Isolation of limb muscle from neonatal mice
1. Neonatal mice by decapitation or CO2 inhalation.
2. Rinse
the limbs with 70% ethanol and remove them with sterile scissors.
Dissect the muscle away from the skin and bone with sterile forceps.
Dissection
is easier if done under a stereo dissecting microscope. Store the
muscle tissue in a culture dish on ice in a drop of sterile PBS as
successive limbs are being processed, maintaining sterility in the
accumulated tissue.
Dissociation of muscle cells
3. Add enough PBS to keep the tissue moist, and mince to a slurry with razor blades in the culture dish.
Do this and all subsequent steps in a sterile tissue culture hood.
4. Add approximately 2 ml of primary cell isolating solution per gram of tissue and continue mincing for several minutes.
For the amount of tissue obtained from 1-5 neonatal pups, 0.5 ml of the enzyme solution is usually sufficient.
5. Transfer
the minced tissue to a sterile tube and incubate at 37°C until the
mixture is a fine slurry, usually about 20 min. Several times during the
incubation, gently triturate with a plastic pipette to break up clumps.
6. If
desired, the slurry can be filtered through a piece of 80 µm nylon mesh
in a sterile funnel to remove large pieces of tissue.
7. Centrifuge
the cells for 5 min at 350 x g. Resuspend the pellet in 2-4 ml of
primary myoblast growth medium depending on the amount of tissue
processed, and plate in a 35-60 mm collagen coated culture dish.
It
is common to see a great deal of debris and very few recognizable
cells; however, after two days, many cells will be evident and the
debris will be rinsed away during the first change of medium.
Supplement for myoblasts
8. Incubate in a 37°C 5% CO2 incubator
and change the medium every 2 days, using primary myoblast growth
medium. Do not leave cells in the same culture dish for more than 5
days, regardless of cell density.
Primary myoblasts grow best
when dense. Do not grow them at less than approximately 10% confluence,
but also do not allow them to become too crowded, or else they may
start to differentiate or die. Split them at no more than 1:5 dilution.
The primary myoblast growth medium gives myoblasts a growth advantage
over fibroblasts.
9. When the cells are ready to be split,
remove the cells from the dish using PBS with no trypsin or EDTA. This
is accomplished by aspirating off the growth medium, rinsing the dish
with PBS, leaving a small amount of PBS in the dish, and hitting the
dish very firmly in a sideways fashion against the edge of a table top
to dislodge the cells.
It can take several minutes of
incubation in the PBS before the cells can be knocked off of the tissue
culture plastic. However, the myoblasts will freely come off during this
treatment and many fibroblasts will be left behind.
10. Preplate
the cells for 15 min on a collagen coated dish before moving the cells
remaining in suspension to a new collagen coated dish.
This tends to
leave behind cells that stick quickly, which are predominantly
fibroblasts. To avoid leaving myoblasts behind, swirl the liquid in the
dish, tilt it towards the pipette, and jostle the dish from side to side
while slowly removing the cell suspension. Failure to do this can
result in a significant number of myoblasts trapped in the meniscus.
11. Repeat steps 9 and 10 for the initial week of culture expansion or until most of the fibroblasts are gone from the culture.
Fibroblasts
tend to be very flat when grown on collagen, whereas myoblasts are more
compact and much smaller in diameter. Myoblasts can also be identified
by immunofluorescent staining for desmin.
12. After the fibroblasts are no longer visible in the culture, the medium can be changed to primary myoblast growth medium.
This
medium allows myoblasts to grow faster and to higher densities.
However, it also removes the selective growth advantage of myoblasts
over fibroblasts, and thus should not be used until after the
fibroblasts are gone. This medium can be changed every three days
instead of every two, but care should still be taken to avoid overgrowth
of cells. Primary myoblasts can be frozen for storage using standard
cell culture protocols.
After approximately one week of growth,
the myoblasts usually go through a crisis period during which a
significant fraction of the population (sometimes the majority) dies.
This tends to occur at the time that the fibroblasts have disappeared
from the culture, although a direct correlation has not been confirmed
in our laboratory. The remaining myoblasts soon repopulate and have a
remarkable proliferative ability (up to 100 doublings) but still retain
the ability to differentiate under the proper conditions.
Differentiate in culture
13. If
desired, the potential of the myoblasts to differentiate in culture can
be assessed by replacing the medium with fusion medium.
The
medium must be changed daily. Start with an approximately 30-50%
confluent dish of cells. Within several days to one week, large
multinucleated myotubes should be quite obvious. After approximately one
week in fusion medium, the myotubes can sometimes be observed to twitch
randomly as the contractile machinery assembles.
Reference
Springer,
M.L., T. Rando, and H.M. Blau (1997). "Gene delivery to muscle." In
Current Protocols in Human Genetics. Unit 13.4, A. L. Boyle, ed. John
Wiley & Sons, New York.