I am trying to culture the MDSCs from human skeletal muscle and the protocol I'm following is the one published by Eberli et al. in 2009 (http://www.sciencedirect.com/science/article/pii/S1046202308002016).
I'm using the same collagenase and dispase digestion, and ending up with a mass of hypercontracted fibres and mostly fibroblasts. Does anyone have any advice with respect to the handling and digestion of the muscle during the isolation so as to ensure myofibre viability? I'd be very grateful for your input!
Hi Sujata,
every lab utilizes its own protocol to isolate myogenic precursor, we use collagenase typeII (Gibco) 100U/ml 45 min a 37°C. The human muscle biopsy is kept in Hank's solution at 4°C not more than 48hours. The muscle is finely minced before digestion ( 45min at 37°C) centrifugation, pellet resuspension in aMEM 20%FBS filtered in cell strainer (100, 70 and 40um) and then plating on plastic at low confluence (1000cells/mm2).
I hope this will help you, good luck.
Hi Cesare,
Thanks SO much for your answer!
The protocol I'm using is quite similar. We finely mince the sample, digest it in Collagenase IV and Dispase, filter it through a 100 micron strainer and then plate it. We also re-plate the suspension at 24 hours. However, in both the first and second platings, we are getting predominantly fibroblasts (typical fibroblastic appearance and very high proliferation rate). Is there anyway to avoid this? After your protocol, are you able to see live muscle fibres out of which the precursors migrate?
Also, thank you for the suggestion to serially filter the suspension. I will try that and see if most of the debris gets removed.
Once again, thank you for your help!
Plating the cells at very low confluence after serially filtering avoid fibrobalst contamination, nevertheless the only way to discard fibroblast is cell sorting either fluorescence or magnetic.
Regarding muscle fibres, I do not really understand what do you mean with "live muscles fibres out of wich..." after micing and enzymatic digestion you will never collect any fibre, just a very small pieces of this. There are othere protocol using different isolation protocol the forecast single fibre plating to isolate coming out satellite cells.
Best luck
Cesare
Hi Cesare,
Thank you again for your answer. I agree with you, even I got very small pieces of fibres after digestion. However, the original protocol as reported by Eberli et al. mentions that after digestion and trituration, single fibres can be confirmed by microscopy. That's why I was checking in case anyone else has any experience with this. I'll try the serial filtering and low density plating, thank you!
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