I'm working on myogenic differentiation with c2c12 and I've noticed a lot of figures (c2c12 myotube) searched on Internet have their cells aligned in single direction (without any messy clumps of cells or myotubes stretching in irregular alignment).
This is my protocol for myogenic differentiation,
1) Change into Diff. media (DMEM, 2% horse serum, 1% Penicillin/Streptomycin) after reaching fully confluence (95~100%)
2) Change diff. media every single day and differentiate cells for 0~6 days.
and I've always had myotubes clumping and stretching out in irregular direction/alignment.
Does full/over confluence disrupts cells aligning in single direction? If so, is it better to start differentiation before your cell culture reaches full confluence?
This is out of my area of expertise but I have read in numerous papers where application of cyclical uniaxial stretch to muscle cell cultures results in the myotubes being parallel to each other. From what I understand, there are commercially-available devices for doing this.
You can also look into microgrooved culture dishes for muscle cells… ive seen collagen and gelatin-based protocols. I’m not sure if they are commercially available, but they’d probably be the least technical option out there…
Have a look at https://www.curibio.com/nanosurface-plates. Very expensive but work really nice to get aligned cells. Good luck!
As suggested above, it may have something to do with the culture well surface but I never faced such an issue. Generally, I have observed unidirectional, huge and contracting myotubes for primary Human and Bovine muscle cells. However, C2C12 differentiated slightly less but had same pattern. I would add diff media (same as yours) at 80% confluence and change media every other day (after 48 hours). This just helps in better differentiation of myoblasts. I also suspect that this could be due to uneven spread of cells in the plate as this will help better fusion and nice parallel myotube formation. I hope this helps, send a pm if you would like to discuss further.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line