Hello,
I have no prior experience with growing cell line nor differentiation toward specific cell type. Currently I have C2C12 cell line which I need to grow and then differentiate it towards skeletal muscle cells.
Can you please provide me with as much as possible information, SOP, tips and tricks?
Thank you
There are commercial kits like Skeletal Muscle Cell Growth Medium available for skeletal muscle maintenance. You can also prepare a Growth medium that proliferates and maintains C2C12 cells in DMEM high glucose medium containing 10% FBS and 1% Pen-Strep. For differentiation; You can use ready-made kits. Bu ı prefer home-made medium to myoblast differentiation. When the cells become over-confluent, I switch the Growth medium to a Differentiation medium which includes DMEM with 2% DHS and 1% Pen-Strep. I can observe myotube formation within a few days.
A trick to the maintenance of myoblast cells; You should not forget to subculture the cells before they become too confluent. Sometimes cells can begin to differentiate themself at high density even if they are not induced by a Differentiation medium. Cells that differentiate into myotubes cannot regain myoblast characteristics and their proliferation stops.
This cell line is really easy to maintain. I hope you can handle it.
Thank you very much Duygu Sevim for your answer.
At which day do cells begin their differentiation, or it is based on looking at them not to become over-confluent? I suppose this will come to me easily after few times of growing them. Also let us say that they begin the differentiation themselves, how can I actually check this, via microscope observation?
Once the cells become over-confluent and switched in the differentiation medium, they differentiate within 4-5 days at most. Of course, since this process varies from cell to cell, I cannot say an exact time. Starting to self-differentiate is a more time-consuming process than being induced by DHS.
When cells differentiate, myotube structures that are easily noticed under the microscope begin to form. I don't think you have a chance of missing the myotube structures. Myotubes look like hollow cylindrical structures.
Duygu Sevim if you have information regarding the thowing, growing and freezing SOP, could you share them ?
Hi Aleksandar Dishkelov
Thanks for the question.
I have been working with the C2C12 cell line for quite a while and I can share my protocol which might help you.
Typically, I prepare two types of media which are as follows:
1. Full media: DMEM + 10% FBS + 1% Pen-Strep + 1% L-Glut
2. Differentiation media: DMEM + 1% horse serum + 1% Pen-Strep + 1% L-Glut
For thawing, Take out the C2C12 vial from the liquid N2 dewar and thaw it in a 37°C water bath for a few seconds. Keeping in mind, not to over-thaw the cells. Take out the cells from the water bath when you start seeing the transition of frozen cells from a solid to a liquid state. Transfer the cells in pre-chilled full media (I usually take 50ml media for a single vial). Centrifuge the media containing cells (1200rpm for 5min at 4°C). Discard the supernatant and re-suspend the cells using fresh full media. Transfer it to the cell culture flask (t25, t75) and let them proliferate in the incubator set at 37°C and 5% CO2. To avoid contamination, remember to perform this procedure in a biosafety cabinet except for the water bath step.
For differentiation, once the C2C12 cells become 50-60% confluent, plate them in the multi-well plate (You can optimise your experiment by plating different concentrations of cells. For me, 5 * 10^5 cells/ml works fine)
a.) On day zero (this is the day when you plate your cells), plate the cells and put the plate in the incubator (37°C and 5% CO2) for the next 24 hours.
b.) On day 1, check the cells under the microscope. If you see 90-100% confluent cells, remove the media, wash the cells twice with PBS and add differentiation media (I explained the composition in the beginning). Put it back in the incubator.
c.) On day 2, check the cells under the microscope. (you'll see some differentiation)
d.) On day 3, remove the media and wash it twice with PBS. Add fresh differentiation media. (small myotubes will be seen)
e.) On day 4, large myotubes will be observed.
f.) On day 5, fully developed myotubes will be seen.
Note: this is my observation with 5 * 10^5 cells/ml. the differentiation time may vary with changes in the concentration of cells.
Cheers,
Anki
Ankush Dehlia Thank you very much for sharing your experience! Much appreciated for dividing the protocols day by day.
Ankush Dehlia Can you please also include which DMEM you are using, if the glucose is high, low or normal?
Thank!
Ankush Dehlia Hi, I have been working on C2C12 differentiation for almost 2 months. Sadly, the differentiation rate is low, can you please give some potential reasons to improve it? The differentiation protocol is totally the same with yours. As far as I observed low-passage cells tended to differentiate in a higher proportion, but still only 20-30% in my experience. I will appreciate your help very much.
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