Islet isolation is a bit of an art. If it goes perfectly, islets purified on a gradient can be washed and put into culture and will be beautiful. If your prep is not optimal, you may need to hand pick anyway. Islets that have been subjected to a gradient tend to look a little rough around the edges at the time of isolation, but usually look better in 24 hours and have normal GSIS. Over-digested islets will not work well.

In my lab, we typically want to maximize yield, so we always hand pick mouse islets. We typically get ~300 islets per mouse. I can pick 300 islets very quickly, so for me it is better to not take time to run a gradient.

We do use Histopaque 1077 for rat isolations, and when done well can yield a very clean prep - we always want a count (of islets, not islet equivalents), so we end up hand picking anyway, but it is much faster to hand pick from a clean prep. I have some tricks to use if the prep is not so clean, which will happen if the gradient is not done well.

If you prefer to avoid using a gradient, or your gradient fails, wash the islets a few times in your buffer. When you resuspend, do so in a 50 ml tube, using 40 ml buffer. Allow the prep to sit on ice for about a minute and then use a Pasteur pipette to suck material from the bottom of the tube. Place this in your dish and add buffer. Proceed to pick islets into clean buffer in a clean tube. Resuspend the contents in the wash tube and repeat until there are no more islets in the wash tube. This helps you avoid what we refer to as "the dust" that is acinar and other cell debris. You can also use the sedimentation method to clean up human islets if you should get a prep of low purity.

Islet isolation is a bit of an art. If it goes perfectly, islets purified on a gradient can be washed and put into culture and will be beautiful. If your prep is not optimal, you may need to hand pick anyway. Islets that have been subjected to a gradient tend to look a little rough around the edges at the time of isolation, but usually look better in 24 hours and have normal GSIS. Over-digested islets will not work well.

In my lab, we typically want to maximize yield, so we always hand pick mouse islets. We typically get ~300 islets per mouse. I can pick 300 islets very quickly, so for me it is better to not take time to run a gradient.

We do use Histopaque 1077 for rat isolations, and when done well can yield a very clean prep - we always want a count (of islets, not islet equivalents), so we end up hand picking anyway, but it is much faster to hand pick from a clean prep. I have some tricks to use if the prep is not so clean, which will happen if the gradient is not done well.

If you prefer to avoid using a gradient, or your gradient fails, wash the islets a few times in your buffer. When you resuspend, do so in a 50 ml tube, using 40 ml buffer. Allow the prep to sit on ice for about a minute and then use a Pasteur pipette to suck material from the bottom of the tube. Place this in your dish and add buffer. Proceed to pick islets into clean buffer in a clean tube. Resuspend the contents in the wash tube and repeat until there are no more islets in the wash tube. This helps you avoid what we refer to as "the dust" that is acinar and other cell debris. You can also use the sedimentation method to clean up human islets if you should get a prep of low purity.

Getting pure islets with a high yield needs a good optimization for any of the isolation protocols.

I have used several different protocols with different enzymes. In all protocols, inflating the pancreas properly and optimizing the digestion time are the two key steps for good yield and purity. Optimization should be done according to the number of pancreases that will be pulled together in one tube. If I need a high yield of islets, I prefer to use 15-20 mice pulled together in 2-4 tubes. This saves a lot of time during the whole isolation process which is very healthy for the islets. If you end the isolation with less tubes, picking them will be very fast.

I use Histopaque-1077 as a gradient, wash 2 times and do 3-4 times self sedimentation. Usually, after the final sedimentation I count them by handpicking. From a 8 week old WT mouse the average is between 200-250 healthy islets. After O/N culture the loss is as low as 5-10%.

I also know other labs where they use 2 or 3 gradients at the same time. With a good optimization they also get very pure islets with high yields. They also use self-sedimentation (3-4min) and handpick islets from the petridish.

Handpicking will provide you with a higher yield with less optimization. It can be important especially if your mice are of little size and expensive.

For example, our transgenic B6 mice yielded only around 150 islets/pancreas by handpicking. Since we had a limited amount of them at a given time we couldn't really pool a big amount of pancreas per assay. So, we chosed handpicking to avoid the losses of islets that one usually has with a gradient.

yield is very poor. According to SOo Young Park et al., 2005 the number of islets in adult mice pancreas  is 3 x 10^3

Hi Raza Ali,

You are correct that the yield is lower than what one might expect of one thinks that one can obtain all of the islets from a given pancreas. The fact is, you cannot obtain anywhere near "all" of the islets via digestion. Smaller ones may get overdigested, some may be left in pieces of underdigested tissue if the protocol is not optimized, mechanical damage also occurs during processing. Some islets dies off or become necrotic overnight. A very good yield from a mouse would by 400 islets in my hands. 800 from a rat. Another concept is that of useable islets. For example, the technique that I use for GSIS involves a mesh that has an opening that will allow very small 25-40 micron islets to fall through, so for this purpose, I cannot use the smallest islets, therefore they do not make it into the count when I pick for GSIS or other static incubation experiments. This is in addition to any lost in the pellet if you do a gradient.

