We have used NB8 in the past and did not find it to yield as well as collagenase from VitaCyte.

Yield of islet isolation may be related to a lot of factors: collagenase type, rat breed, and one of the more important ones age. The younger the animals less adipose tissue and fat you will have and it may help increasing your islet yield. Nevertheless it may help to try a new collagenase type or brand (ex. Serva is very good) and you can also consider replacing Histopaque with Dextran (works better and its less toxic).

We basically avoid a stage of histopaque and separate the islets during cell culture passage

We are using Collagenase from Sigma (Catalog Number : C7657) at 0.65 mg/ml in HBSS + 1.15 ul/ml of DNAse. Digestion time 13 min at 37C

It works very well around 700 islets in 2 month-old wistar rats

I agree with Rita Ribeiro that there are a lot of factors, but often times changing collagenase will help. Since we primarily study mice, I will defer to others as to the best brand/type for rat, but I can say that even lot-to-lot variability from the same manufacturer can impact yield and islet viability. Timing also matters. It is very easy to overdigest (which will damage the islets) or underdigest (which doesn't separate the islets from the exocrine tissue sufficiently). 

One other suggestion that might help you to get a better handle on your problems would be to skip the Histopaque step for one of your preps and instead dilute the "pancreatic goo" following collagenase digestion at 1:10 with PBS or KREBs buffer and take a look under the scope. If there are a lot of large chunks of exocrine tissue with islets still embedded, you likely have underdigested. If the exocrine tissue is broken up into very small chunks and the islets look especially jagged at the edges, you may have over-digested.

Islets generally have an amber color in normal light, with the exocrine tissue looking pale by comparison. I've got a picture of islets in a dish of mostly exocrine tissue that might help (see Figure 1A from this paper):

http://www.biologicalproceduresonline.com/content/pdf/1480-9222-11-1-9021.pdf

I hope this helps. Best of luck!

Good Day Yael.

As has been stated, there are a lot of variable applicable to your isolations.  Working on the assumption that your animals are relatively age matched and that you use the same type each time, that takes care of two important variables. 

One thing that may improve outcomes would be to make sure everything is kept cold during the collagenase injection and harvest.  As with any system, you want to minimize warm ischemia as much as possible.  If you are processing multiple animals at one time, then of course you should keep the distended pancreas/collagenase cold until all are harvested and you are ready to digest.

You didn't mention if you are agitating during digestion.  If so, this should be gentle.  If not, you might want to consider it.

Drawing on Craig Nunemaker's comments, you might want to do a time evaluation to determine if 17 minutes is really an optimal digestion time.  It has been a long time since I did any rodent isolations, but it seems that 17 minutes might be too long a time. 

Similarly you may need to optimize the concentration of collagenase you are using (not noted in your description). 

Why six sedimentations?  Are you pipetting the tissues during this?  Are you using only wide tip pipets?...

As a QA check, you might want to take a count at the end of your digestion phase to determine a theoretical maximum and then relate your final yield to that.

After 17 minutes at 37 ° C, I go straight to the washing with appropriate solution and do the pescagem islet into a magnifying glass with a Pasteur pipette tuned into the fire. Thus, I aspire one by one and put into plate wells.

Hi everyone,

Here is a protocol we published in 1990. If you follow it carefully, it consistently yields close to a 1,000 islets. One modification we introduced over years (which further improved consistency and reproducibility) is that we replaced collagenase type 1 from Sigmawith recombinant Liberate TL from Roche (Ref# 05401020001), it's a combination of Collagenase + Thermolysis, it works great at 0.1mg/ml.

Remember that it is critically important to add extra-Calcium to the HBSS Collagenase solution (6mM CaCl2), or else the Collagenase will not work to its full power. If you also add some DNase (0.1ug/ml) to the Liberase solution, it will prevent clumping of the digested tissue (caused by DNA released from some dying acing/ductal cells), and further improve the separation by Histopaque 1077. 

Here below is the detailed protocol from our publication (Rouille et al., Exp Cell Res, 191:305-312, 1990) Good luck!

Islet isolation. Pancreatic islets were isolated from Sprague-Dawley

rats (weighing 2009240 g) by a modification of the method of Sutton

et al. [7]. Briefly, six overnight-fasted rats were anesthetized with

0.2 mg pentothal i.p./lOO g body wt (Abbot, Cham, Switzerland), their

pancreas was exposed, and a cannula was inserted downstream into

the bile duct. After clamping the pancreatic duct at its duodenal outlet,

16 mg of collagenase (type 1, Cat. No. C-0130, Sigma, St. Louis,

MO), diluted in 6 ml ice-cold Hanks’ balanced salt solution (HBSS),

pH 7.2, containing 6 mM CaCl, was injected through the cannula. The

distended pancreas was immediately dissected out and stored at

2-4°C in a 50-ml plastic tube until all six rats had been processed.

Each pancreas was then washed twice with 50 ml of HBSS, prewarmed

at 37°C. Digestion was for 20 min, under static conditions, at

37°C. The reaction was stopped by adding ice-cold HBSS, containing

0.35% (wt/vol) bovine serum albumin (BSA, fraction V, Cat. No.

