The overall goal of this procedure is to isolate satellite cells from representative muscles originating from the first, second, and fourth brachial arches for their culture on an extracellular matrix. Gel spots. This is accomplished by first dissecting the massacre, digastric and levada vli palatine muscles of a young adult rat.
In the second step, the satellite cells from each muscle sample are isolated by enzymatic, digestion, and duration. The cells are then cultured on the extracellular matrix gels. Ultimately, the dynamic state of myogenesis of the satellite cell cultures can be tracked through staining for the muscle markers of interest.
Cleft lip and palate is one of the most common c craniofacial malformation in humans. When a cleft is present, surgical repair is required in order to close the defect and to allow a normal speech development, Fibrosis, and defective muscle Regeneration, however, can hamper the functional recovery of the soft palate muscles. After cleft palate repair, You can demonstrate an innovative method for the culture of freshly isolated satellite cells using exo cellular matrix gel spot coatings of just millimeter size.
This avoids the requirement for expansion. Anding these cells One day before the isolation place eight well chamber slides into a 100 millimeter Petri dish and transfer the dish to a cold surface. After 10 minutes, use a prefilled micro pipette to dispense a single 10 microliter drop of extracellular matrix gel into each well.
After chilling the slides for another seven minutes completely remove the remaining gel and dry the wells at 37 degrees Celsius overnight the next day. To dissect the massacre muscle, remove the skin from the head of a nine week old rat and use hypodermic needles to fix the head onto its side. On a silicone pad, isolate the parotid gland and the facial nerve and remove the gland.
Next, using a number 15 scalpel blade, carefully remove the facial nerve. Identify the superficial head of the masseter muscle muscle. Next, use a straight forceps to separate the tendon from its origin at the zygomatic process, followed by careful cutting of the tendon with dissecting scissors.
Using the scalpel, dissect the superficial head of the massacre until its insertion at the angle and inferior half of the lateral surface of the ramus of the mandible. To dissect the posterior belly of the digastric muscle, fix the head in the supine position with needles and remove the subcutaneous fat overlying both the sublingual and submandibular glands. Expose the anterior and posterior di gastric muscle bellies then holding the anterior tendon of the posterior belly with a straight forceps cut into carefully dissect the tendon to its origin in the tympanic buller.
To dissect the levada vli palatino muscle, localize the stylo halloid muscle. Pull it laterally and carefully remove the muscle tissue. Next, localize the tendon of the levada vli palatino that inserts that the tympanic buller and dissect it carefully.
Then lift the esophagus from behind the trachea. Identify the area of the soft palate where the levada vli palate is inserted and cut the muscle loose. Then submerge the muscle in 70%ethanol and transfer it into ice cold PBS supplemented with antibiotics.
To isolate the satellite cells, transfer each muscle into individual wells of a six well plate, and to cut the tissues into small two millimeter pieces. Taking care not to mince the muscles too much. Next, carefully incubate the tissue fragments in 2.5 milliliters of 0.1%pronase solution per well at 37 degrees Celsius for 60 minutes with gentle shaking every 20 minute.
When the fiber bundles Exhibit A loosened appearance at 2.5 milliliters of DMEM supplemented with horse serum and antibiotics to each well and transfer the digested tissues to individual 15 milliliter conical tubes. Spin down the tissue slurries and decant the supernatants. Then add five milliliters of medium to each tube and homogenize the tissue with a 10 milliliter plastic pipette.
Spin down the tissues again and transfer the supernatants into new 15 milliliter conical tubes. Then add five more milliliters of medium to the tubes and tri rate the tissue pieces again until the fragments pass easily through the pipette. After another centrifugation, pull the supernatants into the appropriate corresponding tubes and filter the cell suspensions through 40 micrometer strainers into individual 50 milliliter tubes.
Wash the strainers with one milliliter of DMEM for maximal cell recovery. Then spin down the cells, resuspend the pellets in fresh, medium, and count the cells to culture the isolated satellite cells. Next, dilute the cell suspensions to 1.5 times 10 to the three cells per 10 microliters of culture medium.
Then add 10 microliters of cells onto each previously prepared extracellular matrix gel spot, and incubate the gels at 37 degrees Celsius after six hours. Carefully add 400 microliters of culture medium to each gel spot and return the slides to the incubator. After three days, initiate the differentiation of the cells with the addition of the appropriate culture medium immediately after isolation.
More than 90%of the freshly isolated satellite cells express pack seven By day four, pack seven and MyoD are expressed by the satellite cells from all of the muscle groups. My OG is highly expressed at day seven in all muscle groups. Four days after seeding on the extracellular matrix, gel spots, the proliferating cells begin to fuse and form small multinucleated myo tubes, which express myosin heavy chain at day seven and 10.
Long and well-organized myo tubes are evident by day 10. Myo tube twitching can also be observed. This protocol allows the study of satellite cells originating from brer head muscles and offer new possibilities for the treatment of cleft pal.Patients.
