微細と3D微小血管の組立

The overall goal of using engineered micro-vessels is to replicate the structure and function of vessels in the body to study vascular physiology in normal and diseased states. This method can help answer key questions in the field of vascular biology such as how different anathema cells respond to flow, how they influence tissue function, and how vascular diseases initiate and progress over time. The main advantage of this technique is it can be easily modified to mimic different organ systems and disease states.

This can be done by changing the cell types, the matrix and media composition, and other variables. Generally individuals new to this method could struggle before they become aware of common mistakes that can lead to poor vessel quality. Visual demonstration of this method is critical because there are several tricks to successful fabrication during the injection molding and assembly steps.

For this procedure, have the flat and micro-patterned PDMS molds and the PMMA housing pieces prepared in advance. A description of how to make these parts is provided in the text protocol. Autoclave the PDMS molds along with the screws, pins, forceps, and a spatula.

Do not autoclave the PMMA pieces. They must be sterilized in bleach under a hood. After allowing the PMMA pieces to soak in bleach for at least an hour, rinse off the bleach with autoclaved water two times.

Then, place the pieces in sterile dishes. Aspirate excess water and let them air dry. With everything cleaned, start the protocol by treating the inner wells of both the top and bottom PMMA pieces using a handheld plasma treater for approximately one minute.

Turn the light off to help visualize the coverage of the plasma treatment. Next, add 1%PEI onto the inner wells. If the PEI doesn't spread easily, increase the plasma treatment time.

After letting the PEI react for 10 minutes, remove the solution and then rinse the devices off with sterile water and let them air dry. Once dried, apply 0.1%glutaraldehyde onto the treated surfaces and allow the glutaraldehyde to react for half an hour. Then, rinse the pieces with sterile water twice and allow them to air dry.

Now, prepare fresh 0.75%neutralized collagen for vessel fabrication by combining the pre allocated stock collagen volume with the neutralization solution of medium and sodium hydroxide. Stir slowly and carefully with a spatula until the solution is homogeneous and is orange in color. Then, if needed, add cells at the desired concentration and continue to stir the solution until the cells are uniformly distributed.

In preparation for the assembly process, place the micropatterned PDMS mold into a 100 by 20 millimeter dish and treat the mold with plasma for one minute. Next, align the top PMMA piece onto the PDMS so the square reservoirs line up with the inlet and outlet reservoirs. Then, put stainless steel dowel pins into the inlet and outlet reservoir holes so they will remain open after collagen injection.

Now, load the collagen cell mixture into a one milliliter syringe without introducing air bubbles. Then using forceps, gently press down on the top PMMA piece to secure it to the PDMS mold and slowly inject about one half milliliter of collagen cell mixture into the injection port. The collagen must fill the 20 by 20 millimeter area over the pattern and not leak out.

Then, carefully close the dish without jostling the dowel pins. Next, into the bottom PMMA piece place a 22 square millimeter glass coverslip in the inner well. Then, dispense about 0.25 milliliters of collagen evenly onto the glass.

Complete the bottom half by gently lowering the flat PDMS into the collagen so it is flat against the PMMA with no air bubbles in between. Now, carefully place both halves of the device into a 37 degree Celsius incubator and allow the collagen solution to gel for 30 minutes before proceeding. After gelation, add enough PBS to surround the PDMS on the bottom piece.

Then, use forceps to remove any excess collagen around the edges and slowly peel the flat PDMS piece off while keeping the collagen hydrated with PBS. To prepare the top piece, use forceps to lift the PMMA and PDMS assembly and flip it over. Then add a few drops of PBS to the inner face and remove the PDMS mold from the top PMMA piece, peeling quickly and firmly.

Next, with a second pair of forceps, gently remove the pins from the other side of the PMMA housing. Ensure the inlet and outlet are clear of excess collagen by moving the pins up and down before their removal. If the pins fall out on their own, simply use another sterile dowel pin to ensure that the inlet and outlet are clear.

