Can you add here the protocol you have used? 

Hi, Are you sure you plated sufficient live satellite cells? If you have a very few number of viable cells, fibroblasts will grow quickly and easily in serum growth medium. These fibroblasts will dominate in culture and naturally your satellite cell growth will be suppressed and eventually die.

I suggest you use low serum and make sure your growth medium has D-Valine and not L-Valine. Fibroblasts require L-Valine for growth. This may not completely rid you of fibroblasts but should limit them.

Yes Laura, in my protocol I used F-10_Gibco_life technologies with 10% FBS.

Hello Elizabeth, I use 10% FBS in my growth media. Could you tell me low serum mean how much? About Valine, my medium contain L-valine and other protocols that I refered also use F-10 medium and FBS was added then. So I don't know which medium I can use to replace.

Hello Chandra, usually the first 2 or 3 days I don't have too many satellite cells and also fibroblast are just a few. Then from that time, satellite cells start to growth, also beside that fibroblasts they growth crazily. My satellite cells still can growth fast after that but the problem is when i seed the cells, my plates were always contaminated fibroblasts and their number make me worry about my results so I was not confident to continue.

My brief protocol:

1. Sacrifice 2 adult mouse (3-8 weeks) and collect all the muscle from hind limbs

2. Remove all the connective tissue and wash 2 times

3. Chop the muscles into small pieces 

4. Incubate with collagenase II for 1h20 minutes at 37 °C with shaking

4. Remove the enzyme and incubate with enzyme mix collagenase II and dispase II in 20 min

5. Dissociate more by sysinge and incubate for 10 more minutes

6. Centrifuge to remove fiber debris and filter by cell strainer

7. Wash the cells 2 times

8. Plate on regular plate for 1 hour to remove fibroblasts. Do this step twice

9. Plate on type I collagen coated plate 

I think the point is that you have to culture satellite cells for a longer time to get rid of the fibroblasts. I would try to have the culture between 50% and 80% confluency so that the fibroblasts start having problems with space while the satellite cells can still proliferate. Also, I would repeat the preplating on a non-collagen plate every time you split the cells.

But these are only some Ideas since I have the problem that my satellite cells do not even attach on the plate :)

In my experience, after culturing for a long time the cells simultaneously going to differentiation or change the morphology. And they just growth in clump so I can not determine the confluence. I shall consider to repeat the preplating on a non-collagen plate every time splitting the cells.

Thank you a bunch everyone.!!!

Another thing I just realized we use bFGF (5ng/ml)  in the culture that helps the myoblast to proliferate. Could be that this might help to increase the proliferation of the myoblasts over those of the fibroblasts.

Hi

Supplementing basic FGF is well established proliferation factors for many cells including MSCs (which is being called as fibroblast origin by some groups), So definitely bFGF supplementation have no selective advantage.

While complete digestion of muscle with enzyme, the fibroblast contamination is unavoidable. Try to sort the cells using specific markers, if you r more concern about contaminating cells. or

 Try once the fiber culture, have seen showing better cell phenotype than the complete digestion samples.

We have seen after passage 1, if u seed the cells in uncoated flask,(so called preplating) even the satellite cells are adhering to them ( i think in vitro culture leads to satellite cells get property to grow in uncoated flask). .

i m curious like all, to get if any other option is there. ;)

What do you mean with "fibre culture"? Cultivating the myoblasts attached to the fibre? This method can work but you should be aware of the fact that satellite cells you obtain by migrating away from the fibres have other properties since they are activated and might already change their program.

Another option but I have not tried yet is indeed positive or negative selection. negative selection might be more comfortable since you get rid of all the cells except the satellite cells. There are also nice Kits to help you with the isolation e.g.

http://www.miltenyibiotec.com/en/products-and-services/macs-cell-separation/cell-separation-reagents/various-cell-types/satellite-cell-isolation-kit-mouse.aspx

so u mean??? the cell isolated from complete enzyme digestion wont get activated from quiescent. ??? 

If you plate the fiber as well as cells (isolated from complete enzyme digestion) in ECM coated flask, the satellite cells get activated and under go proliferation.

Yes the negative the selection is one option, But the cost of kit is Rs. 2,00,000 (close to $.3000). If u r tried/going to try, please post ur experience, it will be helpful for other.

Thank you Denise and Karthikeyan.!!

Actually, I tried the fiber culture already before. However, as Denise has mentioned, they started to going out of fiber and change their properties. They were not round shape anymore. I think this technique we just should use in determining the cell dividing.

And one more thing, we plan to do sorting but double positive sorting. Hopelly they work well. I will update my experience about that. :-D

Thank you everyone.!!!

Hey so I have done my recent isolation and it's nearly clean of fibroblast. I did it that way: After isolation I plated them for 2h on a non-coated dish. After that the supernatant was transferred to a collagen dish. I waited for another one day and then transferred the supernatant yet on another collagen plate with 1h of preplating . After that my culture was already very clean only very few fibroblasts left. Of course, you loose some cells but this way it works very well for me.

I attached two pictures showing my culture in the moment. Sorry for the quality I just took them very fast with my mobile phone. :)

Hello Denise, 

I'm wondering now how can you still have cells in the supernatant after plating on collagen-coated plate? for me after putting into the collagen plate, just some cells still floating, almost all attach to the plate. 

Hey,

Actually,  they do not attach before 2days after isolation in my culture. I have two cultures in parallel that I finally mix together when I'm done. I attached a scheme of the plan that was given to me and works well for me:

After isolation I preplate for 1-2h on a noncoated dish (1) after that i put the supernatant on a coated plate (2) and put fresh medium on plate (1). The next day I transfer again the supernatant from plate (1) this will be now (3). in parallel I add some fresh medium to plate (2). two days after isolation No (3) gets new medium and the supernatant of plate 2 goes on plate (4).

After that you have to see which plate gives the best results. The next two splitting steps I pre-plate after that nearly all fibroblasts are gone.