The overall goal of this procedure is to generate engineered muscle tissues. This is accomplished by first culturing satellite cells or a myoblast cell line to achieve the required number of cells. The second step is to make the anchoring points.
Next, the cells are mixed with a gel and the resulting mixture is cast between the anchoring points. The final step is to differentiate the cells into muscle tissue. Ultimately, electrical stimulation can be used to induce contractions of the cells and fluorescent staining and fluorescent or confocal microscopy can be used to detect the sarcomeric organization of the cytoskeleton in the differentiated muscle cells.
The main advantage of this technique over existing methods is that muscle cells will align in one direction in these tissues. This technique also has implications for patients that suffer from muscle loss. If we, for example, use patient's own cells in future to make these tissues, these can be used for transplantation purposes.
This method provides insight into the development of muscle and muscle damage, but it'll also provide insight for novel applications like regenerative medicine and e vitro cultured meat. Demonstrating the procedure will be arian Methods for isolating and freezing mirin. Myoblast progenitor cells have been described elsewhere.
Please consult the written protocol for full references. After retrieving the vial of mouse myoblast progenitor cells from liquid nitrogen. Place the vial in a water bath at 37 degrees Celsius to thaw.
Then add 10 milliliters of warmed growth medium to a 25 centimeter squared matric gel coated tissue culture flask. As soon as the cells are thawed, add the contents of the vial to the media. In the tissue culture flask incubate the cells at 37 degrees Celsius and 5%carbon dioxide.
On day three of incubation, harvest the cells with trypsin, ization, and centrifugation. At 1000 RPM then passage the cells into a 75 centimeter major gel coated tissue culture Flask split the cells every three days, and on day six passage the cells into a 150 centimeter squared matri gel coated tissue culture flask. On day nine, transfer the cells from the 150 centimeter squared matri gel coated tissue culture flask into a triple 150 centimeter squared matri gel coated tissue culture flask or three 150 centimeter squared flasks.
On day 12 of incubation, the cells are ready for seeding into a muscle construct. Begin by cutting segments of Velcro of approximately five millimeter squared with one edge in a triangular shape as illustrated by this schematic, only the soft side of the Velcro is used. Then prepare silicon glue by mixing the elastomer with the curing agent in a 10 to one ratio.
Then glue two pieces of Velcro into each well of a six well culture plate, leaving approximately 12 millimeters between the pieces and leave to dry overnight. The next day, sterilize the wells and Velcro by adding 70%ethanol to the wells and incubating for 15 minutes. Next, rinse the wells three times with PBS.
Leave the third wash in the wells and expose the plate to UV light for 15 minutes. Then aspirate the PBS and transfer the plates to the tissue culture incubator until ready to use. Prepare a solution of 3.2 milligrams per milliliter, collagen and sterile 0.02%acetic acid.
Place the collagen solution on ice along with matrigel solution that has been previously thawed in the refrigerator. Next, harvest the cells by trypsin, ization, and centrifugation. Then resuspend the cell pellet in growth medium and perform a cell count according to standard procedures.
Once the cells have been counted, place the tube containing the cells in the tissue culture incubator. The matrix solution is prepared by mixing the solutions on ice in the following percentages, 51.3%Collagen solution 0.2%0.5 molar sodium hydroxide, 8.6%matrix gel, and 39.9%growth.Medium. Ensure that the final solution is pink in color as an increase in pH can result in rapid ation.
The final volume depends on the number of wells to be plated. This table shows the volumes for 10 muscles. Ensure that the cells are fully resuspended and transfer 750, 000 to 1, 250, 000 cells per construct to a fresh centrifuge tube centrifuge at 1000 RPM for five minutes, aspirate the supernatant and resuspend the cell pellet in a small but sufficient volume of the matrix mixture.
Then once the pellet is fully resuspended, add the cells to the remaining volume of matrix mixture. Now after retrieving the prewarm plates from the incubator pipe at 0.35 to 0.4 milliliters of the cell matrix mixture into each piece of Velcro. Then begin to pipe at the mixture from the center between the attachment sites and extend to the Velcro.
Finally, use the remainder of the gel to fill up the gap between the two Velcro anchors and pipette around the Velcro pieces. Cover the plate and leave it room temperature for five to 10 minutes. Then gently tap the dish and visually inspect to see if the gel is rigid.
If so, the dishes can be gently transferred to the tissue culture incubator. After one to two hours in the incubator, gently overlay each gel with four milliliters of prewarm growth medium. Then return the plate to the tissue culture incubator.
After 24 hours, replace the growth medium with differentiation medium. Repeat the media change every two to three days. Mature oriented muscle fibers should be apparent on day seven of culture.
At this point, the fibers can be electrically stimulated. First, sterilize the electrodes from the ion optics plate with 70%ethanol for 15 minutes and dry under UV light for 30 minutes. Then place the sterilized electrodes onto the culture plate, ensuring that the electrodes are placed parallel to the muscle construct as shown in this picture showing the bottom of a plate, the white arrows indicate the electrodes cover the plate with its lid and transfer to the tissue culture incubator.
Connect the ion optic C pacer with the appropriate cables and apply the stimulation protocol here. Four volts per centimeter. Six millisecond pulses at a frequency of two hertz are used.
Electrical stimulation is usually done for a period of 48 hours. Change the culture media every 24 hours during stimulation. This image shows a typical example of a mature muscle construct.
The size of the tissue is approximately eight millimeters long, two millimeters wide, and 0.5 millimeters thick. This image shows a typical example of a muscle progenitor cell in engineered skeletal muscle tissues. Frozen sections of non stimulated mirroring bio artificial muscles or M bms.
At day eight of differentiation were stained for sarcomeric alpha actinin shown in red, sarcomeric myosin shown in green and nuclei shown in blue. The panels on the right are magnification of the boxed areas. The protocol can also be used on C two C 12 cells.
This image shows differentiated C two C 12 cells stained in the same manner as shown in the last image. Sarcomeric alpha actinin is shown in red. Sarcomeric myosin is shown in green, and nuclei are shown in blue.
Again, the panels on the right are magnifications of the boxed areas. While attempting this procedure, it's important to remember to keep everything on ice. Following this procedure, other methods can be performed to answer additional questions like quantitative PCR to demonstrate changes in gene expression levels After its development.
The technique paved the way for researchers to study the role of mechanics in tissue morphogenesis. It also aided in the design of engineered cardiac muscle models. After watching this video, you should have a good understanding of how to generate engineered muscle tissues using a hydrogel based scaffold.
