The objective of this video is to demonstrate a lab on a chip technique for characterizing the inflammatory state of endothelial cells. To begin, human aortic endothelial cells are grown to confluence on a tissue culture substrate, and then inserted into a Conan plate shearing device. The cells are treated with the pro-inflammatory cytokine, TNF alpha, and simultaneously exposed to differential shear stress conditions for four hours by precisely controlling the rotation of the cone above the substrate shear stress modulates a well-characterized inflammatory response to cytokine.
That includes the upregulation of adhesion molecules such as VCA one and ICAM one. The substrate is transferred to a microscope stage where A-P-D-M-S microfluidic device is vacuum sealed directly to the monolayer creating flow channels. Activated monocytic, THP one cells are flown over the preconditioned hs, where they bind in proportion to the level of cell adhesion Molecule expression.
The inflammatory State of the endothelium is determined by quantifying the total number of THP one cells That are firmly arrested. Hi, my name is she D Diverse, and I'm Greg Foster. And I'm Keith Bailey.
We are in the laboratories of professors, pastor and Simon in the Department of Biomedical Engineering at the University of California in Davis. Today we are going to describe the use of a lab on a chip based approach to quantitatively examine endothelial dysfunction and inflammation. These tools provide a platform for us to conduct studies related to the superposition of metabolic stress with hydrodynamic factors and the regulation of endothelial inflammatory phenotype and susceptibility to atherosclerosis.
So we refer to our devices as vascular mimetic, microfluidic chambers, or vmmc. These devices allow us to quantify inflammatory outcomes in endothelial cells and monocytes under conditions that mimic those in atherogenic vessels. The VMMC facilitates the use of small amounts of reagents or materials that may be in limited supply.
They can also be constructed in a number of customizable layouts and geometries, making them very suitable for a vast range of applications. Today we'll demonstrate a representative experiment using our lab on a chip approach. Human endothelial monolayers will be exposed to cytokine activation under well-defined shear stresses.
We will demonstrate the use of VMC to quantify human monocytic cell recruitment to the monolayer in real time as a functional readout of endothelial activation using a lathe three inch circular substrates are milled from 100 millimeter tissue culture dishes. The three inch substrates are washed with distilled water, then sterilized by soaking in 70%ethanol. Sterile substrates are placed in 100 millimeter Petri dishes and allowed to dry.
They're then incubated with type one collagen at a concentration of 100 micrograms per milliliter for one hour at room temperature and washed with PBS. Human aortic endothelial cells or HA are suspended at a concentration of 650, 000 cells per milliliter. One milliliter of cell suspension is placed in droplets on a dried collagen coated substrate and spread Into an even suspension.
Seated substrates Are then placed in a 37 degree 5%CO2 incubator and allowed to attach to the substrate surface. After one hour nine milliliters of E GM two growth, medium is added and the cells are allowed to grow into Monolayers at approximately 90%Co fluency monolayers are ready to be used for subsequent sheer experiments. Vascular mimetic microfluidic chambers are constructed by pouring A-P-D-M-S mold over a master form comprising the desired channel.Geometry.
Silicone base is poured into a plastic way boat, followed by the silicone curing agent at a 10 to one ratio by weight. The PDMS gel is then well mixed, Taking care not to scrape the sides of the way boat, and thereby introducing plastic chips into the mixture. Photoresist masters are cleaned with hexane and prepared to mold the VMMC.
The PDMS Mixture is then poured over the master form, then placed into a vacuum chamber. In the chamber. Air is Removed from the PDMS mixture and rises to the surface after 15 minutes.
The mold is removed from the chamber And air is blown Gently over the PDMS surface to purge remaining air bubbles from the mold. The mold is then covered and placed in a 37 degree oven for one hour where the PMS is allowed to cure. Remove the solidified PDMS gel from the oven and make circular incisions around the chamber using a small scalpel.