Then, drop more PBS onto the micro patterned collagen, flip the piece over, and place screws into the four corner holes. Now, gently lay the top piece on the bottom piece aligning the screws into their holes. Do not let the two pieces slide against each other.

Then, gently screw the pieces together using a spatula and a gentle touch so they are not overtightened. It is critical to be gentle and precise during assembly. By applying an ample amount of PBS, the friction between the inner face is reduced.

While securing the screws, proceed slowly and stop as soon as any resistance is met. Next, aspirate the PBS surrounding the PMMA surfaces and place a small piece of cotton under one edge of the device. Then, aspirate any PBS from the reservoirs.

Now, refill the reservoirs with cell culture medium and incubate the device for at least an hour for gelation. Once the device is assembled, use an endothelial cell suspension at 10 million cells per milliliter to seed the device. Remove the medium in the inlet and outlet reservoirs and using a 20 microleter pipette with gel loading tips, add 10 microliters of the cell suspension into the center of the inlet reservoir.

The cells will immediately flow into and coat the network. Once the cells have filled the network and the flow has equilibrated, add 150 microliters of medium into both reservoirs. Then incubate the device for an hour or more so the cells can spread and attach.

For long term culture, remove the medium every 12 hours and add back 150 microliters of fresh medium to the inlet reservoir and 50 microliters to the outlet reservoir. After 24 hours of culturing, a syringe pump can be used to establish continuous flow conditions. After bringing the materials to the bio hood, remove the tubing from the autoclave bags and place it in a dish.

Then, prepare a sterile flow dish by melting side holes and then threading the tubing through. Prepare a 10 milliliter syringe with culture medium containing 3.5%Dextran. Next, attach the filled syringe to the lure lock at the end of the inlet tubing.

Then, secure the syringe to the pump and profuse medium until any trapped air bubbles have exited the tubing. Once the tubing is filled with medium and is clear of bubbles, transfer the device to a new dish and insert the inlet connector joint into the housing inlet. Then, set the syringe pump to the desired flow rate and begin profusion.

Prepare the outlet by filling the tubing with cell culture media using an 18 gauge needle and a one milliliter syringe. Clamping it and removing the needle. Then, melt a hole in the cap of the outlet collection tube using a soldering iron.

Afterwards, add media to the collection tube, insert the tubing, and attach the connector joint into the housing outlet. Throughout this process, a clamp is used to retain the medium in the tubing. Once completed, transfer the entire setup into an incubator.

Depending on the experimental conditions, the syringe may need to be refilled after 24 to 36 hours. During syringe replacement, ensure bubbles are not introduced into the tubing. Human umbilical vein and ethelial cells were profused through the collagen embedded micro fluidic network to form a patent lumen and confluent endothelium.

Several vessel geometries were used to establish different flow conditions. Continuous flow culture conditions were used to apply constant shear stress on the endothelium. Devices cultured under gravity driven flow conditions also maintained long term viability and patency.

A critical feature of these micro-vessels is their ability to respond to inflammatory stimuli. This was investigated by profusing the micro-vessels with whole blood. Non-stimulated micro-vessels contained non-activated endothelium and remained quiescent.

Stimulated micro-vessels became activated and their response initiated thrombus formation. Another interesting application for the system is to study phenotypic changes in vasculature formed from different endothelial cell populations. For example, micro-vessels formed from unique populations of stem cells exhibited different angiogenic sprouting capabilities.

After watching this video you should have a good understanding of how to fabricate micro-vessels and use them to study vascular biology and to build complex model organ systems. While attempting this procedure it is important to remember that practice will improve with the rate of successful vessel formation and allow for more consistent results. Once mastered, several devices can be made in three to four hours.

Following this procedure, other methods like the profusion of whole blood, Dextran, or pharmaceutical treatments can be performed to assess endothelial quiescence, permeability, drug response, and the general morphology of cells and vessels.