Name: Murine Pancreatic Islet Isolation (Video) | JoVE Journal
Uploaded: 2007-08-22T00:00:00.000+00:00
Duration: 15 min 20 s
Description: Learn murine pancreatic islet isolation using collagenase digestion and Ficoll gradient purification. This method enables researchers to prepare viable islets for transplantation into diabetic NOD mice.

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5 minutes

Collagenase Preparation
<ol>
<li>Weigh out the collagenase into a 50 ml conical and add isolation buffer to make the final concentration, as indicated in the table below. Dissolve the collagenase by vortexing. (12 mice + 2 extra mice = 14 mice x 5 ml/mouse = 70 ml total collagenase solution) (70 ml x 0.5 mg/ml or 0.3 mg/ml = 35 mg or 21 mg of collagenase P total) 
<table style="BORDER-RIGHT: #ddd 1px solid; BORDER-TOP: #ddd 1px solid; BORDER-LEFT: #ddd 1px solid; BORDER-BOTTOM: #ddd 1px solid; TEXT-ALIGN: left" border="0" width="80%">
<tbody>
<tr>
<th style="font-size: 12px; border-bottom: #ddd 1px solid; text-align: left">Strain</th><th style="font-size: 12px; border-bottom: #ddd 1px solid; text-align: left">Age</th><th style="font-size: 12px; border-bottom: #ddd 1px solid; text-align: left">Collagenase P (mg/ml)</th><th style="font-size: 12px; border-bottom: #ddd 1px solid; text-align: left">Time (min)</th>
</tr>
<tr>
<td style="font-size: 12px">C3H, Balb/C, B6</td>
<td style="font-size: 12px">&gt; 12 weeks</td>
<td style="font-size: 12px">0.8</td>
<td style="font-size: 12px">17</td>
</tr>
<tr>
<td style="font-size: 12px">C3H, Balb/C, B6</td>
<td style="font-size: 12px">&lt;= 12 weeks</td>
<td style="font-size: 12px">0.5</td>
<td style="font-size: 12px">13</td>
</tr>
<tr>
<td style="font-size: 12px">NOD, NOR, NON</td>
<td style="font-size: 12px">&gt; 12 weeks</td>
<td style="font-size: 12px">0.8</td>
<td style="font-size: 12px">17</td>
</tr>
<tr>
<td style="font-size: 12px">NOD, NOR, NON</td>
<td style="font-size: 12px">&lt;= 12 weeks</td>
<td style="font-size: 12px">0.5</td>
<td style="font-size: 12px">17</td>
</tr>
</tbody>
</table>
 </li>
<li>Dispense 2 ml of collagenase solution into each glass vial and place on ice. Load each 5 ml syringe with 3 ml of collagenase add 30g needle and place in ice (prepare in hood).</li>
</ol>
Distension
<ol>
<li>Just before dissection, sacrifice animal by cervical dislocation. Spray the whole body with 70% of ETOH, making it completely wet. Make a V-insision starting at the genital area. Rotate the mouse so the tail is facing away from you.</li>
<li>Remove the bowel to the left side of the open mouse. This will expose the pancreas and common bile duct.</li>
<li>Place a hemostat clamp on either side of the small-intestinal, where the bile duct drains, leaving a small pocket for collagenase solution to enter the intestine.</li>
<li>Inflate the pancreas through the bile duct with a 30g needle and 5 ml syringe containing 3 ml of cold collagenase solution, starting at the gall bladder.</li>
