Harvest and culture of mouse primary myoblasts
-written by Matt Springer while in the lab of Dr. Helen Blau at Stanford
Does it matter from what age mice I isolate myoblasts?
You can isolate myoblasts from mice of any age. However, we have had our best results with neonatal mice about 1-2 days old. As they age, the amount of muscle certainly gets larger, but less myoblasts are isolatable from the tissue.
I isolated mouse myoblasts and I've got cells, but instead of being nice and flat, they are all rounded up. Should I throw away the dish?
Nope, that's exactly what you want to see. With time, those balls (which I think of more as truffles because they have the little "skirt" down at the bottom like a chocolate truffle) will proliferate like crazy and later will flatten out a bit and become more like short spindle-shaped cells, sometimes triangular. If they become long needles, that means that they are trying to differentiate; you don't want to see that because it means that they are starving or otherwise unhappy. Keep them in the F10 with 20% FBS and 2.5 ng/ml bFGF until you don't see those fibroblasts anymore, then you can switch to the 50:50 F-10/DME with 20% FBS and 2.5 ng/ml bFGF. If you hit them with the DME/horse serum differentiation medium, they'll differentiate no matter what stage of the isolation they are in.
Hey, I tried using that F10 medium but my primary myoblasts had lots of blebs; they looked really unhappy. What gives?
Yeah, they can look that way sometimes, but it doesn't appear to affect them.
I had this great dish of cells, but then they stopped growing and some of them actually died. They've been sitting there for a week! What should I do?
Just keep your fingers crossed and wait a bit longer. The myoblasts frequently undergo a crisis period after most of the fibroblasts have been removed from the culture. They will stop growing and a large number may die. If this happens, don't be afraid to trypsinize them and move them to a smaller dish accordingly, to prevent them from being too sparse. They usually come out of it. It is possible that this represents the outgrowth of a subset of cells, but that population still grows and differentiates under the proper conditions.
I've been trying to get my primary myoblasts (or my C2C12 myoblasts) to differentiate by putting near-confluent cultures in differentiation medium, but I haven't seen very many myotubes. What can I do?
If your cells are too confluent when you put them in differentiation medium, they are more likely to overgrow and die than differentiate. This is because the cells still grow for a bit after you put them in the new medium. Ideally you want the cells to be about "70% confluent", but this means different things to different people and is not even all that consistent for an individual investigator. The failsafe way to get your myoblasts to differentiate is to split them into all of the wells of the 6 well dish, with the first well being almost confluent, the last well being 10-fold diluted from the first, and the intermediate dishes being at intermediate densities evenly spread from the first to last. That is, if plating 1 ml of your cell suspension makes the first well be almost confluent, then seed 0.1 ml in the last well, and give the others 0.2, 0.4, 0.6, and 0.8 ml respectively. Usually you will see a cell death in the first well, sparse individual needles in the last couple of wells, and some great myotubes somewhere in the middle. You'll also notice that the myotubes in the different wells will start twitching at slightly different times, with the more-dense starting before the less-dense.
By the way, this seems to vary by investigator, but I have found that 2% horse serum in DMEM works well for differentiating C2C12 myoblasts, and 5% horse serum in DMEM works well for primary myoblasts.
I don't REALLY need to grow my primary myoblasts on coated dishes, do I?
YES, you DO. Collagen is our usual standard, but laminin, Matrigel, and "E-C-L" matrix (Upstate Biotechnology, NY) all work as well. Growing them on uncoated plastic is not advised.
Has anyone ever written a song about myoblasts?
Glad you asked that! Check out The Myoblast Song by Matt Springer. Download it and play it for your myoblasts. Show them you care.