It is crucial to do this gradually to avoid contact with the photo resist material as this will damage the master mold. Excise the microfluidic chamber from the mold punch inlet and outlet ports for each channel using a 19 gauge lure lock needle. Additionally punch holes for the vacuum tubes away from the channels.
The Preparation for the VMMC is now complete. Pipette the pro-inflammatory Cytokine TNF alpha into six milliliters of L 15 media to achieve a final concentration of 0.5 nanograms per milliliter. L 15 media is utilized here as the shearing fluid due to its pH buffering capacity in the absence of 5%CO2.
The media is then drawn into a sterile 10 milliliter syringe, which is used To load the shearing device. The Confluent monolayer is then transferred to the cell shearing device, which has been sterilized with ethanol leveled and the cone gap height has been set. The substrate is removed from the Petri dish and placed on the glass gauge block centered and carefully inserted into the bottom of the housing chamber, such that the cone is directly above the cells.
The substrate is secured by evenly tightening stainless steel screws around the gauge block. The shearing media is then loaded into the chamber, taking care not to introduce any air Bubbles. Next, a computer Is utilized to input the desired shear stress program and the micro separate motor is turned on.
Here we utilize an oscillatory shear of zero plus or minus five dines per centimeter squared. After four hours, the shearing program is stopped. Gauge block is removed and the substrate is carefully placed back into the Petri dish.
The cells are now ready for the monocyte adhesion assay. Immediately Following shearing HA monolayers are transported to the microscope station for assembly of the VMMC. The VMMC is attached to the vacuum hose and submerged in DI water to remove any air bubbles trapped within the channels.
With the vacuum valve initially off the chamber is carefully pressed on top of the monolayer and vacuum valve is switched on. It is critical here to ensure that no air bubbles have been trapped within the channels of the chamber. Now, 80 microliters of HBSS with calcium and magnesium are pipetted into a reservoir made from cutting the tip off of a 19 Gauge lure Lock needle fluid is Pushed out of the tip by firmly pressing the top of the reservoir, which is then inserted into the inlet port Of the VMMC.
The whole assembly is placed on the microscope stage and the microscope camera and video monitor are turned on. A 10 milliliter glass syringe is loaded onto the syringe pump. Next, the syringe tubing is connected to the outlet port of the VMMC.
The flow rate is set To induce a sheer stress of one dine per centimeter squared within the channel, and then run in refill mode such that the syringe pulls fluid from the reservoir through the channel. Th P one cells stimulated with SDF one are suspended at a concentration of 2 million cells per milliliter. Within two minutes of flow activation, 50 microliters of cell suspension are added to the reservoir.
Again, taking care not to introduce air bubbles upon fully developing flow. Video is captured at three frames per second and data is enumerated using Image J software.Adherent. THP one cells are defined as moving no more than one half cell diameter in 10 seconds.
Here we demonstrate Data depicting THP one adhesion to ha monolayers stimulated with TNF alpha and conditioned under either a steady high wall shear stress of 15 dines per centimeter squared or an oscillatory shear stress of zero plus or minus five dines per centimeter squared ha monolayers conditioned with oscillatory shear stress exhibited threefold greater TP one cell recruitment compared to monolayers that experienced a high shear stress. Today, we demonstrate the use of customizable in vitro techniques that can be combined to provide important insight into early inflammatory events underlying atherosclerosis. Specifically, we quantify outcomes associated with endothelial dysfunction and cells exposed to inflammatory stimuli under well-defined hydrodynamic conditions.
The use of VMM CS allows us to examine specific mechanisms underlying endothelial and leukocyte adhesion interactions. These devices also have the advantage of being flexible and inexpensive and facilitate the use of materials that may be limited in supply, such as serum derived components from human subjects. Our studies applying these devices have included investigations of cytokine, lipid, and rage induced inflammation in endothelial cells.
We've also developed on-chip assays examining adhesion mechanisms in multiple leukocyte types as well as in whole blood. Ultimately, we envision this technology will lead to ex vivo approaches for assessing an individual's risk of inflammation mediated cardiovascular Disease.
