The overall goal of the following experiment is to perform cell rolling studies with increased throughput under a tightly controlled, physiologically relevant shear flow. This is achieved using a multi-well microfluidic system in which adjacent wells in a specialized plate are connected via a microfluidic channel. The interface of the system connects an electro pneumatic pump to the top of the multi-well plate, and applies a pneumatic pressure that drives the fluid from inside the wells through the microfluidic channels at a defined flow rate.
Once the microfluidic channel is coated with the desired substrate or cell mono monolayer, the cells of interest are introduced into the microfluidic channel to explore their specific rolling interactions with the coated surface, the rolling properties of the cells as they interact with the substrate or monolayer coated surface under different experimental conditions can then be analyzed with the appropriate software. The main advantage of this technique over existing methods like parallel plate flow chamber, is that this technique enables the study of silver rolling properties with a significantly higher throughput under precisely controlled, physiologically relevant tube flow while minimizing reagent and cell consumption. This platform may help advance exogenous cell-based therapies by allowing the rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing.
To coat a microfluidic channel with a protein substrate first add 25 to 50 microliters of the freshly prepared protein solution into the inlet. Well then apply a sheer force of two dine per square centimeter for five minutes to perfuse the solution into the channel. When a bead of liquid becomes visible in the outlet, well stop the flow, incubate the plate for the appropriate amount of time with the protein of interest, and then aspirate the solution from each.Well.
Next, add 200 to 500 microliters of PBS into the outlet well, and then apply a sheer flow of two dines per square centimeter for five minutes to wash the channel to create a cell monolayer inside the microfluidic channel. Begin by gently trypsin the cells of interest from their culture dish for three minutes, stop the reaction with a twofold volume of full media, and then centrifuge the cells for five minutes at 400 Gs and rt. Next, wash the pellet in 10 milliliters of full media.
Then resuspend the cells in one milliliter of fresh full media and count them, adjust the cell solution to the appropriate concentration, and then plate 25 to 50 microliters of the cell suspension into each inlet. Well now place the plate on the microscope stage and apply sheer flow of two dines per square centimeter to flow the cells into the channel until cells are observed to be filling the entire channel. After stopping the flow, fill both outlet and inland wells with 200 microliters of the appropriate cell media, and then let the cells settle and adhere at 37 degrees Celsius in 5%CO2.
After three hours, wash the channel with full media to remove the unattached cells. The cells should now be completely confluent and ready for use to induce the inflammatory activation of endothelial cells in the channels. Add 100 microliters of freshly prepared TNF alpha solution to the inlet well, and then introduce the solution into the channel by applying a sheer flow of two dines per square centimeter for five minutes.
For the control nonactivated endothelial cell channels, add 100 microliters of endothelial cell basal media to the inlet. Well, to block P or E selectin on the endothelial cell surface, introduce five micrograms per milliliter of the appropriate neutralizing antibody into the channel and incubate the plate for one hour at 37 degrees Celsius. Then wash the channels with basal media before starting the rolling assay.
Carefully examine the channels under a microscope to confirm that the channels are properly coated. Then wash the HL 60 cell suspension with basal media two times after the second wash count, and then resuspend the cells in IMDM at five times 10 to the sixth cells per milliliter concentration. After adding 25 to 50 microliters of the cell suspension into the outlet, well put the plate in the 37 degrees Celsius temperature controlled plate holder, and place the plate holder onto the microscope stage.
Introduce the cells into the microfluidic channel. They should be observed within 10 to 15 seconds flowing from the outlet to the inlet. Here, the fluorescently labeled HL 60 cells can be observed, interacting with the P select encoded surface, displaying a rolling response to examine the rolling response as a function of shear stress, reduce the shear to 0.25 dines per square centimeter and acquire 20 to 32nd videos using the stream acquisition function in each desired shear, and by gradually increasing the shear from 0.25 up to five dines per square centimeter.
Finally, use a CCD camera to acquire video of the assay with the stream acquisition of 11 frames per second. For example, here, an HL 60 cell rolling on a monolayer of chope cells is shown. Analyze the rolling paths and velocities with the appropriate compatible software.
