe pump, mount a one-milliliter syringe and attach the tubing to the blunt needle. Draw approximately 20 microliters of the cell suspension into the tubing without letting it into the syringe barrel.
Remove the clamp from the outlet port of the device. Connect the tubing to the inlet port without introducing any air bubble into the channel. Stop the pump with a flow rate of four to eight microliters per minute and observe under the microscope.
Stop the pump when the channels are filled with cells. Clamp the outlet and cut the inlet tubing. Place the device in the incubator for four hours at 5%carbon dioxide and 37 degrees Celsius.
After four hours of incubation, prepare another syringe and fill it with fresh cell culture media. Mount the syringe in a syringe pump and connect it to the inlet port. Remove the clamp from the outlet port.
Pass the fresh media through the device for about five minutes at four to eight microliters per minute to remove the floating or unattached cells. Prepare a syringe by filling it with fresh cell culture media. Mount the syringe in a syringe pump and connect it to one inlet port while keeping the outlet port open.
Place the device in an incubator at 37 degrees Celsius and 5%carbon dioxide. Program the syringe pump for culturing endothelial cells under flow as described in the text manuscript. Using a microscope, check bMFA after 48 hours of culture under flow.
Confocal microscopy indicated that all surfaces of the vascular channels were covered by endothelial cells, forming a complete 3D lumen in bMFA. After preparing three different bMFA devices, load three one-milliliter syringes with cell culture media or TNF-alpha or TNF-alpha combined with PKC delta inhibitor respectively, where TNF-alpha and inflammatory cytokine is used to stimulate endothelial cells and neutrophils. PKC delta inhibitor is a novel anti-inflammatory inhibitor.
Connect the three loaded syringes to three bMFA devices. Using buffer TNF-alpha or TNF-alpha with added inhibitor, treat human lung microvascular endothelial cells for four hours at 0.1 microliters per minute. After isolating the human neutrophils as described in the text manuscript, resuspend the neutrophils in 999 microliters of HEPES buffer and add one microliter of 10-millimolar CFDA SE dye stock solution to the suspension, resulting in a 10-micromolar working solution of CFDA SE and incubate it for 10 minutes at room temperature.
Wash the cells twice with HEPES buffer by centrifuging the solution for five minutes at 315 times G.After counting the cells, resuspend at 2 million neutrophils per milliliter in cell culture media or TNF-alpha or TNF-alpha added with PKC delta inhibitor and incubate at room temperature for 15 minutes. Fill the syringe with one-micromolar of a chemo-attractant, fMLP, prepared in cell culture media. Open one inlet and one outlet port of the bMFA.
Remove the port tubing from the tissue compartment and insert the fMLP tubing. Inject approximately 20 microliters of fMLP into the tissue compartment for all bMFA, leaving the one treated with cell culture media. Cut the tubing and clamp it.
Replete the syringe with approximately 200 microliters of neutrophil suspension and mount the syringe on the syringe pump. After placing the device on an inverted microscope stage, set the flow rate at one microliter per minute and start the pump. Wait until a small drop of the neutrophil suspension comes out of the tubing and insert the tubing into the inlet port.
Fluorescently-labeled neutrophils flow into the vascular channels and interact with endothelial cells and the physiologically-relevant flow conditions. After 10 minutes from starting the experiment, open the image analysis software, switch the objective lens to 10x, and use the stage joystick to center the device under the microscope. To obtain the adhesion map, go to Acquire, click Scan Large Image.
A new window pops up. Choose 10x Objective lens and set the field option, for example, 5 by 3. Click Scan, click File, and save the adhesion map.
To obtain the migration map for migration analysis, center the device under the microscope using the stage joystick. Click View, Acquisition Controls, ND Acquisition. A new window pops up.
Set the Path to save the file and type the file name. Check Large Image function, set Scan Area, for example, 5 by 3. Check Time function, set the Interval as five minutes, and Duration as 60 minutes.
Take time lapse images of the tissue compartment, with one image taken every five minutes during the next hour. CFD simulation shows the laminar flow pattern in vascular channels, except for bifurcation areas where the flow pattern is disturbed. After performing the biomimetic microfluid assay, phase contrast images revealed that the surfaces of the vascular channels were covered with endothelial cells, and aligned in the direction of shear flow after 48 hours of culture.
Fluorescence imaging was carried out using confocal microscopy, indicating that the endothelial cells form a complete three-dimensional lumen in bMFA A neutrophil adhesion map was obtained, which revealed that there is significant adhesion of neutrophils to endothelial cells in bMFA. Neutrophil migration map in bMFA revealed that upon TNF-alpha activation, considerable migration of neutrophils occurs inside the tissue compartment, while no such migration was observed without TNF-alpha activation. An adhesion map correlating the spatial distribution of neutrophils with the shear rate demonstrated that neutrophil adhesion occurred preferentially in vessels with low shear rate and near bifurcation regions.
Further, TNF-alpha treatment significantly increases adhesion, which was inhibited with the PKC delta inhibitor. Analyzing time lapse images indicated that TNF activation of endothelial cells increases neutrophil migration in response to fMLP, whereas treatment with the PKC delta inhibitor reduced migration compared to TNF-alpha treated cells. Thus, bMFA can be used to test the efficacy of a novel therapeutic for treating inflammatory disease.
The bMFA can also be used to study the endothelium integrity by measuring variables such as permeability and trans-endothelial electrical resistance, what's called TEER, and adhesion molecule expression during inflammation. The biomimetic microfluidic assay can mimic the microenvironment of different organs and is not limited to single cell types or species, and can also model cell/cell communication critical to organ function and modeling different diseases.
