This manuscript was supported by a clinical innovation grant from the Mid-America Transplant Foundation.

All procedures involving animals are approved by the Institutional Animal Care and Use Committee (IACUC) at the Washington University School of Medicine Division of Comparative Medicine. Adult Yorkshire pigs ranging from 35 to 35 kg are ideal for this procedure; however, the protocol can be adapted for pigs of different sizes depending on the experimental context. The entire procedure should be performed in a sterile fashion in an operating room setup.
1. Preoperative preparation and anesthesia
<ol>
	<li>Sedate the pig with a cocktail of 4 mg/kg tiletamine and zolazepam, 20 mg/kg ketamine, and 2 mg/kg xylazine injected intramuscularly.</li>
	<li>Establish peripheral venous access via the auricular vein.</li>
	<li>Induce general anesthesia with inhaled 1%-3% isoflurane in 100% oxygen and endotracheally intubate the pig. 
	NOTE: It may be preferred to use propofol or any another intravenous induction agent vs. inhalant for induction for a smoother induction and intubation procedure. The vital signs, end-tidal CO2, oxygen saturation, and cardiac waveform are continuously monitored to assess and maintain the depth of anesthesia. The abdominal surgical plane is confirmed using bedside ultrasound.</li>
	<li>Shave and prep the pig with chlorhexidine gluconate from the sternal notch to the bilateral groins.</li>
	<li>Drape the operative field with a clear view of the entire abdomen (Figure 1A).</li>
</ol>
2. Operative setup and exposure of the retroperitoneum
<ol>
	<li>Perform a midline laparotomy incision (Figure 1B).
	<ol>
		<li>Use a 10-blade scalpel and electrocautery to make a midline laparotomy from the xiphoid process to the pubis. Take care to curve the incision around the inferior abdominal urogenital opening.</li>
	</ol>
	</li>
	<li>Perform a right visceral rotation.
	<ol>
		<li>Externalize the right colon and small bowel, wrapping them in a sterile towel.</li>
		<li>Gently retract the colon and bowel to the left to expose the right retroperitoneum while avoiding bowel torsion or malrotation.</li>
	</ol>
	</li>
	<li>Drain the bladder (optional).
	<ol>
		<li>If the bladder is full and impacting exposure, grip the bladder between two DeBakey forceps and use an 11-blade scalpel to incise the ventral bladder wall.</li>
		<li>Place a Yankauer suction tip into the bladder to drain the urine and decompress the bladder.</li>
	</ol>
	</li>
	<li>Set up an abdominal retractor set to facilitate optimal exposure of the right retroperitoneum (Figure 1C).</li>
</ol>
3. Retroperitoneal dissection
<ol>
	<li>Begin retroperitoneal dissection (Figure 2A).
	<ol>
		<li>Using electrocautery, incise the peritoneum to the right of the midline, from the level of the right kidney to the aortic bifurcation.</li>
		<li>Sharply expose the infrarenal aorta, inferior vena cava (IVC), and right renal artery and vein</li>
	</ol>
	</li>
	<li>Obtain circumferential control of the distal infrarenal aorta with a large vessel loop.</li>
	<li>Ligate the IVC (Figure 2B).
	<ol>
		<li>Obtain circumferential control of the mid-infrarenal IVC.</li>
		<li>Ligate the IVC using a 2-0 silk tie.</li>
	</ol>
	</li>
	<li>Ligate the renal vessels (Figure 2C).
	<ol>
		<li>Obtain circumferential control of the right and left renal arteries and veins.</li>
		<li>Ligate these structures using 2-0 silk ties.</li>
		<li>If there are multiple renal arteries, similarly isolate and ligate each of these vessels.</li>
	</ol>
	</li>
</ol>
4. Intrathoracic dissection
<ol>
	<li>Incise the right hemidiaphragm (Figure 3A).
	<ol>
		<li>Gently retract the liver inferiorly to expose the right hemidiaphragm. Apply two Allis clamps to the membranous portion of the right hemidiaphragm and incise the diaphragm in between the two clamps using electrocautery.</li>
		<li>Extend the diaphragm myotomy to approximately 7 cm horizontally.</li>
	</ol>
	</li>
	<li>Obtain circumferential control of the intrathoracic IVC (Figure 3B).
	<ol>
		<li>Place a 2-0 silk tie around the intrathoracic IVC. Snap the ends of the suture with a small hemostat without occluding the IVC.</li>
