心筋細胞ベースのアクチュエータと自己安定化ビオロボ - 第2部

The overall goal of this study is to populate microfabricated devices with heart muscle cells and to characterize the biochemical and biomechanical properties of the cells on these devices as well as their performance over time. The matter described here can easily be modified for other experiments that require high density cell addition on microfabricated devices. The main advantage of the bioactuator developed in this video is that it can be easily adopted to quantify cell contraction forces of any other cell type.

It can also be used to study the changes in the biomechanical characteristics of stem cells as they undergo differentiation. Visual demonstration of this method is critical as the cell seeding steps are difficult to learn with descriptions from texts. They have to be carried out properly to ensure confluent cell attachment on the cantilever arms which is essential for proper biological function.

Demonstrating the procedure will be Neerajha Nagarajan from my laboratory. With the biorobot, start in the T25 flask. Use a magnet to hold it in place at the bottom of the flask.

The magnet is not needed for the biological actuators as they will not move due to the weight of the glass bead in the base. Roll a small square plastic sheet to use as a funnel and place the sterile funnel over the actuators in the biorobots. Adjust the diameter at the wider end to fit the device and the height so that it fits snugly when the top of the flask is tightened.

Use a separate flask to test and adjust the fit of the funnel. Snug fit of the funnel will ensure that there is no leakage of the cells as mentioned into the rest of the media. This is essential for providing confluent coverage of the cantilever arm.

With the funnel in place, resuspend cardiomyocytes in complete DMEM at a density of 16 million cells per milliliter and slowly drop 400 microliters of the suspension onto the devices through each funnel. Then, slowly move the flasks back into the incubator without disturbing the devices or the funnels within. Allow the cells to attach for 24 hours at 37 degrees Celsius.

After the incubation period, slowly remove the funnel. Gently wash the samples with PBS and refill each flask with 10 milliliters of fresh DMEM complete. Then, remove the magnet from the biorobot so that they're afloat and place the flasks back into the incubator.

Mount a camera equipped with a zoom lens in the incubator to visualize the devices in culture. Then, add an LED light strip to use as a light source. Next, connect the camera to a computer and open the imaging program.

Once in the program, click on the camera image in the File tab to open all camera options and choose the connected camera. From the list of tabs, choose Live and manually bring the image into focus by adjusting the lens dial. Next, select crop of the region of interest from the top panel.

Then, manually draw a rectangle in the video frame enclosing the biological actuator device and the cantilever arms to mark the region of interest. In the case of biorobots, capture the whole screen in order to record the swimming motion of the device. Next, select Camera Settings and set the frame rate by adjusting the exposure and pixel ratio of the live image.

Set the frame rates to about 30 frames per second. Finally, click the record button to start recording the videos. Open the programming software by clicking on the icon.

Once loaded, click File, Open, and select the m script file for running image analysis. Ensure that the recorded TIF images and their AVI versions are in the same folder as the m file and then click Run to run the script. Press Play to start the actual program.

Once running, click the Open button and locate the TIF or AVI file that is going to be analyzed. Then, click the base button. Select the point where the cantilever attaches to the base at the top and hit Enter.

This will place a square marker on the image for each frame to denote the location of the cantilever base. Next, click the scale button and then manually click on one edge of the glass bead. Bring the mouse pointer to the opposite side of the glass bead and press Enter.

This will draw a line that measures the diameter of the glass bead and will relate the diameter to the pixels displayed. Click the Analyze button. Then, click along the cantilever at a short distance from the first square marker that represents the cantilever base.

Continue to click along the cantilever including the tip and press Enter when done. This will place an X on each point clicked on the cantilever. Finally, check if the superimposed circle correctly traces the cantilever profile.

If correct, click on the next frame button. This will switch to the next frame in the TIF file. Repeat this process for all of the frames.

Once all frames have been processed, click on the Export button. Start by opening the image analysis software and loading the swimming biorobot video file. Then, open the spreadsheet software.

In the loaded biorobot video, locate a size reference such as the glass bead. Use the Straight tool to draw a line across the glass bead. Then click Analyze and select Set Scale.

Set the Known distance field to 3, 000 micrometers and click OK.Choose a point on a device that does not wobble between frames to act as a marker and record the X and Y coordinates of this point on the spreadsheet. Switch back to the image analysis software window and press the right arrow key to change to the next frame. Point to the marker again and record the new X and Y coordinates on the spreadsheet.

Repeat this process for all of the frames and use the data to analyze the biorobot's motion as described in the accompanying text protocol. The static and dynamic stresses of the actuator were extracted from the surface stress each day. Static stress is the contractile stress the cells exhibit on the surface at the resting state and dynamic stress is the stress generated by the cells at maximum contraction.

As the cells matured in culture, a gradual increase in static and dynamic stresses was observed over time. They exhibited a maximum cell traction force of 50 millinewtons per meter and a maximum contraction force of about 165 millinewtons per meter on day six. Paralleling the static and dynamic stresses, markers for cardiomyocyte maturation, growth and spreading, and cell interconnectivity also increase until day six.

Based on the video analysis, the comparison of the average swim velocity, beating frequency, and distance moved per stroke for three biorobots with different swimming profiles is shown here. Although the vertical biorobots have the smallest beating frequency, they exhibited the highest swim velocity and distance traveled. After watching this video, you should have a good understanding of how to seed heart muscle cells on microfabricated devices that could perform as biological actuators and biorobots.

The process of image acquisition and subsequent data analysis described in this video demonstrates a convenient way of characterizing the cell behavior as well as the device performance. While attempting this procedure, it is important to remember that the lifespan of the cardiomyocytes is limited and the activity of the cells peak on the sixth day. After which, it decreases over time.