<li>Remove the pancreas from the body and place it in a siliconized vial containing 2 ml of collagenase solution. This step should be done QUICKLY and as cleanly as possible, minimizing collection of fat and connective tissue. </li>
<li>Place the vial on ice and repeat step 3 with the next mouse.</li>
</ol>
Digestion
<ol>
<li>Seal each vial and place it into a 37°C water-bath. Incubate for 13-17 minutes (time varies with strain and age of the animal).</li>
<li>After time has elapsed, shake the vial vigorously. The pancreas should fall apart.</li>
<li>Pour each digest through a large sterile sink strainer into a sterile 1000 ml beaker and forcefully pipette off the screen with washing buffer. Dispense the digests into 50 ml conicals tubes (3 pancreata/50 ml tube: if n=12, use enough washing buffer to make the final volume 200 ml and dispense it to 4x50 ml tubes). The dilution should be done rapidly and on ice to avoid unwanted islet digestion.</li>
<li>Spin down: Start the centrifuge. When it reaches 1300 rpm, turn it off.</li>
<li>Aspirate the supernatant, leaving ~5 mL. Be very cautious not to aspirate the pellet.</li>
<li>Resuspend pellet by tapping vigorously with your hand, then add 50 ml of wash buffer.</li>
<li>Spin down: Start the centrifuge. When it reaches 1300 rpm, turn it off.</li>
<li>Resuspend the pellet and wash with 5 ml of washing buffer.</li>
<li>Transfer the 5 ml to 15 ml conical and spin down, as above.</li>
<li>Aspirate the supernatant as completely as possible. (Remaining buffer might cause the change of the density of the Ficoll.)</li>
</ol>
Ficoll
Perform quickly. Long-term Ficoll exposure is TOXIC to islets.
<ol>
<li>Make sure pellet is completely broken up prior to adding ficoll (unbroken tissue is difficult to resuspend in ficoll).</li>
<li>Resuspend the pellet in 7 ml of ficoll, density 1.108, by vortexing vigorously.</li>
<li>Layer on top of each density of ficoll 2ml of each of the remaining densities in this order: 1.096, 1.069, then 1.037.</li>
<li>Spin for 15 minutes at 1800 rpm at 4 degrees, with break OFF!</li>
<li>Pick islets from the second layer using spoid (sterile plastic eyedropper). Transfer all collections to a 50 ml conical containing ~25 mL of cold buffer.</li>
<li>Wash, as above, 3 times with washing buffer (repeating step 12-15).</li>
<li>Resuspend Islets in 20 ml RPMI-1640 (containing 10% FCS and penicillin and streptomycin, HEPES, MEM-NEAA) and mix gently. Remove 100 ul of sample for counting.</li>
<li>Transfer 100 ul to 35 mm Grid-plate containing 1 ml of media and 1 ml dithizone.</li>
<li>Incubate remaining islets in RPMI 1640, in a 37°C, 7% CO2 incubator, in 160 mm plates with a total of 30 ml of media/plate. <img src="/files/ftp_upload/old/255_2008_8_24_3/255_Figure-01.png" alt="Density gradient centrifugation diagram, islet layer separation process, labeling 1.037-1.108." width="220" height="194" data-altupdatedat="1787630408000" data-alt="Figure 1"></li>
</ol>