	</ol>
	</li>
	<li>Extend the diaphragmotomy to the left hemidiaphragm (Figure 3C).
	<ol>
		<li>Use two Allis clamps to retract edge of the diaphragmotomy inferiorly and extend to the left hemidiaphragm. 
		NOTE: Avoid entering the pericardium during diaphragmotomy.</li>
	</ol>
	</li>
	<li>Expose the descending thoracic aorta in the left posteromedial chest region.</li>
</ol>
5. Infrarenal aortic cannulation, visceral isolation, and perfusion
<ol>
	<li>Systemically anticoagulate the pig.
	<ol>
		<li>Administer systemic heparin (100 U/kg) via peripheral venous access. Wait 3 min before proceeding with aortic cannulation.</li>
	</ol>
	</li>
	<li>Access the infrarenal aorta using an 18 G micropuncture needle (Figure 4A).</li>
	<li>Pass a j-wire through the needle into the aorta and remove the needle over the wire.</li>
	<li>Place a 10Fr sheath over the wire into the aorta and remove the wire (Figure 4B).</li>
	<li>Connect IV tubing to the 10Fr sheath and in a sterile fashion, infuse UW solution through the sheath. Infuse UW solution at a rate of 40-60 drops/min. 
	NOTE: Flow will not begin until the thoracic aorta is crossclamped. Ensure the intravenous tubing is not on tension to avoid displacement of the intraaortic sheath.</li>
	<li>Perform visceral isolation and perfusion in rapid succession.
	<ol>
		<li>Clamp the infrarenal aorta using an aortic clamp.</li>
		<li>Clamp the descending thoracic aorta using an aortic clamp.</li>
		<li>Tie off the intrathoracic IVC with the previously placed 2-0 tie.</li>
		<li>Administer 100 mg/kg KCl for euthanasia via peripheral venous access. 
		NOTE: Cardiac death commences with the administration of KCl.</li>
		<li>Make a venotomy in the infrarenal IVC, superior to the 2-0 tie, and insert pool-tipped suction through the venotomy (Figure 4C).</li>
		<li>Perfuse the viscera with 2-3 L of cold UW solution.</li>
		<li>Proceed with dissection for total pancreatectomy during the perfusion. 
		NOTE: The viscera, and pancreas in particular, should become pale as the UW solution is perfused into the solid organ region (Figure 5A).</li>
	</ol>
	</li>
</ol>
6. Total pancreatectomy
<ol>
	<li>Release the small bowel from retraction as the UW solution is perfusing.</li>
	<li>Begin dissection of duodenal, connecting, and splenic lobes of the pancreas during the visceral perfusion. Retract the stomach superiorly to expose the pancreas. With a combination of blunt dissection and electrocautery, dissect the pancreas from its omental attachments. 
	NOTE: The body of the pancreas effectively wraps around the portal vein. Take care to avoid the portal vein running posteriorly to the duodenal lobe and then anteriorly to the bridge and connecting lobes.</li>
	<li>Isolate the pancreatic duct (Figure 5B). Locate the pancreatic duct where it enters the duodenum. Ligate the duct using two 3-0 silk ties and transect the duct in between the ties.</li>
	<li>Complete pancreatectomy (Figure 5C).
	<ol>
		<li>Dissect the remainder of the duodenal lobe from the C-loop of the duodenum until it is freely mobile.</li>
		<li>Separate the pancreas from its remaining attachments. Carefully ligate and divide the small vessels from the superior mesenteric artery to the pancreas.</li>
		<li>Separate the pancreas from the portal vein. Dissect the body of the pancreas and the connecting lobe from the portal vein. Ligate any venous branches from the portal vein to the pancreas, including the splenic vein.
		<ol>
			<li>Transect the bridge of the pancreas between the connecting lobe and splenic lobe to allow free mobilization from the portal vein. 
			NOTE: Take care not to disrupt the pancreatic duodenal lobe from the splenic lobe.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Immerse the entire pancreas in cold sterile UW solution, placing the container immediately on ice. Transport the pancreas to a sterile hood for islet isolation. Proceed with pancreatic islet isolation (Figure 6).</li>
</ol>
7. Pancreatic duct cannulation
<ol>
	<li>Place the pancreas on a sterile tray on ice inside the biosafety cabinet.</li>
