血管由来のマイクロ流体ネットワークの画像誘導、レーザーベースの試作

The overall goal of this protocol is to describe how to use a confocal microscope equipped with a femtosecond pulse laser to generate three dimensional vascular derived biomimetic microfluidic networks embedded in polyethylene glycol diacrylate hydrogels using image guided laser based degradation. A significant advantage of this technique is the ability to generate three dimensional biomimetic microfluidic networks that accurately recapitulate the density, tortuosity, size range, and overall architecture of in vivo vasculature in synthetic constructs. This microfluidic fabrication approach will advance our ability to generate more in vivo like tissue engineered constructs, biomimetic disease models, and ultra high density on a chip devices.

Visual demonstration of this method is critical because proper setup and implementation of the microscope software is not intuitive, but is crucial in forming the desired microfluidic networks. Many of the preparatory steps are covered in the text protocol, including generating the virtual masks, configuring the microscope, and uploading the virtual masks to the microscope software. To photopolymerize the PEGDA hydrogel, first attach a ring of double sided adhesive to a 60 millimeter petri dish that has a 20 millimeter hole cut out of the bottom.

Next, in a fume hood, add one milliliter of HBS with TEOA to a foil covered two milliliter amber centrifuge tube. Then, add 10 microliters of one millimolar Eosin Y disodium salt to the tube. Now, pipette a small volume of NVP into a glass beaker using a glass pipette.

From this volume, take 3.5 microliters of NVP and transfer it to the mixture. Next, add 10 milligrams of 3.4 kilodalton PEGDA to a second foil covered tube. Then, add 0.2 milliliters of the mixture to make a 5%weight by volume PEGDA prepolymer solution.

Now, vortex the prepolymer solution until the PEGDA is fully dissolved. Then, pipette 60 microliters onto a PDMS mold without introducing any bubbles. Now, center an acrylated 40 millimeter number 1.5 glass coverslip over the solution on the PDMS spacers.

Then place the assembly under a white light source calibrated to deliver 95 milliwatts. Cure the polymer for three minutes. Next, flow distilled water between the mold and the coverslip to separate the hydrogel from the PDMS and rinse off the hydrogel.

Then, carefully pat dry the edges of the coverslip without wicking water from the hydrogel. Now, adhere the coverslip to the prepared petri dish. A little pressure helps.

Then, submerge the hydrogel in distilled water. Set up the laser scanning confocal microscope as described in the text protocol and start in the hydrogel visualization configuration. Then, fit the hydrogel and petri dish to the stage insert and place it on the stage.

Next, fill the petri dish with at least five milliliters of distilled water and lower the water immersion objective to just above the hydrogel without making contact with it. Now, click live to turn the argon laser line on. Then, focus on the hydrogel until the Eosin Y signal can be seen on the top side of the hydrogel.

To help find the hydrogel, try temporarily increasing the percent power or pinhole size. Mark the locations of the top and bottom of the hydrogel, and of the bottom of the well. Then use the joystick to move transversely and find the edges of the well.

In the focus window, drop the plane of focus down 150 microns within the hydrogel using the down arrow next to the Z position box. The next step is critical. Zero the X and Y location in the stage window, then delete all other marked locations and mark only the zeroed location.

Now, to start the process of forming an inlet channel, first snap an image and then using the rectangle button in the regions window outline the region that will be the inlet to the vascular network. Then move to the channel formation configuration. Details are provided in the text protocol.

Once there, in a Z stack window, click the center buttons at the top and above offset to center the Z stack at the current position. Then depending on how large the inlet needs to be, set the number of slices with an interval of one. Now, check that the speed is set to three and that the percent power is set to 100.

Then proceed with saving the configuration and click the start experiment button. To visualize the hydrogel during degradation of the channel, either increase the gain master in the channels window if the region in the field of view is black, or decrease the gain master if the region is white. After the degradation, ensure that the inlet was formed by going back to the hydrogel visualization configuration and clicking on live.

First, under the regions window, load any ovl file from the Z stack of virtual masks and click the arrow button to see the ROIs in the field of view. Then, click live to scan the hydrogel visualization configuration and use the joystick or the stage window to position the hydrogel so the inlet connects to the correct location within the vascular network. Next, drop the plane of focus to the first position in the microscope macro which is down 50 microns from the previous centered Z location using the step size in the focus window and clicking the down arrow.

Again, it is critical to now re-zero the X and Y locations, delete all other marked locations, and mark only this location as the new zero point. Be sure to save this new configuration. Now, under the channel formation configuration, select only the time series and bleaching windows.

Next, open the macro and select the saving tab. Select the previously made recipe and click apply. Be sure to not click store, or the recipe will be overwritten.

After verifying the settings, save the channel formation configuration again and then click update in the microscope macro. Click no to overwrite current location list, and then click start in the microscope macro to begin the recipe. After the degradation process is complete, use the hydrogel visualization configuration at a lower laser power to enable viewing of FITC-labeled dextran, which has a brighter signal than the native Eosin Y signal of the hydrogel.

Before introducing the fluorescently labeled dextran, wick some water from the well in the hydrogel. Then pipette in 10 microliters of filtered FITC-labeled dextran. The described procedure was used to convert a large volume of microvascular images into a series of virtual masks for image guided microfluidic network formation.

The resulting microfluidic network was compared with the in vivo vasculature it was derived from. The image guided laser based hydrogel degradation enabled the fabrication of 3D biomimetic microfluidic networks that recapitulate the size, tortuosity, and complex architecture of in vivo vasculature. Pressure heads and syringe pumps were both used to initiate fluid flow in these embedded microfluidic networks.

Fluorescently labeled 70 kilodalton dextran and 10 micron polystyrene beads were observed moving from left to right by a pressure head induced flow. Alternatively, syringe pump driven flow was initiated by photopolymerizing a hydrogel inside of a secondary microfluidic device. Using the same protocol described here, laser based degradation was then implemented to fabricate a 2D vascular derived network in a housed hydrogel for syringe pump driven flow and profusion of 65 kilodalton dextran.

After watching this video, you should have a good understanding of how to use a laser scanning confocal microscope equipped with a femtosecond pulse laser to generate biomimetic microfluidic networks using image guided laser based hydrogel degradation. Once mastered, this technique can be done in two to three hours if performed properly. While attempting this procedure, it's important to remember that the laser parameters including the power, wavelength, and scan speed need to be adjusted and optimized based on the hydrogel formulation being degraded.

Thank you and good luck with your experiments.