We were used to isolate Wistar rat (8 to 12 weeks old) islet with collagenase V and XI (from Sigma) with relatively good islet yields and quality in the lab.
Briefly, our protocol consists in inflation of 10mL collagenase solution (from 0.5-1mg/mL for Collagenase V depending on the batch, around 0.5mg/mL for Collagenase XI, in HANK’s Hepes without BSA), digestion during 9min (for Collagenase V) and 9 up to 18min (for Collagenase XI) at 37°C in a static water bath. After manual shaking (30sec), digestion is stopped by cold HANK’s Hepes 0.5%BSA addition. After centrifugation to wash the dissociate tissue, and filtration to remove big debris, islet separation on Histopaque 1077 gradient is performed before wash steps and then islet handpicking.
But, since about two years we have a lot of problems of yield and quality islet despite of concentration and digestion time optimization with the news batches tested (200-400 islets maximum, instead of 500-800 before, and a lot of little and/or damaged islets).
Maybe the problems come from also other factors such as quality BSA batch (which apparently can contain some amounts of endotoxin), Histopaque 1077 batch (seems to possibly contain endotoxin too...), quality of HANK’S Buffer (homemade), too much adipose tissue (despite its removing during dissection, rat being already at 480g at 12 weeks old in our lab, seems elevated…).
But we’d like to try enzyme as Liberase TL or Vitacyte CIzyme which could avoid lot testing and to improve yields and islet quality.
As anyone already try one of these enzyme, and which one would you advise us to order?
Thanks by advance for your advice which could be very usefull to us!
I have no experience with Vitacyte CIzyme, some with Liberase TL and considerable experience with old-fashioned Collagenase V (Boehringer Mannheim).
As you expect, Liberase TL will likely take away some of the enzyme related batch to batch differences at a considerably higher cost but if you are using only a few animals (hand-picking), it will be worthwhile. [We used to test multiple individual lots and after identifying one that worked the best, used to buy in bulk - $10-20k but then I was using as many 80 rats a day!]
Single most helpful thing in your case would be to use smaller rats, if you can. I would suggest using ~250gm or lower weight.
Best,
Thanks for your feedback Tausif!
We're doing only 4 rats at the same time, that's why maybe it could be usefull to pay twice the price if we have twice more islet, and much less optimization experiments.
Yes we sould use rat with lower weight, but it seems that the yields can decrease if the rats are too young. Also, according to the growth curve of our rat, we should use them at maybe at 6-7 weeks old, and I don't know if it's not too young to study adult rat islet (but I thinks there's a problem here with maybe the food or temperature that makes rat more fat). But this must be a part of the problem too of course.
It is a matter of how you define adulthood for rats, they breed at 5-6 weeks! ;)
I concur with your cost-benefit analysis.
Hello, I have been using Liberase TL for a few years, now. I too, had used Collagenase V for over 10 years, with good results, but I tried several different lots a few years ago with no luck. I could not optimize the digestion times, taking into consideration the Wunsch/mL. I compared nine different lots, before, finally switching and trying the Liberase TL. Although Roche, says that their enzyme is more stable and there is less variability, I did not find this to be the case. I tested three lots and they were each slightly different than the other. The lot I decided to purchase gave me the best results and I bought the whole lot to last 3-5 years...I would suggest finding out how much of a lot is available to purchase prior to testing it, then if it works, buy it to last. Because the lot was available in small vial aliquotes only (5mg vials), I had a large quantity of small vials, but this worked out to be ideal. We now use Liberase TL. The vials I purchased are in 5mg quantities which we dilute with 1ml cold Hank's Balanced Buffer Salts to dissolve, then further dilute 1:40 in HBSS prior to distention. We also have suspended the Liberase in (5mg in 1ml) buffer and re-froze the vial several times and tested it with good results with multiple freeze, thaws; therefore, it appears to have good enzyme stability (although, this is likely not the best practice). Please be aware that I have been using the enzyme for "mouse" islet isolations. I have not tested this lot on rats, but our rat and mouse isolation digestions are quite similar. Also, be aware that both our mouse and rat isolation/protocols involve light shaking through the digestion incubation. I agree that yields do decrease, if the animals are too young. We typically use 250-300g rats on a 4% fat chow, but I have isolated from older larger rats in the past as large as 750g with good yields. Best of luck; hope that helps.
Rena, in regards to difference in yield in rats of smaller size, I am curious if your comment is based on body weight-adjusted numbers of if it is absolute number. If the latter, reason is obvious.
Hello Rena,
Thanks you very much for your answer, it's quite reassuring to see that I'm not the only one to have problems with all the last batches of this collagenase... which is very unfortunate because we used too have very good yields with it, whatever the age of the rats. We both suffer the same numerous and unsuccefull steps of optimization...
And thank you for your advice to test several vials because I was believing their promise of batch stability...
Can you just give me your approximate yield for mouse with the liberase TL just to have an idea? (we will also isolate mouse islets with it).
Hello Blandine, The yields we get are absolute numbers. In mice, we isolate from mice greater than 10 weeks. We typically find that less than 10 weeks old 150-200 islets per donor (islets >75um in size at approximately 95% purity). Please note, we do not individually weigh each donor mouse or donor pancreas and these are average yields. We typically isolate 20-30 mice and do not isolate from one individual mouse at a time; therefore, we do not weigh each mouse or pancreas we distend. Like Tausif mentioned, buy bulk and certainly if you get good yields with a 250gm rat versus the same yield or a negligible increase with a 750gm rat, go for the smaller one if your per diems are less. With mice, especially, regardless of age, size will matter because the smaller the animal, the more difficult to cannulate the duct for distention. Collagen within the tissue has been the suspected reason for the increased yield in the older animal model, where the synthesis of collagen decreases with age and its degradation increases; therefore, its easier to digest during the isolation process to free the islets thereby increasing yields. In human and pig models, we take body weight-adjusted numbers (islet equivalents) into effect.
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