	<li>Dissect excess fibrous tissue and blood away from the pancreas.</li>
	<li>If necessary, cut the duct transversely to better visualize the lumen. Locate and advance the cannula into the duct with an appropriately sized catheter (16 G).</li>
	<li>Secure the catheter in place with a 2.0 silk tie but avoid occluding the lumen.</li>
	<li>Prepare three decontamination solutions and submerge the cannulated pancreas in each decontamination solution for 60 s.</li>
</ol>
8. Perfusion and distention of the pancreas
<ol>
	<li>Prepare the enzyme digestion solution (Figure 6).
	<ol>
		<li>Mix 100 mg of commercially available collagenase 1 and 2 solutions (see Table of Materials for further details) with the desired amount of 1x HBSS and 10,000 U Heparin solution. If the isolated pancreatic tissue is between 100 g and 200 g, mix the enzyme with 200 mL total of 1x HBSS and Heparin solution.</li>
	</ol>
	</li>
	<li>Infuse the pancreas with the enzyme solution.
	<ol>
		<li>With controlled moderate force, infuse enzyme solution in 60 mL boluses into the pancreatic duct. Continue this process until the entirety of the enzyme solution is infused into the pancreas. 
		NOTE: Effective infusion should cause the pancreas tissue to distend.</li>
		<li>If some enzyme solution leaks out of the pancreas tissue into the sterile tray, re-inject this remaining solution into the pancreas in step 8.3 or place it in the Ricordi chamber in step 9.2.</li>
	</ol>
	</li>
	<li>Directly inject any remaining enzyme solution into any poorly distended pancreatic parenchyma segments using a 22 G needle.</li>
</ol>
9. Digestion of the pancreas
<ol>
	<li>Place circuit reservoir containing 500 mL of 1x HBSS in a water bath set to 47 &#176;C to preheat the reservoir solution. 
	NOTE: The enzyme solution functions optimally at 36-38 &#176;C and this temperature needs to be reached within 5 min of starting the peristaltic pump. The temperature of the contents of the Ricordi, including the enzyme-infused pancreas, is initially 4 &#176;C. By pre-heating the circuit reservoir to 47 &#176;C, the contents of the reservoir and the Ricordi chamber will mix, bringing the entire circuit temperature up to 36-38 &#176;C within 5 min of starting the peristaltic pump.</li>
	<li>Remove and discard the catheter from the pancreatic duct and cut the pancreas into ~1-3 cm pieces.</li>
	<li>Prepare the digestion circuit. The peristaltic pump will circulate solution from the digestion circuit reservoir, through the heating coil, and into the Ricordi chamber (Figure 6).</li>
	<li>Place the pancreas pieces with nine 1.5 cm silicon nitride marbles inside the Ricordi chamber and fill to the filter level with the remaining enzyme solution.</li>
	<li>To seal the Ricordi chamber, place the mesh filter and rubber gasket, followed by the lid onto the Ricordi chamber. Secure the lid of the Ricordi chamber by tightening the screws and ensure that the chamber is well sealed before proceeding.</li>
	<li>Place the heating coil in a 47 &#176;C hot water bath.</li>
	<li>Place the inflow and outflow cannulas into the digestion circuit reservoir containing 500 mL of 1x HBSS.</li>
	<li>Initiate the pump at a flow rate of 450 mL/min until the entire chamber is filled with the digestion solution. Decrease the flow rate to 100 mL/min as the fluid circulates within the circuit.</li>
	<li>Manually shake the digestion chamber to agitate the tissue with the silicon nitride marbles.</li>
	<li>Starting with 8 min of digestion, take a 1 mL sample from the circuit reservoir for Dithizone (DTZ) staining to monitor digestion progress.
	<ol>
		<li>To prepare DTZ staining, dissolve 10 mg of DTZ in 2 mL of Dimethyl Sulfoxide (DMSO).</li>
		<li>Add 10 &#181;L of the prepared DTZ stain to 1 mL of the digestion sample. Inspect the sample under 4x magnification. Islets will appear dark red to black with DTZ staining. 
		NOTE: Digestion is complete when most or all the observed islets are &#39;free&#39; and intact, with the acinar tissue dispersed into smaller clusters.</li>
	</ol>
	</li>
