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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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><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 &amp;deg;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&amp;nbsp;</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&amp;trade; (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.

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...

Author Spotlight: Techniques and Challenges in Islet Purification for Xenotransplantation

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

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 ...

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

Research

JoVE Journal

Medicine

1000 Views.  Washington University School of Medicine. Ons lab richt zich op micro-inkapseling voor xenotransplantatie van pancreaseilandjes. Dit vereist een betrouwbare isolatie en zuivering van eilandjes. We pakken deze uitdaging aan door de alvleesklier te isoleren zonder deze te beschadigen door warme ischemie of mechanisch letsel.En dit biedt strategieën voor een effectieve vertering en een hoge eilandjesopbrengst. Het isoleren van eilandjes van de alvleesklier is een grote uitdaging. Zowel de opbren...

Video: Auteur in de schijnwerpers: technieken en uitdagingen bij eilandjeszuivering voor xenotransplantatie

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

Developmental Biology

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Biology

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Isolation and Decellularization of a Whole Porcine Pancreas

Tissue engineering of the whole pancreas can improve current treatments for diabetes mellitus. The ultimate goal is to tissue engineer pancreas from an allogeneic or xenogeneic source with human cells. A demonstration of methods for the efficient dissection, decellularization, and recellularization of porcine pancreas might benefit the field. Akin to human pancreases, porcine pancreases have a special anatomical arrangement with three lobes (splenic, duodenal, and connection) rounded by the duodenum and small intestine. The duodenal lobe of the pancreas connects to the duodenum by several small blood vessels. Tissue engineering of the pancreas is complicated because of its exocrine and endocrine nature. In this paper, we show a detailed protocol to dissect the whole porcine pancreas and decellularize it with detergents while saving its structure and some extracellular matrix components. To achieve complete perfusion, the aorta is chosen as inlet and the portal vein as outlet. The other blood vessels (hepatic artery, splenic vein, splenic artery,&#160;mesenteric artery and vein tree) and bile duct are ligated. To prevent the formation of thrombus, the pig is heparinized and, immediately after dissection, the organ is flushed with cold heparin. To inhibit the action of exocrine enzymes, the pancreas decellularization is set at 4 &#176;C. The decellularization is performed by perfusion of Triton X-100, sodium deoxycholate, and deoxyribonuclease, with an intermittent and final extensive washing. With a successful decellularization, the pancreas appears white, and a histological evaluation with hematoxylin and eosin shows an absence of nuclei with a preserved extracellular matrix structure. Thus, the proposed method can be used to successfully dissect and decellularize whole porcine pancreas.

Tissue engineering of the whole pancreas is a challenge because of its exocrine and endocrine functions. We show a method for the dissection of an intact porcine pancreas and the process of successful decellularization by perfusion of detergents Triton X-100, sodium deoxycholate, and deoxyribonuclease.

Tissue engineering of the whole pancreas can improve current treatments for diabetes mellitus. The ultimate goal is to tissue engineer pancreas from an ...

Bioengineering

Surgical Tips and Tricks for Performing Porcine Pancreas Transplantation

Despite the promising results of pancreas transplantation in type 1 diabetes mellitus and metabolic syndrome, the biggest concern around this state-of-the-art technique remains the paucity of organs deemed fit for transplantation. High intravascular resistance, delicate intraparenchymal capillary framework, and complex lobular anatomy around the mesenteric vasculature are what make this organ more susceptible to injury and less tolerant to trivial trauma compared to organs such as the liver and kidney. Meticulous surgical dissection and judicious tissue handling form the cornerstone of the entire exercise of pancreas transplantation. Owing to morphological similarity between the anatomy of the porcine pancreas to the surrounding mesenteric vessels and the organs when compared to the human anatomy, demonstration of the technique in the porcine model could help to most accurately extrapolate this to a human setting. The present article aims to outline the essential surgical tips and tricks that need to be followed, in order to ensure a higher success rate of transplantation of this highly susceptible organ in a porcine 3-day survival model.

The video article summarizes the technique of pancreatectomy and pancreas allotransplantation in a porcine 3-day survival model with a step-by-step description of the method and emphasis on the surgical tips and tricks to deal with the precarious and delicate porcine visceral anatomy.

Despite the promising results of pancreas transplantation in type 1 diabetes mellitus and metabolic syndrome, the biggest concern around this ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Adult Pig Islet Isolation

Type 1 diabetes mellitus (T1DM) is caused by autoimmune destruction of pancreatic &#946; cells, which results in little or no insulin production. Islet transplantation plays an important role in the treatment of T1DM, with the improved glycometabolic control, the reduced progression of complications, the reduction of hypoglycemic episodes when compared with traditional insulin therapy. The results of phase III clinical trial also demonstrated the safety and efficacy of islet allotransplantation in T1DM. However, the shortage of pancreas donors limits its widespread use. Animals as a source of islets such as the pig offer an alternative choice. Because the architecture of the pig pancreas is different from the islets of mice or humans, the pig islet isolation procedure is still challenging. Since the translation of alternative porcine islet sources (xenogeneic) to the clinical setting for treating T1DM through cellular transplantation is of great importance, a cost-effective, standardized, and reproducible protocol for isolating porcine islets is urgently needed. This manuscript describes a simplified and cost-effective method to isolate and purify adult porcine islets based on the previous protocols that have successfully transplanted porcine islets to non-human primates. This will be a beginners guide without the use of specialized equipment such as a COBE 2991 Cell Processor.

This detailed protocol covers the methodological steps of adult pig islet isolation from the digestion phase via purification to the final functional assessment of the islets. This outline can be used as a guideline for adult pig islet isolation in research institutions.

Type 1 diabetes mellitus (T1DM) is caused by autoimmune destruction of pancreatic &#946; cells, which results in little or no insulin production. Islet ...

Surgical Perfusion and Isolation of the Porcine Pancreas for Islet Isolation

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