You are lucky to be in a place where there are very many islet isolation experts - the surgery department has a lab that does nothing but isolate islets all day, or at least they did when I was there!

Completely agree with Elizabeth. Getting an average of 200-250 islets every time is actually very good. I know that it is 150-175 in most of the labs. 400 is amazing. Maximum I got was 360.

Thanks

Yeah, 400 is my personal best. I am happy with 200.

Can anyone share your protocol with those who are struggling with yielding decent amount of islets (including me)?

For rat also please.

HOW DO YOU CALIBRATE THE DIGESTION TIME?

How do you Hand Pick?

How do you know the number of isolated islets?

Hi Lina,

After density gradient separation and couple washes, I pour it into a 60mm petri-dish and I handpick them, under a dissecting microscope (20X), with a 200 ul pipette from one petri-dish to another containing cell media. This way, we can count the number of islets and purify the islets from "the dust" at the same time.

Hi Yik,

Thanks a lot for your answer. May I ask you another question about islet viability? I want to use FDA/PI method to measure islet viability after 48h incubation. The islets will be stick to the petri dish. Do you know how should I remove the islets from the petri dish and mix well the islets before FDA/PI measurement?

Thanks.

Kind regards,

Lina

Hi Lina,

I presume you are working on flow cytometry method.

Removing whole islets should be easy. But I would also like to see if its what everyone else doing.

I do it under a dissecting microscope. Flushing the islets with cell media gently with cell media until all islets are physically detached from the dish.

Hope this helps and if someone could confirm this or add comments to improve this would be even better!

Hi Yik,

Thanks for your answer. I heard from a friend that they use trypsin to remove the cells from petri dish. But I don't know whether the trypsin will digest the islets or not. Therefore, I asked you about this. Flush the islet gently will have less damage to the islets. Do you use microscope to check whether the islets were all detached from the dish?

I plan to use fluorescent microscope to measure the islets viability this time and  will try flow cytometry method later.

Thanks again for your suggestions.

Lina

Hi Lina,

I have seen people using trypsin for detaching cell lines from culture dish, but detaching whole islets with flushing is easy, fast, observable and less invasive. Trypsin will break down islets into single cells if you give them enough time and a right temperature. I use trypsin to breakdown the islets into single cell suspension for flow cytometric analysis.

Using trypsin for detaching whole islets from culture dish is unnecessary. 

Yes I do check them under a dissecting microscope, pick them up with a pipette, transfer them into a new tube with washing buffer. and so on.

Yik 

To isolate mouse islets I am using collagenase type V, at a concentration of 0.3 -0.5 mg / ml. incubating at 37 ° C for 10 min. But I do not get intact islets Can you help me with some protocol to isolate islets please?

I found out that if you use the simplest petri dish for microbiology and NOT for cell culture the islets will not stick to the plate. 

How long are isolated islets in culture still usable? for how many days can they be kept without compromising GSIS?

Why do I have a lot of islets with necrotic centers? Any suggestions to prevent this?

Most of my isolated islets stick to the Petri-dish after an overnight incubation even on simple non-tissue culture treated dishes. Is this necessarily bad? Any suggestions to prevent this from happening?

Im using 0.84mg/ml type XI collagenase in RPMI 1640,  Ca+ and separating them on Histopaque 1077.

@Orlando Mamani sorry it took me 7 months to reply you. Have you figured out yet?

if you didn't get any islets, there could be many factors like degree of pancreas inflation with collagenase, collagenase concentration, digestion time, methods for separating islets from other tissues which were not specified in your question.

@Yael Shushlav there are a lot of protocol available online such as this:

http://www.bio-protocol.org/e1840

@ Jay Patankar Hi, I haven't cultured islets myself before, but I have read some journals that say their islet survived up to 20 days (unbelievable). sorry I am not sure how long they can survive and still be responsive to GSIS. Islets tend to undergo necrosis after isolation, but some process during the isolation and purification may speed up the process such as aggressive handling, prolonged exposure to histopaque, insufficient wash after histopaque exposure, etc. I am not completely sure if sticking to the petri-dish is a bad sign, but I believe it could be a good sign that they are settling down like other beta cell lines which attach to the plate when they are growing happily. 

You may also try swirling incubation overnight to keep them close together, hopefully they live happier in this way, like they used to be in a compact pancreas.

I am grateful with you @Yik Hei Ng and everybody. All is going to well.