	<li>When digestion is judged complete, transfer the coil to the ice bath, remove the inflow cannula from the reservoir, and increase the pump speed to 210-250 mL/min. Let the digested tissue drain from the Ricordi chamber into the circuit reservoir. 
	NOTE: It is typical to see very little tissue remaining in the Ricordi chamber after digestion. Typically, &#60;10% of undigested pancreatic tissue will remain, although this may vary due to specimen-specific factors.</li>
	<li>Weigh the remaining tissue to determine the efficiency of the digestion. Transfer the digested tissue from the circuit reservoir into 250 mL conical tubes that are maintained cool on ice.</li>
</ol>
10. Collection of pancreatic tissue
<ol>
	<li>Centrifuge the digested pancreatic tissue in 250 mL conical tubes at 250 &#215; g for 1 min at 4 &#176;C without brake.</li>
	<li>Remove the tubes from the centrifuge with caution and aspirate the supernatant using a serological pipette while avoiding disruption of the pellet.</li>
	<li>Resuspend the pellets in each conical tube gently and combine them into one 250 mL conical tube. If there is &#8804;50 mL of tissue, resuspend the pellet to a total volume of 100 mL in Islet Collection Solution. If there is &#62;50 mL of tissue, resuspend the pellet to a total volume of 200 mL in Islet Collection Solution. In the case that over 100 mL of tissue is collected, run two Cell Processor purification cycles.</li>
	<li>Resuspend the tissue in RPMI medium to make a total volume of 100 mL (assuming 50 mL of tissue was collected) and load in the Cell Processor.</li>
</ol>
11. Islet purification using the continuous gradient method
<ol>
	<li>Prime the Cell Processor per manufacturer&#39;s instructions.</li>
	<li>Load 125 mL of High Density gradient solution into the continuous gradient former, then load the High Density gradient solution into the Cell Processor using a pump set at 200 mL/min.</li>
	<li>Start the Cell Processor centrifuge function at 1,000 RPM.</li>
	<li>Release the air from the system and re-prime with High-density Gradient Solution.</li>
	<li>Add 125 mL of High-density gradient solution and 130 mL of Low-density Solution to the gradient former.</li>
	<li>Load the 100 mL of digested tissue suspension.</li>
	<li>Increase the Cell Processor speed to 2,000 RPM for 3 min.</li>
	<li>Prepare 16 50 mL conical tubes for purified islet fraction collection. Add 25 mL of RPMI medium to each tube and arrange all tubes in ascending order.</li>
	<li>Collect the purified islet fractions in the prepared collection conical tubes.</li>
</ol>
12. Islet assessment and staining
<ol>
	<li>Collect 100-200 &#181;L aliquots from each of the purified islet fractions and place them in a 24-well tissue culture plate for DTZ staining to assess islet content and from each purification fraction. Inspect each fraction using light microscopy with 4x magnification. The islets will appear dark red to black with DTZ staining.</li>
	<li>Once the islets are identified, select the fractions with the highest content of islets. Centrifuge the purified fraction at 250 &#215; g for 1 min at 4 &#176;C without brake.</li>
	<li>Carefully aspirate the supernatant and resuspend the islet pellet in islet growth media. Transfer the purified islets to 1-4 T-125 culture flasks.</li>
	<li>Following purification, evaluate the islets for quantity, viability, and function.
	<ol>
		<li>Use an islet cell counter to evaluate the islet number and size distribution (Figure 7A). An entire pancreas will typically yield 1-2 million islet equivalents.</li>
		<li>For viability staining, mix 2 &#181;L of calcein AM and 2 &#181;L of ethidium homodimer-1 (CaAM/EthD-1) with 1,000 &#181;L of PBS (Figure 7B). Allow the islets to be stained for 30 min, then evaluate them with under 4x or 10x magnification. Viable islets will stain green while nonviable islets will stain red.</li>
	</ol>
	</li>
</ol>
13. Cell culture of pancreatic Islets
<ol>
	<li>During the initial 48 h, culture the islets in recovery medium at 37 &#176;C to provide sufficient nutrients (~1,000 islets/mL of recovery medium).</li>
	<li>From days 2-21, culture the islets in standard growth medium at 37 &#176;C.</li>
</ol>