<table><tbody><tr><td>Dithizone</td><td>reagent</td><td></td><td></td><td>Dissolve 5mg Dithizone in 5 ml of DMSO in a 50 ml conical tube. Add 45 ml of normal saline and mix gently. Filter with a 60 cc syringe and a 0.22 &quot;m syringe filter into a new 50 ml tube. Store at 4 OC, until use. Add 1 ml to each 35 mm counting Grid-plate, when needed.</td></tr><tr><td>PBS</td><td>Buffer</td><td></td><td></td><td></td></tr><tr><td>HBSS</td><td>Buffer</td><td></td><td></td><td></td></tr><tr><td>Isolation soln.</td><td>Buffer</td><td></td><td></td><td>HBSS containing 10 mM of HEPES, 1 mM of MgCl2, 5 mM of Glucose, pH 7.4</td></tr><tr><td>Wash soln.</td><td>Buffer</td><td></td><td></td><td>Isolation buffer containing 1mM CaCl2</td></tr><tr><td>Collagenase P</td><td></td><td>Boehringer Ingeheim</td><td>1213873</td><td></td></tr><tr><td>Dissection tools</td><td></td><td></td><td></td><td>Hemostat clamp (n=2), Forceps (n=2), Scissors (for skin incision an for taking out pancreas from adjacent tissue)</td></tr><tr><td>Syringe</td><td></td><td></td><td></td><td>sterile, 5ml, 1/mouse.</td></tr><tr><td>30 G needles</td><td></td><td></td><td></td><td>sterile, 1/syringe</td></tr><tr><td>siliconized glass vials w/Teflon cap</td><td></td><td>Fisher Scientific</td><td>213-018-54</td><td>sterile, 1/mouse</td></tr><tr><td>large sink strainer</td><td></td><td></td><td></td><td>sterile</td></tr><tr><td>1000 ml Beaker</td><td></td><td></td><td></td><td>sterile</td></tr><tr><td>Gauze pads</td><td></td><td></td><td></td><td>4x4, 2/mouse</td></tr><tr><td>plastic eye dropper</td><td></td><td></td><td></td><td>sterile</td></tr><tr><td>Mice</td><td>Animal</td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

