This procedure begins with the harvest of skeletal muscle tissue from Neonatal Lewis Wraths. From this tissue we isolate myoblast cells. These myoblasts are cultured on tissue culture dishes for two days and then harvested and mixed into a collagen based tissue construct mixture.
This mixture is carefully pipetted into a mold submerged in culture medium and incubated at 37 degrees Celsius. Hi, I am Christina Pesach from the laboratory of Douglas Cowan in the Department of Anesthesiology at the Children's Hospital, Boston and Harvard Medical School. Today we will show you a procedure for creating myogenic engineered tissue.
We use this procedure to fabricate an engineered tissue for insertion in the heart. Our goal is to create an electrical connection between the upper and lower chambers of the heart as an alternative therapy to cardiac pacemaker implantation in children with complete heart block. So let's get started.
In The construct, casting mold is used to provide a form for the surgically implantable collagen based tissue construct. In this video, the process of creating engineered tissue constructs containing skeletal myoblasts is described in an accompanying video. The cardiac implantation of the cellularize tissue made in this protocol is demonstrated.
A tissue mold can be made into virtually any shape or size to meet the physiological requirements of any heart for which the tissue is designed. In this demonstration, the mold will form a piece of tissue that can be sliced to fit into the AV groove of an adult rat heart. Begin constructing the mold by using a razor blade to cut the silicone tubing into three centimeter long pieces and then slice each piece in half lengthwise.
Cut a small piece of polyester mesh. Next place a drop of implant grade RTV silicone adhesive on the inside of the very end of the tubing. Try to keep the amount of adhesive deposited to a minimum because it is somewhat toxic to cultured cells.
Quickly place a small piece of polyester mesh on the silicone adhesive drop and align it with the end of the tubing. This will provide a slightly raised and flat surface for construct attachment. Repeat the process for the other end of the tubing.
Allow the mold to dry at room temperature for three days. It is helpful to make 20 to 30 molds at a time as these are essentially disposable. Once the adhesive is completely dry to help sterilize the molds, store them in a container filled with 70%ethanol until they are needed with the molds prepared.
Next, determine whether all the required solutions are also ready to use To prepare for cell isolation. Make a diluted laminin solution of one milligram laminin in 250 milliliters 0.2 micron. Filter sterilized PBS containing antibiotics and a fungicide.
Next coat, 150 millimeter tissue culture plates with four milliliters of the diluted laminate solution and incubate them at 37 degrees Celsius for at least four hours before use. Two other solutions must also be prepared. The myoblast medium and the skeletal muscle digestion solution.
Refer to the text portion of the protocol for their formulations.Filter. Sterilize the digestion buffer by using a 0.2 micron filter disc fitted onto a 10 milliliter syringe. Aliquots of the digestion buffer can be stored at minus 20 degrees Celsius.
Estimate that you will need five milliliters of digestion buffer per Lewis rat litter. Incubate the required amount of tissue digestion buffer, and 10 milliliters of myoblast medium at 37 degrees Celsius. And proceed with dissecting the rat pups.
First, obtain at least one litter of sacrificed neonatal Lewis rats using forceps and small scissors. Remove paraspinal muscles from dead neonatal Lewis rats by slicing down the back along the spinal cord, peeling back the skin and fascia layers and then excising the muscles. Place the excised muscles in a 50 milliliter conical tube filled with 40 milliliters of one X-H-B-S-S on ice.
Allow the muscle strips to settle to the bottom of the tube and carefully remove most of the liquid and blood remnants by vacuum suction with a sterile pasture pipette in a culture. Next, pour the harvested muscle pieces onto a 150 millimeter culture dish and aspirate away as much of the remaining liquid as possible. Using two single edged razor blades, mince the tissue to a thick paste.