Purification of Porcine Islets by Surgical Perfusion of Pig Pancreas

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R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cost+and+scalability+analysis+of+porcine+islet+isolation+for+islet+transplantation:+comparison+of+juvenile,+neonatal+and+adult+pigs.">Cost and scalability analysis of porcine islet isolation for islet transplantation: comparison of juvenile, neonatal and adult pigs.</a> Cell Transplant. 28 (7), 967-972 (2019).</li><li>Weegman, B. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+profiles+of+different+cooling+methods+in+porcine+pancreas+procurement.">Temperature profiles of different cooling methods in porcine pancreas procurement.</a> Xenotransplantation. 21 (6), 574-581 (2014).</li><li>Hart, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+an+adequate+perfusion+pressure+for+continuous+dual+vessel+hypothermic+machine+perfusion+of+the+rat+liver.">Determination of an adequate perfusion pressure for continuous dual vessel hypothermic machine perfusion of the rat liver.</a> Transpl Int. 20 (4), 343-352 (2007).</li><li>Tullius, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+crystal+deposits+in+abdominal+organs+following+perfusion+with+defrosted+University+of+Wisconsin+solutions.">Accumulation of crystal deposits in abdominal organs following perfusion with defrosted University of Wisconsin solutions.</a> Am J Transplant. 2 (7), 627-630 (2002).</li><li>Ching, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reliable+method+for+isolation+of+viable+porcine+islet+cells.">A reliable method for isolation of viable porcine islet cells.</a> Arch Surg. 136 (3), 276-279 (2001).</li><li>Lu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+pig+islet+isolation.">Adult pig islet isolation.</a> J Vis Exp. (176), (2021).</li><li>Taylor, M. J., Baicu, S., Greene, E., Vazquez, A., Brassil, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+isolation+from+juvenile+porcine+pancreas+after+24-h+hypothermic+machine+perfusion+preservation.">Islet isolation from juvenile porcine pancreas after 24-h hypothermic machine perfusion preservation.</a> Cell Transplant. 19 (5), 613-628 (2010).</li><li>Iwanaga, Y., Sutherland, D. E., Harmon, J. V., Papas, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreas+preservation+for+pancreas+and+islet+transplantation.">Pancreas preservation for pancreas and islet transplantation.</a> Curr Opin Organ Transplant. 13 (4), 445-451 (2008).</li><li>Pratschke, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+death+and+its+influence+on+donor+organ+quality+and+outcome+after+transplantation.">Brain death and its influence on donor organ quality and outcome after transplantation.</a> Transplantation. 67 (3), 343-348 (1999).</li><li>vander Windt, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+islet+damage+after+direct+exposure+of+pig+islets+to+blood:+has+humoral+immunity+been+underestimated.">Early islet damage after direct exposure of pig islets to blood: has humoral immunity been underestimated.</a> Cell Transplant. 21 (8), 1791-1802 (2012).</li><li>Wright, M. J., Cavanagh, T. J., Fetterhoff, T. J., Wile, K. 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The authors report no conflicts of interest.

Pancreatic islet xenotransplantation using porcine donors is a promising strategy for the treatment of Type I diabetes. Islet isolation is challenging, and final islet viability and yield are highly susceptible to hypotension and tissue ischemia encountered during organ procurement16,17. To optimize pancreas procurement and preservation, this protocol presents a new method for aortic cannulation, visceral isolation, and perfusion during the procurement of the por...

