We have been trying to isolate cardiomyocytes from type 2 diabetes model OLETF rats using conventional procedure for adult rat hearts but we always fail from obtaining viable cells. The rats are also 52 weeks old. We assumed that the heart at this age would be more resistant to collagenase treatment, so we increased the treatment time, but kept the collagenase concentration at 0.1 mg/mL. Of note, the heart starts to change its color from an old rose color to a pale flesh about 4 minutes into collagenase treatment. We have tried adding BSA to it but the heart continued to show the same results. Any insight on this would be of great help. If more information is needed, I would be more than happy to expound on it.
Bulletin of Experimental Biology and Medicine
9-2005, Volume 140, Issue 3, pp 370-373
A Simple Method for Isolation of Cardiomyocytes from Adult Rat Heart
M. V. Egorova, S. A. Afanas'ev, S. V. Popov
Abstract
A simple, economic, and sparing method for isolation of cardiomyocytes from adult rat heart is proposed. Ultrastructure of cardiomyocytes from suspension of freshly isolated cells was studied by light and transmission electron microscopy. The isolated cardiomyocytes were viable, had characteristic shape and size, and retained their normal structure.
Bulletin of Experimental Biology and Medicine
9-2005, Volume 140, Issue 3, pp 370-373
A Simple Method for Isolation of Cardiomyocytes from Adult Rat Heart
M. V. Egorova, S. A. Afanas'ev, S. V. Popov
Abstract
A simple, economic, and sparing method for isolation of cardiomyocytes from adult rat heart is proposed. Ultrastructure of cardiomyocytes from suspension of freshly isolated cells was studied by light and transmission electron microscopy. The isolated cardiomyocytes were viable, had characteristic shape and size, and retained their normal structure.
Hello
You can see also the following article :
Am J Physiol. 1984 Dec;247(6 Pt 2):H1018-26.
A new method for preparation of isolated single adult myocytes.
Bkaily G, Sperelakis N, Doane J.
However, the best way to obtain adult cardiomyocytes with all their differentiated characteristics and morphology is the Langendorff method. For that see our article :
Biol Cell. 1990;70(3):121-7.
An efficient isolation procedure of Ca-tolerant ventricular myocytes from ferret heart for applications in electrophysiological studies.
Bouron A1, Potreau D, Besse C, Raymond G.
Best regards
JP Gomez
Hi,
Not sure about the diabetic scenario. When using a Langendorff / Enzymatic digest isolation protocol for young and old sheep hearts, we used to find that the old hearts required relatively higher concentrations of collagenase. Presumably due to collagen accumulation.
Not sure if this will be an issue in old rats but may be worth considering.
Best,
Dave
we had a project about aging and our experience as follows,
you can increase amount of collegenase(roche)-protease and/or increase your temperature about 1 Celcius as to increase collegenase activity due to collagen and other ECM accumulation.
Using collegenase+protease would increase your viability.According to our experience using collegenase 0.7 mg/ml+protease 0.06mg/ml,(digestion time :18-20 minutes) yields best viabilitiy
best isolation achieved with alteration of "collegenase+temperature"
good luck:)
Double check pH of your collaganese solution. As Yusuf forgot to mention...
I totally agreed with Nazmi( our lab chief and promoter). In detail,check pH at 25 0C in order to ensure room temperature(adjust all solution in RT). As aforementioned, pH of collagenase solution plays vital role.
Other possible way is adding some chemicals into enzyme solution like insulin,bdm,creatine,taurine etc. Creatine and taurine serves energy reservoirs that needed during and after isolation. BDM(butandione monoxime, washable) is prevents actin-myosin cross-bridge and make cells calcium tolerant. thus, the biggest problem druing the adaptation has been achieved, whereby obtaining calcium tolerant and high yielded cells..
I do not have any special experience with old and diabetic hearts. The Langendorff perfusion is the best way to obtain cardiomyocytes. The perfusion time may vary wiuth different kind of hearts and also depends on the enzyme batch.
Thank you all for your responses!
It appears I have failed to mention how we isolate cells, and yes it is through Langendorff perfusion. We have tried to change the collagenase brand and were able to get better cells using Worthington collagenase (335 u/mg) instead of Yakult (800-1030 u/mg).
As mentioned by Dr. Greensmith, we did try to increase the treatment time of collagenase (30 minutes); however, what we consistently encounter is the immediate change in color of the left ventricle, which looks like an infarction, and usually results in almost 0 cell yield. For this problem, as I have previously mentioned, I changed the collagenase brand and was able to get cells as in the photo attached with this post. However, this was done with a 9-week old Wistar rat and not with a 52-week old rat, so I cannot really tell whether it was just the collagenase problem or there should be a modification when working with aging/diabetic models.
As per Dr. Olgar's suggestion, I shall try to do the temperature change during collagenase treatment to see how it could make my cell yield and viability better!
Again, I am grateful for all of your help! Cheers!
Circ. Res. 2007;101;185-194.DOI: 10.1161/CIRCRESAHA.106.146670
Knock-In Mouse Model of Dilated Cardiomyopathy Caused by Troponin mutation
Cheng-Kun Du, Sachio Morimoto, Kiyomasa Nishii, et al.
Best regards
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