The overall goal of this procedure is to isolate and culture single live fibers and their associated stem cells from the EDL muscle of adult mice. This is accomplished by first dissecting out the EDL using a tendon to tendon dissecting procedure that does not damage the fibers. Next, a muscle digestion is performed to unbundle the fibers while preserving the myofiber stem cell Association, which is a unique step to this protocol.
In the final step, individual myo fibers are gently separated from each other while debris or hyper contracted fibers are removed by sequential washes. Ultimately, myo fibers isolated by this technique can either be kept in a semi quiescent state with minimal activation stimuli or be cultured in high serum medium for studying of satellite cells activation and differentiation. In recent years, the use of cultured mire fibers has been a very powerful approach to study the, the activation, proliferation, and differentiation of satellite cells in a relevant physiological niche.
This approach really has allowed us to make great headway in, in understanding of the, of a function of these cells at the molecular and cellular level. For each mouse from which EDL are isolated, prepare five plastic 60 millimeter Petri dishes with a coat of horse serum pipette, four millimeters of horse serum onto a dish and swirl it for even coating. Then remove the horse serum aspirating any leftover serum, and let the dish dry for at least 30 minutes.
After half an hour, add four milliliters of washing media to four dishes. Label the dishes as follows, muscle after digest, wash one, wash two, and wash three. Add four milliliters of fiber culture media to the remaining fiber culture dish.
Keep the dishes at 37 degrees Celsius in a 5%carbon dioxide incubator prior to use. Next, for one fiber isolation, prepare two sterile pipettes under a microscope. Make a large bore pipette by cutting a small bore pipette with a diamond pen so that the opening is large enough for the entire muscle to be flushed through.
The second pipette is a small bore pipette for MyFi manipulation. Now flame the pipettes to smooth the edges and for sterilization by using the flame curve, the tip of the small bore pipette. This will help handling single fibers during the isolation.
Then coat both pipettes in horse serum. Lastly, be sure to clean the microscope station and dissecting tools with 70%ethanol. For this demonstration, eight week old transgenic SV 1 29 mice.
Were euthanized. Spray the hind limbs with 70%ethanol. Then pin the animal to a support board face up.
This is needed to provide a better grasp of the hind limbs. Next, cut through the entire length of the limb and expose the underlying muscle. Remove the skin as well as any hair or fur.
Using a fine scissors, cut through the thin fascia that surrounds the muscle without damaging the underlying tissue. Expose both TA and EDL distal tendons using sharp cohan Vana. Spring scissors.
Cut both EDL and TA distal tendons. Next, with the help of forceps, hold both TA and EDL muscles by their tendons and delicately pull the muscles up towards the proximal end. At this point, you should be able to clearly see the EDL muscle just underneath the TA muscle.
Now separate the EDL from the TA muscle by pulling the two tendons in opposite directions. Avoid stretching the EDL muscle while performing this operation. As this will damage the myo fibers.
Expose the EDL tendon. At this point, it may help to remove the TA muscle to better visualize the proximal tendon. It may also help to cut off some of the connective tissue around the knee.
Once the tendon is clearly visible, use sharp cohan Venus Spring scissors to free the EDL muscle. If the procedure has been done correctly, if EDL should be able to maintain its elongated form, once removed from the limb, holding the EDL muscle through its tendons, transfer it to a prewarm two milliliter collagenase solution, and incubate in a water bath at 37 degrees Celsius. Repeat the process to isolate the second EDL and transfer it to the same tube of collagenase to an uneven digestion.
The isolation of the second EDL should be completed within five minutes of isolating. The first EDL Incubation time may need to be adjusting depending on collagenous activity, muscle size, animal age, or muscle condition. For example, muscle with a lot of fibrotic tissue may require a longer incubation time.
Also, note that agitation during this time has been shown to activate S cells During the digestion time regularly check the muscle to avoid over digestion. Stop the digestion when the muscles start to loosen up and the myo fibers become visible. Using the large bore pipette, carefully transfer both muscles to a prewarm Petri dish.
With four milliliters of washing media over digestion must be avoided as it results in hyper contracted myo fibers. To release the myo fibers, use the large bore glasss pipette to flush the muscle with warm medium until the fibers naturally start being released. Do not T tritrate the muscle as this will inevitably result in damaging fibers.
Continue releasing the myo fibers until the desired number is reached. Do not leave the dish at room temperature for more than 10 minutes. Return it to the incubator for five minutes to re equilibrate before working with it any longer.
The culture currently contains a mixture of live single myo fibers, which are long and shining tubular structures, hyper contracted fibers, which are dark and short and debris. Now using the small size bore pipette, transfer live single MYOP fibers to a new pre wormed dish. Handle each myop fiber individually.
Instead of transferring the bulk of myo fibers all at once, if necessary, return the dish to an incubator for 10 to 15 minutes to re equilibrate the medium. Transfer the single myop fibers twice more to remove all of the dead myo fibers and debris by the end of at least three consecutive washes. Only single live myo fibers should remain in the dish for culture or downstream analysis.
Live fibers have a long morphology. On the contrary, hyper contracted fibers are shorter and thicker. Now, incubate the isolated myo fibers in washing media for at least an hour prior to switching to culture medium.
In a new prewarm dish, high medium will allow satellite cell activation for long cultures. Matrigel could be appropriate regardless of the chosen medium. Change it every other day at the time of the isolation.
The satellite cells on each MyFi appear as tiny protuberances on the myofiber surface when observed under a light microscope. If the myo fibers are maintained in serum rich medium for extended periods of time, fully differentiated myo tubes are predominantly observed in the culture. Reporter mouse strains such as the MIFF five Cree Rosa YFP line are optimal tools to study the process of muscle regeneration.
Myo tubes derived from a Miff five Cree Rosa YFP fiber culture expressed the MIFF five reporter YFP. This suggests that muscle regeneration requires the activation of Miff five. The PAX seven Cree TD tomato mouse strain is another reporter line.
TD tomato fluorescence signal is detected only by satellite cells while myON nuclei are clearly negative. This suggests that the transcription factor PAX seven is uniquely expressed by muscle stem cells and not by their differentiated progeny. Myo fibers and their associated satellite cells can be fixed for downstream.
Immunofluorescence PAC seven positive myo D positive satellite cells are classically considered proliferative committed muscle progenitors. They will either complete the differentiation program by DOWNREGULATING PAC seven while maintaining my OD expression or return to quiescence by downregulating my OD and maintaining PAC seven expression. The single fiber isolation technique has the unique feature of preserving the physiological Myofiber Satellite Cells Association.
The most critical step of this procedure is the dissection of the EDL muscle from tendon to tendon. Minimal fiber manipulation also allows the preservation of fiber integrity and viability. Successful isolation of single myop fibers depend on several factors such as collagenous activity, muscle condition, or age.
Most importantly, the isolation of the muscle from tendon to tendon allows to maintain fiber integrity. Also, minimal fiber manipulation throughout the procedure allows for higher survival rate.