murine pancreatic islet isolation

Murine Pancreatic Islet Isolation

Hi, I am Greg Zott. I'm with the UCSF Diabetes Center in the Bluestone Laboratory, and today I'm gonna show you the process for isolating mouse eyelets. The procedure involves the injection of a collagenase solution into the pancreas of a C3 H donor mouse, where we will then remove that pancreas, digest it away, purify the eyelets on a fial gradient, and then handpick them for ultimate transplant into the kidney capsule of a diabetic NOD mouse, Which will be our recipient.Before we start this procedure, we diluted our ATE solution in a modified Hanks buffer and we have adjusted the concentration specific for the strain of mouse that we're using the C3 H.We found digests better at a 0.8 mgs per mil collagenase solution, and we will use that to inject into the common bile duct to distend the pancreas.Performing. The procedure today Will be Pavo codre of the diabetes center. He will inject, open up the mouse, expose the the intestines, and then inject into the common bile duct.We will spray down the mouse with ethanol to keep the hair down, and then we will also verify that the mouse has been sacrificed properly with a toe pinch. Pavo will now open up the mouse and exposing the intestine and the pancreas with a midline incision. The diaphragm will also be cut to verify that the mouse will be sacrificed.Before we get started, I'd like to point out some of the important structures. The stomach is lying right here with the pancreas attached underneath into the duodenum and above. This is the liver where we'll find the gallbladder and the common bile duct, which you can see right here.So what Pavo will do next is he will isolate the small intestine, find where the common bile duct enters the small intestine. Once the intestine is exposed, we clamp on either side of the common bile duct with mosquito clamps. We find that this allows more collagenates to enter the pancreas while other groups try to direct the clamp on top of the common bile duct.We find that this is a better technique in allowing enzyme to enter the pancreas. This is a gentle procedure. You don't want to tear the bowel.You wanna allow the clamps to lie gently on either side of the common bile duct. Now we will find a gallbladder, and once the gallbladder's exposed, we will use that as a guide to injecting the collagenase into the common bile duct. Powell's using a 30 gauge needle with a five cc syringe containing three mills of collagenase solution.As he's entering the common bile duct, he's distending that duct with collagenase solution and then he travels down the common bile duct till he reaches near the intestine. And as he's going down, the enzyme solutions being pumped into the injected into the pancreas. Now that the pancreas is inflated with collagenase solution, Powell's gonna remove the hemostats and then remove the pancreas from the the mouse carefully tearing it away from the intestine.He removes it from the stomach. Okay, and then using the spleen as a handle, he lifts up the pancreas and then slowly snips it away in the common bile duct, and that's it. Pancreas is laid out and then the spleen is removed, and there he's removing the meso enteric tissue, and then the pancreas is placed in a two mill collagenase solution and a sterilized glass vial and it's placed back on Ice.The isolated pancreas that we put into the glass vials will now be placed into a 37 degree water bath for approximately 17 minutes. We placed them into a 50 mil conical rack so that they don't float up. Once during the bath, the timer starts and the digestion begins.Once the digestion time is completed, vials are removed, and then they are shaken and disrupted gently. And once the tissue starts falling apart, the vial is then placed on ice. A sterile screen is placed over a 400 mil beaker that contains washing buffer.The pancreas digest is then carefully poured over the screen into the wash buffer. For demonstration purposes, the hood has been turned off and the glass of the hood has been raised so we could see what we're doing. Once the pancreas digest is poured into the beaker, the vials are rinsed with the same washing buffer and the wash buffer placed over the screen.We have a syringe filled with washing buffer. We will use that to forcefully wash the tissue through the screen. The idea is to release any loose eyelets that are unattached to the matrix, but leaving the membranes and the undigested tissue on the screen.And when you're finished washing a tissue, this is what you usually See at the end of the procedure. The Pancreas tissue that's in the bottom of the tube will now be transferred to two 50 mil conical, and the tissue will be washed, the tissue's mixed evenly, and then distributed evenly so that we have a equal pellet volume in each tube. The beaker is now rinsed with more washing buffer.The sides are rinse so that you get all tissue that is entered the beaker again. Then it's equally distributed between each two, both 50 mil. Conical then are topped off with wash buffer.The tissue is inverted several times and then placed into a centrifuge. This is a quick spin. The centrifuge is allowed to reach a thousand RPM and then is turned off.The tissue at this time is very S eyelets are very sensitive, and you don't want to over compact them. The tubes are removed and we will do this wash step three more times. This is the end of the third wash.The components of the 50 mil conical tube are transferred to the 15 conical tube and centrifuge for a thousand RP M and then stopped. The supernat will be removed and the first part of the FI Call will be started. We are at the step where we Have our pelleted di pancreas digest.We'll resuspend that tissue so it's nice and loose to the pellets. We'll add the first density gradient, which is 1 1 0 8. We'll add five mils to each tube.The pellet is then mixed thoroughly in each of the gradients. It's important that you get good tissue distribution in your heaviest gradient. Now we will layer two mils of 1 0 9 6 density.We will then on top of that, layer two mils of 1 0 6 9 density, and then on top of that 1 0 3 7 density. It's important while you're layering the densities that you don't mix. Then all the layers have been added to the tube.And you can see this is the top of 1 1 0 8, the top of 9 1 0 9 6, the top of 1 0 6 9 and the top of 1 0 3 7. And you could see the tissue is already starting to move to its proper density even without centri fusion. The fial gradients are brought up to speed at 1800 RPM for 15 minutes, and remember to keep the brake off after the tubes are removed from the centrifuge.You can see between the 0 0 9 6 and 0 0 7 layer, there's a nice band of eyelet tissue Pablo's. Now removing the 0 3 7 layer to remove any tissue debris and DNA that might have been released during the processing. Using a sterile plastic pasture pipette, he will now remove the islet layer and transfer them to a 50 mil conical containing 25 mils of wash buffer.You want do this step quickly because fol is toxic to eyelets. We actually removed the entire 0 6 9 and 0 9 6 layer. Pavo cuts the top of the plastic pasture pipette and rinses it with wash buffer.The eyelets are then centrifuge like we centrifuge previously. Centrifuge is brought up to a Thousand RPM and then we stop it and We aspirate off. The supernatant eyelets will be washed three times with wash media.So we just completed the eyelid Isolation process we took from the digest to the fol gradient to the purified eyelets after our third wash with wash buffer, we resuspended the eyelets in our RPMI media and we played it in a tissue culture plate. These eyelets are now ready for in vitro assays or in vivo preps like eyelet Transplant.

Research

JoVE Journal

Biology

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