心筋細胞ベースのアクチュエータおよび自己安定化バイオロゴ - パート1

The overall goal of this video is to describe how to develop and characterize a biological actuator using flexible PDMS cantilevers in cardiomyocytes. These actuators are then incorporated with a modified base to form a self-stabilizing swimming biorobot. The verification method described here is unique, wherein the mechanical base structure is modified to form a self-stabilizing swimming biorobot whose position can be also determined externally with magnets.

The main advantage of this technique is that the base properties can be easily altered to tune the buoyancy of the device and for self-stabilization. The method can be applied to develop other novel devices which would require self-stabilizations and flotation. Generally, individuals new to this method will struggle because mechanically deforming the cantilevers is challenging.

Due to their thin size, they can easily be torn or stuck in odd shapes. Visual demonstration of this method is critical as steps and assembly are difficult to learn without visual aid. To begin, place a silicon wafer on a photo resist spinner.

Add a thin photo resist to the wafer and spin coat at 2, 000 RPM for 20 seconds to obtain a target thickness of 1.2 micrometers. Next, use wafer tweezers to transfer the coated wafer to a hotplate preheated to 120 degrees Celsius. Cover the wafer with a shallow Petrie dish and bake it for 10 minutes.

Weigh out six grams of a PDMS base and combine it with 0.6 grams of a PDMS curing agent. Mix the PDMS components thoroughly for five minutes. Place the PDMS mixture into a vacuum chamber and pull a vacuum of 100 millibar for 30 minutes.

When finished, break the vacuum and remove the container. Keep the container covered until the PDMS is needed. Once the wafer has cooled, place it back onto the photo resist spinner.

Slowly pour the entire degassed PDMS mixture on the wafer so that no new bubbles are introduced. Then spin coat the wafer at 1, 200 RPM for five minutes. Using wafer tweezers, remove the wafer from the spin coater and place it in a preheated oven.

Bake the wafer overnight and then cool the wafer to room temperature. Next, prepare a laser engraver to pattern the thin film PDMS layer. Start by turning on the laser engraver and its exhaust system.

Then turn on the computer connected to the laser engraver and open the laser engraver software. Under File, open the biological actuator design file. Press the Settings button, click on Blue, and change the power setting to 3%and speed to 4%Click Set, click on Red, and change the mode to Skip.

Then click Set. Do the same for Black. Press the Apply button to finish the settings.

Next, select Activate the Engraver. Then press Relocate to move the design into the center of the screen. Next, press the Focus View button and click on the edge of the design to move the guiding laser dot to the corresponding point.

Carefully align the wafer with the guiding laser dot using gloved hands. Once aligned, select Start Engraving the Prior Job to start the engraving process. In order to fabricate the biological actuator base, a container for baking is needed.

First, place that container on a scale and zero the scale. Then, pour premixed PDMS and place cleaned 3 millimeter diameter glass beads onto the PDMS mixture at regular intervals. Leave at least 5 millimeters of space around each bead for the biological actuator base.

Place the container into a vacuum chamber, reduce the vacuum to 100 millibar and turn off the vacuum pump. After 30 minutes, break the vacuum and remove the container. Keep the container covered until it is needed.

Next, heat a hotplate to 40 degrees Celsius and carefully place the container of PDMS with the glass beads on the hotplate. Cover the container and bake it overnight. Once cooled, use a razor blade to cut 5 millimeter cubes out of the bulk PDMS so that each cube contains one glass bead in its center.

Then dip the tip of a needle into freshly prepared liquid PDMS and place a drop of the PDMS on the engraved base area of the wafer. Smear the droplet so that it completely covers the 5 millimeter by 5 millimeter base area. Finally, use a pair of tweezers to place the cube containing the glass bead onto the base area that is covered with liquid PDMS.

Repeat this process for each device. Once finished, transfer the wafer to a 40 degree Celsius hotplate and cover to bake overnight. Use a pair of sharp tweezers to peel the cantilever from the wafer and attach it to the sidewall of the base.

Then carefully detach the entire assembly from the wafer by pulling the base. To begin, repeat the fabrication process used for the biological actuators through the laser engraving step. However, for the fabrication of biorobots, use the biorobot design during laser cutting.

Next, prepare the micro-balloon PDMS mixture and the nickel powder PDMS mixture as described in the accompanying text protocol. Carefully pour the solutions into separate containers using a scale. Place both containers into a vacuum chamber and reduce its pressure to 100 millibar for 30 minutes.

Once degassed, place the containers onto a hotplate at 40 degrees Celsius, overnight. Use a razor blade to cut the biorobot bases out of each of the polymers with dimensions respective to each biorobot size as shown here. Place the nickel PDMS base onto a metal plate with a clean room tissue in between.

Then turn on the corona discharger and bring the tip of the discharger 1 centimeter above the bases. Move the tip around the base for 15 seconds to treat the surface. Repeat this process to treat the surface of the laser engraved bases, for the same duration.

Then use tweezers to place the nickel PDMS treated side onto the treated side of the film. Let the device sit for five minutes as the parts bond together. Next, use a pair of sharp tweezers to peel the biorobot cantilever from the wafer and place it on the bottom of the nickel PDMS base.

Then remove the entire assembly from the wafer. To attach the micro-balloon PDMS to the assembly, first, place a small drop of uncured PDMS on top of it. Then use a pair of tweezers to place the side of the nickel PDMS having the thin film PDMS onto the drop of uncured PDMS.

Place the entire assembly in a plastic Petrie dish and then place this on a hotplate at 40 degrees Celsius to cure overnight. To prepare the devices for cell seeding, first place 100 microliters of a Fibronectin solution into the center of a separate T25 culture flask for each device. Then place the biorobot or the biological actuator facing down over the droplet of Fibronectin solution.

Ensure that the cantilever is unfolded and immersed within the droplet, and then incubate the flask at 37 degrees Celsius for 30 minutes. After the incubation, remove the Fibronectin solution and wash the devices twice with PBS. Then remove the PBS and fill the flask with 10 milliliters of DMEM supplemented with 10%FBS and 1%penicillin.

Incubate the device at 37 degrees Celsius for one hour to degas the PDMS. Then use a magnet to hold the biorobot on the bottom of the flask, and place the flask with the samples in an ultrasonication bath for five minutes, to remove any bubbles. To determine the right thickness of each composite layer, various mixing ratios were tested with the mixing ratio of nickel and micro-balloons in PDMS.

The nickel PDMS layer allows for magnetic control and stability of the robot. Additionally the micro-balloon PDMS layer enables the biorobot to float and swim steadily. The combination of the two layers produces the self-stabilizing framework of the biorobot.

Using a computer script, the stability was numerically analyzed and proven to have a strong restoring moment using this two-layered method. It is important to remember to keep the cantilever clean throughout this procedure, and to maintain the correct ratio of PDMS mixture and thickness of the cantilever. After watching this video, you should understand how elastomeric materials can be formed into a thin cantilever to support cardiomyocytes and how they can be modified to attain new properties such as buoyancy or magnetism for advanced functionalities.

This verification procedure can be modified by incorporating other techniques like patterning alignment cubes, in order to promote cell orientation and increase fourth generation. Don't forget that working with laser engravers can be extremely hazardous, and precautions such as wearing appropriate protective clothing and eyewear should always be taken while performing this procedure.