The muscle paste will be digested in pre-warned digestion solution. Pour some of the digestion solution onto the muscle paste and transfer the solution and muscle paste mixture to a fresh 50 milliliter tube. Using a 10 milliliter pipette generally for efficient digestion of muscles from three liters of Lewis Rats, we use 10 to 15 milliliters of solution.
Place the tube on a rocking platform and digest at 37 degrees Celsius for about 30 minutes. During the enzymatic digestion, the tissue is occasionally trid without bubbling. With a 10 milliliter pipette fitted onto a cordless pipette.
Once the muscle is liquified, pass the solution through a 70 micron cell strainer. Then centrifuge the filtered cells at room temperature for 10 minutes at 600 G with a pasture pipette and suction, aspirate as much of the liquid as possible and resuspend the pellet with 10 milliliters of warm myoblast.Medium. Prelate the cells on an uncoated 150 millimeter tissue culture plate for 15 minutes to help remove any fibroblasts as they attach faster than myoblasts.
Place the plate in a humidified incubator set at 37 degrees Celsius with 5%carbon dioxide. Remove the plate from the incubator and transfer the cells that have not yet attached to the Lamin coated culture. Wear and dilute them in myoblast medium so that there is one plate for every one to two rat pups.
If the cells are plated too densely, they will differentiate into myo tubes. Lastly, allow the cells to grow in a humidified set at 37 degrees Celsius with 5%carbon dioxide. After one to two days of incubation, the cells will be ready for casting into tissue constructs.
After one to two days of incubation, remove four of the 150 millimeter myoblast tissue culture plates from the incubator. Do not wait longer than 48 hours or fibroblasts will overwhelm the myoblast population. Aspirate the medium from the cells.
Then rinse the cells twice with one XPBS warmed to 37 degrees Celsius to detach the myoblast from the plate and four milliliters of warmed 0.05%tryin EDTA solution to each plate and return the plates to the incubator for five minutes. Harvest the myoblast in the culture hood and collect the liquid in a 50 milliliter tube. Use 10 milliliters of myoblast medium to rinse all of the plates and combine this with the cells in the centrifuge tube.
Pellet the cells at room temperature for 10 minutes at 600 G and aspirate off as much of the excess liquid as possible. Now re suspend the myoblasts in 1.4 milliliters of myoblast medium and place them on ice. Using forceps carefully, remove a construct mold from the ethanol bath and rinse it twice by submersion into one XPBS aspirate all traces of PBS with a pasture pipette before placing the mold in an empty well of a six well tissue culture plate.
At this time, a solution must be formulated. Collect the following reagents on ice. 10 x hams F 10 fungi zone penicillin streptomycin.
Type one collagen cells resuspended in myoblast, medium sodium bicarbonate and gel. Combine the reagents according to the written protocol. Vortex the mixture for 20 seconds it will turn pink.
Note that the varieties of type one collagen that are commercially available have a variable effect on mixture, solidification, and consistency. In the final product. Return the tube to the ice bucket for a few minutes to clear the solution of bubbles.
It is that the type one collagen is not irradiated or it will not gelatinize. Here we use a product derived from ovide skin. Before the mixture solidifies carefully cast the mixture into the mold in the culture plate.
Each mold will hold approximately one milliliter of the viscous liquid. Try to avoid introducing air bubbles during this procedure. Carefully transfer the plate containing the construct molds to the 37 degrees Celsius incubator.
As the mixture solidifies, it will change from a dark pink to a light pink in color while the mold solidify warm some myoblast medium to 37 degrees Celsius. After about half an hour of incubation, carefully submerge each tissue construct in warmed myoblast medium and return the plate to the incubator. When this protocol is properly executed, the myoblast containing tissue construct is ready for in vivo implantation or for further analyses.
After two days of incubation, We've just shown you how to fabricate a myogenic tissue engineered construct. When doing this procedure, it's important to remember to keep all of the reagents necessary for the construct solution on ice to prevent premature solidification. So that's it.
Thanks for watching and good luck with your experiments.
