Hi,

Different batch of Collagenase needed? Too long in Ca++ free solution making them calcium intolerant? If all the cells die, it is probably underdigested.

All the best,

Michiel

Hi Michiel, thanks a lot for your answer!

I use Worthington collagenase at now, but I have also old Sigma collagenase and would try it. Anyway, I do not really believe that the problem comes from the collagenase, otherwise I would have difficulties with rat cardiomyocytes (but I haven't). I did shorten the period of perfusion with Ca-free solution and this also did not help. As well, I have used low Ca (200 uM) with the same unsuccessful result.

I will check whether I have really underdigested tissue, thanks!

As you know, guinea pig myocytes are more fragile than rat myocyte. It is still possible that it is the batch of collagenase. Did you ask Christian? He is the expert of course.

Michiel, I did not contact with Christian yet, but have asked Gentaro about the problem. It seems I do the protocol accordingly to his advises and also similarly to that described in the papers of yours, Christian and Gentaro, but still have no living myocytes. So, I posted the question to look if I do something really wrong (or do not important thing) in my isolation and someone can find a critical error. Thank you for your feedback!

Oleg,

I don't think it's any one thing you're doing.  There are several possibilities.

First, is there a reason you need the heart to beat initially?  If you're examining a performance-based parameter, the answer's obviously "yes".  If not, you might try removing all the calcium from the start of the prep (that is, use a calcium-free version of your medium).  Our medium is a Krebs-Ringers-phosphate, but the Krebs-Henseleit you seem to be using works OK as well.   We use a 5-7 min calcium-free period to start, then add the collagenase.  We add back calcium to 1 mM after we have cells.  This works for both rats and guinea pigs (or really most animals).

Second, a 5-10 min digest period sounds very fast.  As Dr. Helms indicated, your heart may not be digested all the way.  Conversely, a concentration of collagenase able to digest in that time is too much.  The cells get a trypsinized look (pretty much what you describe).  Our digestion period is 30-35 min with Worthington class II, 0.4 mg/mL, perfusion pressure 40 mmHg (your 8 mL/min sounds about right for that pressure).

Do you mince the hearts after digestion? We do, then shake them in a water bath for 20 minutes.  Just letting the heart pieces slosh around helps dissociate more gently than using the pipette straight off.

Again, as Dr. Helms indicated, guinea pigs don't seem to handle calcium the same way as other rodents, making the cells look fragile.  I don't know what you do about that, except do everything slowly and gently.

Good luck -- T. Geisbuhler

Timothy, thanks a lot for the information!

1) Do you think that the increase in total perfusion time (due to the Ca-containing perfusion) may affect the viability of the cells? I use this step just to see whether the heart is in its "normal" contractility and also to promote better perfusion through small vessels (when the heart is contracting, this allows extensive propelling of blood through the vessels). Also, I have done several attempts to test the stability of heart contractility - it seems the total time of perfusion is not important if the heart is perfused well (~1 hour in Ca-solution).

The second thing (increase in the perfusion time with collagenase) is really good candidate to solve my problem, as you mentioned and Dr. Helmes as well. I will test this possibility as soon as I will have new animals.

Last, the mincing - I do mince the heart to small-sized pieces, then do further dissociation by pipette after several minutes. It works very well for rat cardiomyocytes, but I can see now that I was wrong with this step for guinea pigs. So, I will try to do as you suggested.

OK, the main thing I should remember in the future - more gentle manipulation with GP cells is necessary for success. Thank you for your help!

According to my experience one of the most important sources of variability in the cardiomocites isolation is the size and age of the animal.

I used to work with a mixture of collagenase type II (2mg/ml), trypsin (0.014%) and 12.5µM CaCl2. The perfusion time varies between 7 and 12 min, depending on the age of the animal and the species.

Currently I'm using a different protocol (collagenase type II 0.5mg/ml + 0.025mg/ml Protease type XIV). Again the perfusion time depends on the age and strain of the animal. Usually a initial perfusion only with collagenase for 5-7 min and then 2 to 5 min with protease and collagenase.

In the first protocol y mince the tissue after enzimatic digestion to get single cells; in the second protocol I only do dissociation by pipetting up and down or directly by agitation of tissue in KB solution.

Important to note that in both cases is stop enzymatic digestion using calcium free solution and FBS or KB.

Jose, thanks for your feedback! I understand now that the size of a heart (possibly not size of an animal or age) is critical for the perfusion time. Do you weigh a heart before cannulation? I would use some free available "weight-time" tables (but for rats) to choose appropriate time. Also, I do tried trypsin in a few cases but without significant changes in the outcome of the cells. Again, I can see that my total time in collagenase is too short, so I think this is the main step I will check in future.