Hi Umar,

In my experience, hyper contraction of the fibers is always linked to the collagenase digestion. If the digestion is too strong the fibers exiting the muscle are/become hyper contracted. On the contrary, if the muscle is not digested enough you damage the fibers when trying to dissociate the fibers with the Pasteur pipet and the fibers become hyper contracted. To know if the digestion is ok, you should see alive fibers coming out spontaneously from the digested muscle.

I don't know if your muscles are too/not enough digested but you may consider to modify the time of digestion (30->90min) and/or your incubation conditions (agitation in water bath strongly increases the efficiency of the digestion). After the digestion, I also used to transfer the muscle in DMEM 10% FBS and to keep it for 1h in the incubator (an advice I got from Zammit lab). 

I have also heard that high concentration of serum may induce hyper contraction of the fibers. I don't know if it's true but I have always let the fibers in DMEM 10% FBS for few hours before changing for a proliferation medium (DMEM with 20% FBS, 1% chicken embryo extract).

Good luck

From my experience, if you touch ANY part of the muscle during the dissection, the fibers will end up hyper contracting. Additionally, when you transfer the individual fibers, coating pasteur pipettes in 5% BSA. Good luck!

Ultimately, the reason is that you are being too rough with the fibres at some stage in the procedure. As Alyson says, the commonest point at which this happens is with pipetting and the commonest mistake is to pick up the fibre in such a way that it bends in the pipette.  You should always pipette very gently - one of the major unrecognized stresses is cavitation as the fluid leaves the pipette tip.

if you continue to have trouble, then you may save a lot of time and money by visiting Peter Zammit's or Jenny Morgan's lab in London.