Type 1 diabetes is caused by the autoimmune destruction of pancreatic beta cells1.&#160;Consequently, patients are dependent on exogenous insulin, placing them at high-risk glycemic fluctuations with episodes of hyper- and hypoglycemia2. Islet allotransplantation is a potential cure for type I diabetes; however, the limited availability of pancreatic organ donors remains a major barrier to widespread adoption of the procedure2,3. Islet xenotransplantation from porcine donors is a promising alternative as these animals are readily available. Optimization and scaling of porcine pancreatic islet isolation will be critical for progressing islet xenotransplantation.
Although several porcine pancreas procurement techniques have been previously published, many of these procedures describe pancreatectomy after induction of cardiac death or exsanguination4,5,6,7,8,9,10. One major disadvantage of these techniques is the variable warm ischemic time (WIT) occurring between the time of cardiac death and the initiation of intraarterial and/or intraductal infusion of preservation solution. Even 10 min of WIT will negatively impact islet yield and viability5. Minimizing WIT requires prompt perfusion of the pancreas with a preservation solution. During abdominal organ procurement, aortic cannulation followed by intracorporeal perfusion with University of Wisconsin (UW) solution is crucial for removing blood, preventing intravascular thrombosis, protecting against ischemic damage, and minimizing cellular injury11,12.
Previous studies have demonstrated that during porcine pancreas procurement, low-pressure flushing of the celiac trunk and superior mesenteric artery with UW solution improves islet yield and purity4. However, previously published methods for porcine pancreas procurement include open aortic cannulation, which can be technically challenging, particularly for smaller pigs13,14,15. In this manuscript, we present a detailed, step-by-step protocol with accompanying visual aids for the surgical perfusion and procurement of the porcine pancreas, followed by islet isolation and purification. This new technique of aortic cannulation during pancreatic isolation was developed specifically to minimize WIT and allow the pancreatic procurement to be performed in a bloodless field as a means of maximizing islet isolation yield and viability.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 M NaOH, 100 mL</td>
			<td>Millipore Sigma</td>
			<td>1310-73-2</td>
			<td></td>
		</tr>
		<tr>
			<td>10x HBSS, 500 mL (15 to 30 &#176;C)</td>
			<td>Thermo Fisher</td>
			<td>14065056</td>
			<td></td>
		</tr>
		<tr>
			<td>1x D-PBS, 500 mL</td>
			<td>Thermo Fisher</td>
			<td>14200075</td>
			<td></td>
		</tr>
		<tr>
			<td>1x HBSS, 1 L</td>
			<td>Thermo Fisher</td>
			<td>14025076</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL Conical Tubes</td>
			<td>Millipore Sigma</td>
			<td>CLS430236-6EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B [50 mg]</td>
			<td>Millipore Sigma</td>
			<td>PHR1662</td>
			<td></td>
		</tr>
		<tr>
			<td>Antiseptic Povidone Iodine 10%</td>
			<td>Millipore Sigma</td>
			<td>25655-41-8</td>
			<td></td>
		</tr>
		<tr>
			<td>BioRad Gradient Former</td>
			<td>BioRad</td>
			<td>395</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride dihydrate</td>
			<td>Thermo Fisher</td>
			<td>10035-04-8</td>
			<td></td>
		</tr>
		<tr>
			<td>COBE Cell Processor</td>
			<td>Ebay</td>
			<td>317690&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Digestion circuit reservoir, Gibco bottle, 1 L</td>
			<td>Thermo Fisher</td>
			<td>10341001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide (DMSO)</td>
			<td>Thermo Fisher</td>
			<td>85190</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithizone</td>
			<td>Thermo Fisher</td>
			<td>60-10-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Gradient Former Connection Kit</td>
			<td>BioRad</td>
			<td>1652008</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin [1000 U/mL]</td>
			<td>Emergency Medical Products</td>
			<td>0409-2720-01</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffer (1 M), 100 mL</td>
			<td>Thermo Fisher</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Serum Albumin 25%, 100 mL</td>
			<td>Celprogen</td>
			<td>HSA2001-25-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Covetrus</td>
			<td>Item#:11695067772</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Patterson</td>
			<td>Item #:07-894-8462</td>
			<td></td>
		</tr>
		<tr>
			<td>Liberase (100 mg)</td>
			<td>Millipore Sigma</td>
			<td>5401020001</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Viability/Cytotoxicity Kit</td>
			<td>Thermo Fisher</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>Lympholyte 1.1 Media 500 mL</td>
			<td>Cedarlane Labs</td>
			<td>CL5020</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex LS 16 Tubing</td>
			<td>Masterfle</td>
			<td>96419-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex Peristaltic Pump</td>
			<td>Masterflex</td>
			<td>07522-30</td>
			<td></td>
		</tr>
		<tr>
			<td>PenStrep (2 mg/mL)</td>
			<td>Celprogen</td>
			<td>PS-30-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulmozyme (1 mg/mL, 2.5 mL/vial)</td>
			<td>Dornase Alfa</td>
			<td>8931278</td>
			<td></td>
		</tr>
		<tr>
			<td>Recovery Medium, PIM(R)</td>
			<td>Prodo Laboratories, Inc.</td>
			<td>PIM-R001GMP</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Millipore Sigma</td>
			<td>R8758-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Culture Medium, PIM(S)</td>
			<td>Prodo Laboratories, Inc.</td>
			<td>PIM-S001GMP</td>
			<td></td>
		</tr>
		<tr>
			<td>Tzed&#8482; (tiletamine and zolazepam) Injection C</td>
			<td>Patterson</td>
			<td>Item #:07-894-6240</td>
			<td></td>
		</tr>
		<tr>
			<td>University of Wisconsin (UW) Solution, 3 L</td>
			<td>Global Transplant Solutions</td>
			<td>1000-0046-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Waterbath</td>
			<td>Sigma Alderich</td>
			<td>CLS6783-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Patterson</td>
			<td>Item #:07-891-6165</td>
			<td></td>
		</tr>
	</tbody>
</table>

