Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

The overall goal of this procedure is to create a microfluidic device coated with endothelial cells. This is accomplished by first fabricating the microfluidic mold. The second step is to create the PDMS channel.

Next, the endothelial cells are seeded into the device. The final step is to culture the cells within the device. Ultimately, endothelial cell coated microfluidic devices are used to show cell to cell interactions in vitro.

The main advantage of this technique over existing methods is that it provides the researcher with tight controls over the biological and biophysical conditions and is compatible with fluorescence microscopy. Though this method can provide insight into the biophysical cell cell interactions in hematologic diseases, it can also be applied to phenomenon such as leukocyte biology, cancer metastasis, and hematopoietic stem cell biology. Generally, individuals new to this method will struggle because conditions have to be tightly controlled for successful cell seating.

In preparation for fabrication creates a photo mask by submitting a computer assisted design image of the Microfluid device to an outside mask vendor. Once received, the mask is composed of a chrome layer on soda lime glass. Here the micro fluidic channel width is 30 microns.

To begin fabrication of the microfluidic device, clean a bare silicone wafer with piranha for 15 minutes, then rinse with deionized water for 10 seconds. Then immerse the silicone wafer in hydrofluoric acid for 30 seconds, and rinse with deionized water for approximately 10 seconds. Next, using a spin coter spin micro chem SU 8 20 25 photo resist onto the wafer to a height of 30 microns using a spin speed of 3000 rotations per minute.

Place the SU 8 20 25 coated wafer onto a hot plate set at 95 degrees Celsius for five minutes to drive off excess solvent. An oven is not recommended for this step. Now, place the photo mask over the wafer and expose to UV light in a mask liner to cross cross-link the SU 8 20 25 photo resist.

After cross-linking, place the wafer back on the hot plate at 95 degrees Celsius for an additional five minutes. To further accelerate the polymerization of SU 8 20 25 photo resist. Then immerse the wafer in SU eight developer, which is composed primarily of P-G-M-E-A for four minutes.

This will remove the non cross-linked SU 8 20 25 photo.Resist. Rinse the newly developed wafer with 100%isopropyl alcohol for 10 seconds, then dry using pressurized nitrogen, or by allowing the solvent to evaporate from the wafer in a clean fume hood for several minutes. Now, tape the dried wafer into the center of the Petri dish, being careful to place the tape at the edges of the wafer.

Finally, pipette 500 microliters of sigma coat onto the wafer. Cover the Petri dish with the lid, and then swirl the dish to ensure complete coating of the wafer. Remove the lid and allow the wafer to dry for several minutes until all of the solvent has evaporated.

After mixing PDMS polymer and curing agent at a 10 to one weight per weight ratio, remove air bubbles using a vacuum desiccate for several minutes to one hour, depending on the strength of the available vacuum system. After all air bubbles have been removed, pour the mixture into the dish so that the wafer and the surface of the dish are covered. To create a sheet of PDMS that is approximately five millimeters thick.

Also create a thin featureless piece of PDMS one millimeter thick. Then cure the mixture in a 60 degree Celsius oven overnight. The next day, use a knife or scalpel to cut around the wafer and remove multiple PDMS microfluidic devices.

Then cut the sheet to isolate individual devices. Cut the sheet of PDMS slightly larger than the microfluidic device. Now use a pin vice Harris unicorn or similar device to create inlet and outlet holes in the microfluidic device.

Next, clean the surfaces of the microfluidic device and the sheet using scotch tape. Then use a plasma cleaner to expose the surfaces of the microfluidic device and PDMS sheet to oxygen plasma for 30 seconds. The oxygen plasma creates reactive species on the exposed surface of the PDMS, which bond when brought into physical contact.

Next, fill a one milliliter syringe fitted with a 20 gauge blunt point needle with 50 micrograms per milliliter. Fibronectin from human plasma in PBS then fit a length of tubing over the needle. Insert a piece of thinner tubing into the first piece of tubing to create a friction fit between the two pieces of tubing.

Then attach the other end of the thinner piece of tubing to the inlet of the microfluidic device. Apply pressure to the syringe to fill the channels completely with fibronectin solution and to create a small 100 microliter drop at the outlook port to ensure that the channels stay wet. Now, incubate the microfluidic device in a humidified incubator at 37 degrees Celsius for 40 to 60 minutes.

Following the incubation, connect a fresh syringe filled with PBS to the blunt point needle. Apply pressure to flush the microfluidic device with PBS. Prepare 500, 000 to 2 million cells per milliliter of human umbilical vein endothelial cells, or hve in endothelial growth media with 8%dextran.

The addition of dextran increases the likelihood that the cells will adhere and culture successfully within the microfluidic device. Filler clean syringe fitted with a blunt point needle and wider tubing with the cell suspension and connect a longer length of tubing approximately one meter in length. It is critical to ensure that the tubing is a tight fit to eliminate leakage between any connections.

Prime the tubing with cell solution, ensuring that there are no bubbles present in either the syringe or any of the tubing To optimize the performance of the system. It is critical at this point to prevent any leaks or bubbles from entering the system. Any leakage of solution or presence of bubbles in the media will prevent the successful seating of endothelial cells.

Once all bubbles have been eliminated and the whole length of tubing is primed with cell suspension, place the syringe on the pump and attach the other end to the microfluidic inlet. The syringe pump is set at the same height as the microfluidic device. The long length of tubing is carefully coiled within the incubator to ensure that the cells and media are adequately warmed before coming into contact with the device.

Then the cell suspension is infused into the microfluidic device at a volumetric flow rate of 1.23 microliters per minute for two hours at 37 degrees Celsius and 5%carbon dioxide In our system. The volumetric flu rate of 1.23 microliters per minute corresponds to a midstream velocity of one millimeter per second in the smallest channels. This approximates a wall shear stress of one dime per centimeter square in those channels using whole blood.

Once the cell suspension infusion is complete, use the same syringe pump and long tubing with a larger syringe to infuse fresh growth media for two to eight days at a rate of 1.23 microliters per minute. When the cell monolayer is at the desired level of confluence, inject blood or cell suspension into the system for experimentation. Here, the micro fluidic device is shown before endothelialization.

The microfluidic device has been infused with food coloring to illustrate the size, scale, and overall design of the device that mimics the physiology of the vasculature. Brightfield microscopy reveals endothelialization of a microfluidic device. This image was acquired less than 48 hours after seeding using the described protocol, the optimized perfusion technique allows endothelial cells to conf fluently culture the entire inner surface of the microfluidic system.

Within 24 to 48 hours of cell seeding. As shown in this confocal image, the microvascular tumor on a chip with micro channels approximates the 30 micron size of post capillary veles the site of most sickle cell microvascular obstructive events. In this experiment, whole blood from healthy patients is infused into a micro vasculature on a chip device in deoxygenated conditions.

This movie shows that the flow of whole blood in our endothelial microfluidic system is regular, smooth, and continuous with no areas of microchannel obstruction. Here, whole blood from patients with sickle cell disease is infused into a micro vasculature on a chip device. In deoxygenated conditions, significant changes in the blood flow is seen with complete occlusions present in several channels.

While attempting this procedure, it's important to remember not to introduce air bubbles when switching tubing. The finished device is compatible with fluorescence microscopy and can be adapted to traditional fluorescence assays. This technique paves the way for experimental hematologists to conduct experiments using single cells in a controlled biophysical and hemodynamic environment.

No Bubbles.