I use langendorff perfusion method and collagenaseII enzyme for digestion.
In most cases, I get high percentage of dead cells (round/oval in shape). Please advise me on what could be possible cause(s) of cell death.
And please say something about Ca2+ tolerant cardiomyocytes. What does this term mean? Why do we need Ca2+ tolerant cardiomyocytes?
And how do we condition the cells or produce Ca2+ tolerant cardiomyocytes?
I will appreciate any useful inputs or materials for reference.
Dear Jerry,
Here is one of my old recipies that I used.
Dissect heart tissue from newborn Wistar rats at postnatal day (P) 5 to 11 following numbing and decapitation. Wash the cardiac tissue in Hanks balanced salt solution, then cut into smaller pieces and incubate at 37°C in 3 ml collagenase dissolved in Hanks at 300 units/ml for 30 to 45 minutes depending on the age of the animal. Transfer the tissue homogenate to a falcon tube and spin for 20 seconds at 2000 rpm at room temperature. Wash precipitated cells twice with Hanks similarly. Followed by dissociation of precipitated cells in 1 ml Dulbecco’s Medium at 37°C. Plate the cells onto uncoated glass coverslips and grow in Dulbecco’s medium with 10 % fetal bovine serum and 1% penicilline and streptomycin overnight (up to 2 days) at 37°C.
Best wishes,
Refik
It sounds like you are trying to isolate adult myocytes from cardiac tissue. I've published the particulars of this method in a couple of places [for example, Geisbuhler and Rovetto (1990) J Mol Cell Cardio 22: 1325-1335, and Geisbuhler and Schwager (1996) J Mol Cell Cardiol 28: 1857-1866]. These methods are pretty much what you've described, a Langendorff setup using crude collagenase to digest the heart. We used to make a Krebs-Ringers-Phosphate salt solution to do this , but have found that Joklik-modified Minimal Essential Medium from Sigma works just as well. These media do not contain bicarbonate, but use phosphate for pH control instead. Gassing uses 100% oxygen. (If you use a bicarbonate-based medium, be sure to use 95% oxygen/5% carbon dioxide to gas the medium; the pH shifts will kill your cells otherwise). The media are nominally calcium-free. So, at the end of the prep, you have suspended cells in medium containing maybe 50 µM calcium.
Usually, problems with morphology or viability come from five places:
1) age of the rat (250-350 g for adults, older animals have more collagen and don't digest as well);
2) strain of the rat (Spragues of Wistars work best; Fishers have more collagen, and give poor myocytes);
3) quality of the water used for reagents (we filter deionized water through mixed-bed and carbon filters; reverse osmosis or double distillation in glass should work also);
4) quality of the "collagenase" (we lot-test batches of class 2 from Worthington Biochemical). "Collagenase" is in quotes because class 2 collagenase is actually a mixture of proteases (clostripain, trypsin, etc). I've always suspected that a "good" lot of collagenase had the proper balance of these enzymes.
5) perfusion pressure of about 40 mmHg (lower pressures let the heart become ischemic, much higher pressures cause vascular failures at inopportune times during the prep).
Calcium tolerance is mostly a test of your myocytes. After much pushing and pulling back in the 1970s (yeah, I'm that old), everyone agreed that so-called "competent" myocytes should be able to "tolerate" 1 mM calcium (more-or-less the level in interstitial fluid) without rounding up. This assures that the cells in your preparation are metabolically competent, and not leaking badly. The test is to add CaCl2 back to the medium (I do it in 100 µM increments) until you achieve 1 mM, then re-settle the cells. The cells that survive the test are "calcium-tolerant", and should be mostly rod-shaped.
Hope this helps.
T. Geisbuhler
Dear Jerry Machuki,
Unfortunately cadiomyocytes isolation is not a process which we could give you a recipe that will work for sure... Probably you will need to make some modifications in the procedure to get good results.
I think that a good first step should read this paper:
"Methods for isolating atrial cells from large mammals and humans"
Obviously it is focused in atrial cells isolation, however there you will get a lot of insights about methods and, specially about the Ca2+ paradox.
I hope this helps.
Jair T Goulart
Hi to you all,
I am grateful for your valuable advice. I usually obtain good cardiomyocytes from rats but I am particularly having problems with ADULT MOUSE. It appears that maybe the amount of collagen in heart of mouse is more than it is in rats?
Secondly, since the heart of a mouse is very small than that of a rat, I usually take up to 4 minutes to locate the Aorta, cut it and mount it on the cannula. I suspect that that duration is slightly long for the heart to survive without perfusion?
Again,I am subsequently going to make appropriate modifications and incorporate your advice to troubleshoot my method. I will also pay attention to the quality of water and perfusion pressure.
this paper "Methods for isolating atrial cells from large mammals and humans" is very helpful.
I look forward to share more with you.
thank you all
Jerry Machuki,
I've worked with mice ventricular myocytes once and we'd gotten better results when we used a method and setup that we normally used to isolate newborn rat cardiomyocytes. So, I think should be a good idea try to review method to isolate neonatal cardiomyocytes.
About the time you've been spending to cannulate the heart I really think it's too long. A good advise should be try to train more your technique in order to do it in less than 1 minute. I know it's very difficult, but each second worth.
I hope you be able to establish a good method! Good luck!
Best wishes
Dear Refik,
Which type of collagenase do you use to isolate the cells from newborn rats? And is this time of digestion (30-45 min) normal for membrane receptors? As i understand, this time of digestion (40 min or more) in a cannulated heart of an adult rat destructs some membrane receptors, so we don't receive any reactions to certain drugs.