Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca²⁺]_i and Nitric Oxide Production

The overall goal of this procedure is to develop a microfluidic device that allows endothelial cells to grow under physiologically relevant shear flow and to measure the agonist induced changes in their calcium and and nitric oxide production. This message can help advance the future of microvessel research, by validating the in vitro microvessel model and bridging the gaps between in vivo and the in vitro studies. The main advantages of this technique are the versatile design of the microchannels to mimic various vasculature and they improve cell culture environment that is closer to in vivo than the conditional static to that original cultures.

Demonstrating the procedures will be Sulei and Xiang, the two from my laboratory. Before beginning, use standard photolithography to create a master mold and soft lithography to fabricate the PDMS microchannel devices as described in the text protocol. To prepare the microchannel devices, first cover the inlet and outlet with a thin piece of PDMS and place the device inside a Petri dish with a wet tissue.

Then, wrap the dish with Parafilm and sterilize the device under a UV light for at least three hours. The next step is to apply the fibronectin. First, place a drop of 30 to 50 microliters of PBS at the inlet.

Create a vacuum at the outlet to rinse the device. Next, place a drop of 100 micrograms per milliliter fibronectin at the inlet. Create a vacuum at the outlet to load the fibronectin solution.

Next, cover the inlet and outlet. Wrap the dish with Parafilm again and incubate the device overnight at four degrees Celsius. The next day, manually rinse the device with PBS three times.

Then, load it with 30 to 50 microliters of cell culture media and warm up the device in an incubator at 37 degrees Celsius for at least 15 minutes. Begin by mixing HUVECs into HUVEC culture media with eight percent Dextran at two to four million cells per milliliter. The Dextran is needed to increase the viscosity and thus improve the cell seeding.

Next, put 10 to 20 microliters of cells over the inlet and introduce them using gravity or by using capillary action from a glass pasteur pipette at the outlet. Next, incubate the device for 15 to 20 minutes. Then, check the seeding status under a microscope.

If necessary, load more cells to achieve a desired cell density. Once enough cells are loaded, gently rinse the device with normal, warm cell culture medium to remove the Dextran. Then, culture the device without any media perfusion for six hours.

Next, set up the tubing connections for the long term perfusion. Tape the device to a Petri dish and then connect the inlet tubing to a syringe with a needle. Insert tubing into both the inlet and the outlet of the device.

Connect the outlet tubing to a waste collector. Now, place the dish and waste container in an incubator. Connect the syringe with a long inlet tube to an external perfusion pump that passes through the incubator door.

Then, set the perfusion rate according to the experimental design. With a well controlled perfusion, the culture in the microvessel network can be maintained up to two weeks. Therefore, it can be extended to longer term studies mimicking the cellular and the hemodynamic environment on the different physiological and the pathological condition.

To begin the immuno-staining, such as VE-cadherin labeling, first fix the cells by perfusing with two percent paraformaldehyde for 30 minutes at four degrees Celsius. After a series of perfusions to block, permeabilize, and stain with antibodies, keep the device at four degrees Celsius until the cells are imaged. To image the calcium concentration in the cells, perfuse the device with 10 milligrams per milliliter of albumin in Ringer solution for 15 minutes at 37 degrees Celsius.

Next, perfuse the device with five micromolar Fluo-4 AM for 40 minutes at 37 degrees Celsius. Then, wash out the lumen bound Fluo-4 AM with albumin Ringers for 15 minutes at 37 degrees Celsius. Next, start up the confocal system for intercellular calcium level imaging with Fluo-4 AM.Acquire images of the Fluo-4 loaded microvessels.

Collect the baseline images for 10 minutes. Then, perfuse the device continuously with 10 micromolar ATP and record the changes of fluorescence intensity for 20 minutes. To image nitric oxide production in the cells, perfuse the device with albumin Ringers for 15 minutes at 37 degrees Celsius.

Then, perfuse the cells with 5 micromolar DAF-2 DA for about 35 to 40 minutes at 37 degrees Celsius. Next, set up the fluorescence imaging system to measure ATP induced nitric oxide production. Record the baseline for 5 minutes, then perfuse the device with 10 micromolar ATP and collect the images for 30 minutes at one minute intervals.

For the cell nitric oxide measurement, it is important to understand that the detach adapter for intensity profile represents a cumulative nitric oxide production with time and not a nitric oxide concentration. Manually select the region of interests from the collected images at the individual cell level. Each ROI covers the area of one individual’s cell which can be indicated by the floresense outline.

Quantify the ATP induced changes in endothelial calcium and nitric oxide. By calculating the changes in the floresense intensity of Fluo-4, less the tissue background. Quantify the nitrous oxide production rate by conducting the first differential conversion of the floresense intensity of DAF-2 over time.

The microchannel pattern in the device has a three level branching network. A 3D numerical simulation was performed to estimate the shear stress distributions under the flow rate of 0.35 microliters per minute. Endothelial cells were seeding into the network as described.

Then, F-actin and nuclei were labeled. Similarly, VE-cadherin was also stained. The results were similar to the VE-cadherin expression in an intact venule.

Next, ATP induced increases in intracellular calcium and nitric oxide production were examined. Calcium levels were examined using Fluo-4 AM as described in the protocol section. Nitric oxide production was monitored using DAF-2 DA as described in the protocol section.

The results were comparable to those derived with individually perfused intact venules. The advanced was easy and tightly controlled in biological conditions and the dynamic fluid environments, which would not have been possible with conventional microskill techniques. After watching this video, you should have a good understanding of how to fabricate the device, and perform calcium and nitric oxide measurements.

After this technique paved the way for researchers, not only to investigate underlying agonist induced but also to open broader applications for bio medical research.