surgical perfusion and isolation of the porcine pancreas for islet isolation

Pancreatic islet transplantation is an emerging treatment for type I diabetes; however, it is limited by donor matching and availability. Porcine islet xenotransplantation offers a promising alternative to allotransplantation, with the potential for large-scale production of on-demand, functional islets. The yield and viability of isolated islets is highly susceptible to the quality of the donor pancreas and the method of procurement, particularly the duration of warm-ischemia time. To improve organ preservation and subsequent islet yield and viability, we have developed a protocol for surgical perfusion and resection of the porcine pancreas. This protocol employs direct infrarenal aortic cannulation and organ perfusion to both minimize warm-ischemia time and simplify the procedure for operators who do not have extensive surgical expertise. Subsequent arterial perfusion of the pancreas via the aorta flushes stagnant blood from the microvasculature, thereby reducing thrombosis and oxidative damage to the tissue. This manuscript provides a detailed protocol for surgical perfusion and resection of the porcine pancreas, followed by islet isolation and purification.

Author Spotlight: Techniques and Challenges in Islet Purification for Xenotransplantation

Surgical Perfusion and Isolation of the Porcine Pancreas for Islet Isolation

Our lab focuses on microencapsulation for pancreatic islet xenotransplantation. This requires reliable islet isolation and purification. We tackle this challenge by isolating the pancreas without damaging it from warm ischemia or mechanical injury.And this offers strategies for effective digestion and a high islet yield. Isolating pancreatic islets is very challenging. Both the yield and purity depend on every phase of the process being executed effectively and efficiently from the organ procurement and digestion to the islet purification.Furthermore, ethical and humane considerations are important when planning animal procedures that will facilitate optimal outcomes while reducing any animal suffering. So here we demonstrate that we can percutaneously cannulate the aorta to selectively perfuse the pancreas. This technique allows us to efficiently preserve the pancreas and then isolate the islets.So a major advantage of our protocol is the use of Seldinger technique for aortic cannulation. This helps maintain a bloodless operative field and preserves the dissection planes. This technique can also be easily adapted for smaller pigs simply by using a smaller sheath.Our lab is currently investigating methods for creating an on-demand supply of pancreatic islets. Specifically, we're interested in seeing if encapsulating islets in a hydrogel can prolong their lifespan and improve their function in vitro and in culture. The ultimate goal is to reliably isolate porcine islets to use it in transplantation research, or even for xenotransplantation.

The operative setup and midline laparotomy is shown in Figure 1. The laparotomy incision should be curved to avoid the urogenital opening (Figure 1B). When setting up the retractor, attach the post for the Omni or Bookwalter retractor to the left, inferior corner of the table. Ideal retraction includes two retractor blades for the right abdominal wall, two for the left abdominal wall, and 1-2 for the right colon and small bowel wrapped in a sterile towel (<stron...

This protocol provides a step-by-step guide for the procurement of a porcine pancreas for islet isolation and purification.

