Hi Sasha,

We have isolated tenocytes from mouse tendon and human tendon routinely. I am enclosing 2 documents.. That should do your job. If you have any question, you may send message, to me,

Good luck,

Subhash

Hi Alexandra,

When doing my PhD we would dissect the tendon as follows:

Fresh rat tendon was harvested from 8 week old, adult Sprague-Dawley (SD) which were sacrificed in accordance with University policy. The rat Achilles tendons were then surgically removed from both hind legs. The tendons were then placed in a universal bijou bottle containing a solution of PBS supplemented with 1000 IU/ml penicillin, 1000μg/ml streptomycin and transported to the laboratory. The tendon was then removed from the transport solution in a Biological Safety Cabinet and placed into a sterile petri dish were the tendon was then subsequently dissected into smaller sections and minced using a scalpel. The petri dishes containing the tendon samples were then placed into an incubator for one hour, allowing the tendon samples to loosely adhere to the petri dish surface. DMEM media supplemented with 1% L-glutamine, 1% NEAA, 10% FBS was then carefully added drop wise to the petri dish until the tendon samples were immersed. After, approximately five days, cells were observed to migrate out of the tendon onto the surface of the petri dish.

The cells in the petri dish were then removed by trypsinisation (1% trypsin/PBS solution) for five minutes, pelleted by centrifugation (three minutes, 200g), then re-seeded into two T-25 tissue culture flasks. One flask was then cultured at 2% O2 and one flask in 21% O2 (Keele University). All flasks were cultured at 21% O2.

Hope that helps.

well there are 2 or 3 methods you can use

Method 1 is the outgrowth from dissection that takes 3-7 days as I described earlier. I know this works 100%.

the method you are using I would not change the media as any cells would be in the media if the tissue has digested enough.

You could utilise a cell strainer to separate the remaining tissue and sound cells in the solution? and also place the remaining tissue onto 6 well plates utilising the method i described earlier.

A method I tried yesterday which yielded 125 million cells from 5 g of tendon is as follows.

0.4% pronase digest for 2 hours followed by an overnight digest in collagenase 0.08%

pronase step

80 mg pronate, 400ul hepes, 40ul gentamicin, then take to 40ml with DMEM.

place on roller at 37oC for 2 hours.

remove digest solution:

then place tissue overnight on roller at 37oC in collagenase solution:

24mg collagenase, 400ul hepes, 2ml FBS, 40ul gentimicin and top up to 40ml with DMEM.

post overnight digest I utilise a 40um cell strainer (falcon) to remove debris then I seed at 1000 cells per cm2.

are you after tenoprogenitors or mature tenocytes?

I then profiled utilising tenomodulin and thrombospondin-4 antibodies to confirm (based on Jelinksy paper ).

Hope that helps.

Richard

Another method is cell out growth from small tissue samples