HL 60 cells are considered gold standard rollers as they express a variety of homing ligands, including the rolling ligands, P select and glycoprotein ligand one and CI Lewis X.To test the capabilities of the multi-well plate microfluidic system, numerous microfluidic channels were coated simultaneously with different substrates and the rolling interactions of HL 60 cells. With those substrates were analyzed, the cells exhibited a robust rolling behavior on the P select encoded surface with the cells first captured from the flow, followed by a distinct rolling movement. As illustrated in this graph and consistent with the literature, HL 60 cells exhibit a similar rolling behavior on e and p selectin surfaces, but not on FiberInc coated substrates.
Cell velocity as analyzed via compatible software was plotted against sheer stress showing a robust rolling response of the cells on p and e selectin with an average velocity between one and 12 microns per second. To assess the feasibility of this microfluidic system in efficiently testing the interactions between the cells of interest and a cell monolayer coating the surface CHO P cells, which were transfected to stably express P, but not e selectin, were used as shown here. HL 60 cells display a significant rolling response on a CHO P cell monolayer to test whether the rolling movement of HL 60 is indeed mediated by pectin.
The monolayer was pre incubated with blocking antibodies for either P or E selectin prior to the profusion of HL 60 cells into the channel. As shown in this graph, blocking the CHO p monolayer with a P selectin antibody resulted in a significant decrease in the number of HL 60 cells rolling on the surface, demonstrating that P select and indeed mediates HL 60 rolling. As previously described, the multi-well microfluidic plate consists of numerous separate microfluidic channels, allowing higher throughput testing of multiple different conditions.
This advantageous design was used to plate endothelial cells inside the microfluidic channels for rapid analysis of the interactions between HL 60 cells and endothelial cells under a variety of experimental conditions to simulate inflammatory conditions, endothelial cells were pretreated with the pro-inflammatory cytokine TNF alpha, resulting in upregulation of E, but not P selectin on the endothelial cell surface. Interestingly, the HL 60 cells did not interact with the inactivated endothelial cells, and the cells were not observed to roll on this surface. On the contrary, the HL 60 cells displayed a robust rolling behavior on TNF alpha activated endothelial cells with an average velocity of five to 15 microns per second to explore the involvement of p or E selectin in the rolling interactions between HL 60 cells and activated endothelial cells.
TNF alpha activated endothelial cells were pre incubated with P or E select in blocking antibodies, and the rolling of HL 60 cells was analyzed as illustrated in the graph blocking selected on TNF alpha endothelial cells resulted in a significant decline in the number of rolling cells on the activated endothelial monolayer. In contrast, using an isotope control or an antibody against pectin, which is not expressed on activated endothelial cells, did not have a significant effect on HL 60 rolling on the activated endothelial layer. These data demonstrate the direct involvement of S selectin in HL 60 rolling on TNF alpha activated endothelial cells consistent with previous reports.
Analysis software permits the tracking of the paths of individual cells as they interact with the surface. Thus, the paths of individual cells as they interacted with TNF alpha activated endothelial cells with or without e select and antibody blocking were specifically tracked As illustrated in this figure, the number of rolling cells on unblocked activated endothelial cells was significantly higher than on elect and blocked activated endothelial cells. Furthermore, it appeared that the rolling movement of the HL 60 cells on unblocked endothelial cells was continuous and robust while the rolling paths of the cells on the elect and blocked endothelial cells were fragmented.
Consistent with this finding, the rolling velocity of HL 60 cells on unblocked TNF alpha activated endothelial cells was significantly lower than the rolling velocity on e select and blocked endothelial cells. This study demonstrates the use of a multiple microfluidic system to efficiently perform cell rolling experiments with increased throughput of up to 10 rolling gases per hour under tightly controlled tube flow. Overall, this microfluidic system emerges as a powerful technique for studying cell rolling, a key aspect of cell hoing.
For instance, our laboratory is currently using this system to screen for conditions to enhance the homing of mesenchymal stem cells as a strategy to improve their therapuetic impact.