Pancreatic islet transplantation is an emerging treatment for type I diabetes; however, it is limited by donor matching and availability. Porcine islet ...

surgical-perfusion-isolation-porcine-pancreas-for-islet

Research

JoVE Journal

Medicine

Dissection and Surgical Perfusion of Porcine Pancreas for Islet Isolation

Begin by preparing for the surgery. To perform retroperitoneal dissection, incise the peritoneum to the right of the midline from the level of the right kidney to the aortic bifurcation using electrocautery. Sharply expose the right kidney, right renal vein, right renal artery, and inferior vena cava, as well as the aorta.Using a large vessel loop, obtain circumferential control of the distal infrarenal aorta. Then, obtain circumferential control of the mid infrarenal IVC. And ligate the IVC using a 2-0 silk tie.Next, obtain circumferential control of the right and left renal arteries and veins. Ligate these structures using 2-0 silk ties. Proceed with intrathoracic dissection by retracting the liver inferiorly to expose the right hemidiaphragm.Apply two Allis clamps to the membranous portion of the right hemidiaphragm, and incise the diaphragm in between the two clamps using electrocautery. Extend the diaphragm myotome horizontally to approximately seven centimeters. Next, obtain circumferential control of the intrathoracic IVC.Place a 2-0 silk tie around the intrathoracic IVC and snap the ends of the suture with a small hemostat without occluding the IVC. Then, use two Allis clamps to retract the edge of the diaphragmotomy inferiorly. And extend it to the left hemidiaphragm.Administer systemic heparin to the pig for anticoagulation via peripheral venous access, and wait for at least three minutes before proceeding. Using an 18-gauge micropuncture needle, access the infrarenal aorta. Then, pass a J wire through the needle into the aorta and remove the needle over the wire.Place a 10 French sheath over the wire into the aorta and remove the wire. Next, connect IV tubing to the 10 French sheath, and infuse sterile UW solution through the sheath at a rate of 40 to 60 drops per minute in a sterile fashion. Then, using an aortic clamp, clamp the infrarenal aorta and the descending thoracic aorta.Tie the intrathoracic IVC with the previously placed 2-0 tie, and administer potassium chloride via peripheral venous access for euthanasia. Then, isolate the pancreatic duct by locating it at the entry of the duodenum. Ligate the duct using two 3-0 silk ties and transect the duct between the ties.Complete the pancreatectomy. To infuse the pancreas with the enzyme solution, pass 60-milliliter boluses into the pancreatic duct until the entire enzyme solution is infused. Placed the pancreas in the Ricordi chamber along with the mesh filter and rubber gasket.And secure the lid onto the Ricordi chamber to seal it. Then, place the heating coil in a hot water bath set to 47 degrees Celsius. Insert the inflow and outflow cannulas into the digestion circuit reservoir containing 500 milliliters of 1X HBSS.Initiate the pump at a flow rate of 450 milliliters per minute until the entire chamber is filled with the digestion solution. Then, decrease the flow rate to 100 milliliters per minute as the fluid circulates within the circuit.

Continuous Gradient Method for Porcine Islet Purification

To begin, set a pump to a flow rate of 200 milliliters per minute and load 125 milliliters of high-density gradient solution into the continuous gradient former, and then into the cell processor. Start the cell processor centrifuge function at 1, 000 RPM. Release air from the system and rep-rime it with a high-density gradient solution.Then add 125 milliliters of high-density gradient solution and 130 milliliters of low-density solution to the gradient former. Load 100 milliliters of digested tissue suspension. Increase the cell processor speed to 2, 000 RPM for three minutes.To prepare 16 50 milliliter conical tubes for collecting purified islet fraction, add 25 milliliters of RPMI medium to each tube and arrange them in ascending order. Collect the purified islet fractions in the prepared conical tubes. To stain the islets, collect 100 to 200 microliter aliquots from each purified islet fraction, and assess islet content using DTZ staining in a 24-well tissue culture plate.Inspect each fraction using light microscopy at 4X where the islets will appear dark red to black. Using this protocol, pancreatic islet isolation was performed on three pigs. Brightfield images showed purified pancreatic islets, which were characterized for viability using live/dead staining.Representative islet size distribution after the islet isolation showed size distribution around 50 to 100 microns. Further, yield and purity were evaluated using an islet cell counter, indicating efficient porcine pancreatic isolations.

647 Views.  Washington University School of Medicine.  This protocol provides a step-by-step guide for the procurement of a porcine pancreas for islet isolation and purification.

Purification of Porcine Islets by Surgical Perfusion of Pig Pancreas

Summary