A-2153, Sigma) (HBSS-BSA). The digested pancreas was then homogenized

by five passages through a 14.gauge (3” length) syringe

needle and the homogenate was washed 2X with buffer. Grossly undigested

pancreatic tissue was removed by filtering through a plastic

tea-strainer. The filtrate of each pancreas was split into two 25.ml

plastic tubes and centrifuged for 10 s at 450g. The pellet was then

taken up in 10 ml Histopaque 1077 (Sigma) and overlaid with another

10 ml HBSS-BSA [8]. The purified islets were recovered at the interface

after a 20.min centrifugation (15”C, 1OOOg) and washed 3~ with

ice-cold HBSS-BSA, pH 7.4. They were finally hand-picked with a

Gilson pipet under the stereomicroscope and kept at 4°C. The yield

was about 800-1000 islets/rat.

Dr. Yael,

Manishma!

Selection of enzyme and species is important. It is important to choose an enzyme and species it pairs well with. For Rats (Wistar, Lewis, SD) we have found Collagenase XI (Sigma) at 1.5 mg/mL in HBSS works well. 

Some common steps to check:

1. Pancreas distension is full, indicating injection was into common bile duct not hepatic artery. Hepatic artery injection will result in islet being preferentially digested instead of the exocrine.

2. Digestion time optimization (12-15 mins). You will probably due 3 rats at once. So, the first one at 12 min digestion, take out conical tube and with one hit about 80-100% of the pancreas should fall apart. If it does not, then digest a little and then stop reaction. You will know to extend your digestion time for the other tubes.

-You can also take dithiozone, and take a sample of the digested pancreas extract to see if intact islets are staining purple. If small fragments are staining, then you have overdigested. If you see trapped islets, then you are not digesting enough. Dr. Nunemaker's comments about islet roughness also apply.

3. Gradient purification tips. Following washing cells, pour out wash into gradient beaker. Then place conical tube, inverted, onto paper towel and let drain for about 30 sec. This will help remove excess water from tissue and help your purification.

4. Did gradient purification work as intended. Following gradient purification with histplaque, collect "other" supposedly non-islet fraction. Stain with dithizone to see where islets are ending up.

 Hi Vincenzo Cirulli.

I have some questions to you:

1. Do you still inject 6 ml with the Liberate? 

2. I add 40ul of CaCl to 40 ml of HBSS+HEPES+ NB8 and DNAse. is that enough?

3. Do you wash the pancrease in warm HBSS in petri dish and then put them in a tube? 

4. I work with RPMI  & NewBorn Calf Serum and not HBSS for washing. Do you think it differ?

I never hand pick but I will try! you use a simple pipete or sterile plastic pipete that has a wider openning?

Hi Yael,

Yes, 6ml of Liberase (but it works decently well with 4ml too, depending on the age and size of the rat).

As for the Cacl2, you need to add 150ul of a 1M stock solution of Cacl2 in 6ml of HBSS to get sufficient Ca for the Liberase to work well. Remember that the Liberase solution does NOT have any other proteins (no BSA, No serum), or else they will inhibit the reaction.

Washing and prewarming doesn't matter much, but you need to check after 15 minutes if the tissue starts to be digested (Liberase acts a bit faster than conventional collagenase).

RPMI is fine, but I don't understand why wasting expensive medium+serum for such a quick procedure. You can wash the islets at the end with your RPMI/Serum.

After you harvest the islets that have migrated at the interface between the Histopaque and HBSS (using a 14 gauge needle), you can do a couple of sedimentation steps in HBSS/0.2% BSA, and the islets will just go down, while single cells, acinar, and other small contaminants will float.

Once you have your nice pellet of islets, remove the supernatant and do a couple of washes in sterile medium.

Good luck!

Vin 

Hi again

Now I'm able to yield 550 islets but most of them are very small - less than 50um.

I hope it's all a matter of incubation time but I will love to read your thoughts..

Thanks

Yael

Hi,

You can reduce incubation time,use prewarmed collagenase solution. If you have 2ml of minced pancreatic tissue add 5ml or 10ml collagenase solution(Prewarmed).Histopaque separation the centrifuged pellet clearly dispersed with wide mouth pipette and slowely add other gradients.Do not use aged rat.

regards,

soosai

Thank you.

But I'm doing a large scale production of 32 rats per hour.

We harvest the pancreases during this hour so I need to put them on Ice until incubation.

I saw a protocol for mice that after Histopaqe the centrifuge the islets and pour off the washing solution and blot tubes on a paper tissue. Did anyone tried it with rat pancreas?

And also I saw an article about Improved islet purity by the hypertonic-hypotonic method. I want to try it after Histopaque. Sometimes I get a lot of acinars and I want to clean it more. Did someone try it?

Hi Yael,

In our rat islet isolation protocol, we actually have a step with a very carefully return of the tube after a 10 second at 400g centrifugation (to have a solid pellet) and to discard all the supernatant.But this step just precedes the FICOL step, and we directly resuspend the pellet in 20 mL of FICOL before slowly filing it under a 20 mL HANKS layer in a 50mL conical tube.

I never heared about this hypertonic-hypotonic method but I would be interested in knowing if it works....

I hope you have succeded in having better islet yields?

We're now trying Liberase TL and it seems to be more efficient compared to our last batches of Collagenase XI (even if it didn't give incredible yields but it's still better).

Regards,

Blandine

So I have tried it today AFTER the histopaque.  During the sedimentation time I left 10 ml of RPMI/NCS and added 10 ml of 400 mmol\l ending with 200mmol/l glucose. I put it on slow rotation for 10 min. Then centrifuged and discard as much solution as I could. 

I have started  islets isolation from mice just recently. I follow this protocol

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

I am annoyed of the last handpicking of the islets step to get clean islets and it takes a really long time and I can still see non islets dispersed cells in the medium. I want to know if you use another way to get clean islets in the end?