This study was financed by a grant from the Swedish Government LUA ALF to S.S.H.

The dissection of a porcine pancreas and the decellularization procedure presented here follow the ethical guidelines of the University of Gothenburg.
1. Preparation of the Decellularization Set-up
<ol>
	<li>Using 3 x 5 mm silicone tubes, connect in series the detergent inlet container to the peristalic pump and then to the pancreas in the organ chamber via the degasser (see Figure 1). Connect a male luer to the free end of tube in organ chamber.</li>
	<li>Using another 3 x 5 mm silicon tube, connect the organ chamber to the detergent outlet&#160;container via the peristaltic pump to collect the perfused detergent.</li>
	<li>Connect a 2 ml unlabeled pipette to the free ends of tubes in detergent inlet container and detergent outlet cointainer.</li>
	<li>Keep the whole set-up&#160;at 4 &#176;C.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58302/58302fig1v2.jpg" style="opacity: 0.9; font-size: 14px;" /> 
Figure 1: Preparation of the perfusion set-up.&#160;Using a 3 x 5 mm silicone tube, as shown in the set-up, connect in series the detergent inlet container to the peristaltic pump, the degasser and the organ chamber. The black arrows show the flow direction from detergent inlet container to organ chamber. For the detergent outlet, use another 3 x 5 mm silicone tube and connect the organ chamber&#160;via&#160;the peristaltic pump to the detergent outlet container. The red arrows show the flow direction from organ chamber to detergent outlet container.&#160;<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58302/58302fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Preparation of Decellularization Solutions
<ol>
	<li>Solution 1 (phosphate-buffered saline [PBS]): Add 8 g of (137 mM) sodium chloride, 0.2 g of (2.7 mM) potassium chloride, 1.44 g of (10 mM) sodium phosphate, and 0.24 g of (1.8 mM) potassium phosphate to 1 L of ultrapure water and stir until dissolved. Adjust the pH to 7.4 with hydrochloric acid (HCl). In total, 3.2 L of this solution is required.</li>
	<li>Solution 2 (PBS + heparin): To 1 L of Solution 1, add 3.4 mL of heparin (17 international units [IU]/mL). Prepare this solution fresh and keep it on ice until it becomes cold. In total, 1.2 L of this solution is required.</li>
	<li>Solution 3 (ultrapure water + sodium azide + disodium ethylenediaminetetraacetic acid [EDTA]): To 1 L of ultrapure water, add 1.86 g of (5 mM) EDTA and 200 mg of (0.02%) sodium azide. Stir until the salts are dissolved. Cool the solution to 4 &#176;C before use. In total, 22 L of this solution is required.</li>
	<li>Solution 4 (PBS + sodium azide + EDTA): To 1 L of Solution 1, add 200 mg of (0.02%) sodium azide and 1.86 g of (5 mM) EDTA. Stir until the salts are dissolved. Cool the solution to 4 &#176;C before use. In total, 1 L of this solution is required.</li>
	<li>Solution 5 (ultrapure water + sodium azide): To 1 L of ultrapure water, add 200 mg of (0.02%) sodium azide. Stir until the salts are dissolved. Cool the solution to 4 &#176;C before use. In total, 260 L of this solution is required. 
	NOTE: EDTA forms a precipitate with SDC and was excluded from Solution 5 since it will be used for immediate washing before and after the SDC treatment.</li>
	<li>Solution 6 (SDC + Triton X-100): To 940 mL of ultrapure water, add 40 g of (4%) SDC, 60 mL of (6%) Triton X-100, 200 mg of (0.02%) sodium azide, and 69.6 mg of (0.4 mM) phenylmethylsulfonyl fluoride (PMSF). Stir until dissolved. Cool the solution to 4 &#176;C before use. Add PMSF before use. In total, 9.6 L of this solution is required.</li>
	<li>Solution 7 (DNase): Add 10,000 Kunitz units of DNase-I in 250 mL of (40 Kunitz units/mL) Dulbecco&#39;s PBS containing CaCl2 and MgCl2. Prepare fresh and use immediately. Warm the solution to 37 &#176;C before use.</li>
	<li>Solution 8 (PBS + sodium azide): To 1 L of Solution 1, add 200 mg of (0.02%) sodium azide. Stir until the salts are dissolved. Cool the solution to 4 &#176;C before use. In total, 1 L of this solution is required.</li>
</ol>
3. Dissection of the Porcine Pancreas
NOTE: In this study, porcine pancreases were dissected from euthanized, heparinized (400 IU/kg) female pigs weighing 45 kg from a farm.
<ol>
	<li>Place the pig on the dissection table in the supine position.</li>
	<li>Make a midline incision from the xiphoidal process to the pubic bone (approximately 40 cm) using a scalpel, exposing all abdominal organs.</li>
	<li>Locate the splenic, duodenal, and connection lobes.</li>
	<li>Locate the level of the major duodenal papilla and ligate the duodenum orally from that site using two separate sutures.</li>
	<li>Ligate the inferior&#160;esophagus with two separate sutures and cut between the ligatures with scissors to take the stomach out.</li>
	<li>Separate the connective tissue from the colon to reach the small intestine.</li>
	<li>Separate the connective tissue of the colon that attaches to the splenic lobe of the pancreas.</li>
	<li>Remove the colon from the small intestine after ligating the arteries.</li>
	<li>Find the inferior mesenteric vein and the inferior mesenteric artery tree&#160;and ligate with one suture where they appear caudally of the pancreas.</li>
	<li>Ligate the splenic artery and vein together with one suture, close to the spleen, in the hilum, and cut distally with scissors to remove the spleen.</li>
	<li>Follow the duodenum until the duodenal and connection lobes are cleared and ligate the duodenum at the end with two separate sutures.</li>
	<li>Dissect the portal vein,&#160;ligate it with one suture to prevent any blood leakage from the liver and cut proximally to ligature. The portal vein serves as an outlet during the decellularization.</li>
	<li>Dissect and ligate the bile duct and hepatic artery with two sutures. Cut distally to sutures.</li>
	<li>Find the aorta under the renal vein and dissect it in the cranial direction from muscle and connective tissue until it reaches the pancreatic area.</li>
	<li>Flip the pancreas gently over and dissect the aorta, keeping the SMA and the CT intact, and cut the aorta superior to CT and inferior to SMA&#160;with scissors.</li>
	<li>Cut the remaining surrounding tissues with scissors and take the pancreas out.</li>
	<li>Using a 50-mL syringe connected to 4-mm arteriotomy cannula, flush the organ through the aorta with Solution 2 until it perfuses the whole organ or until the whole organ becomes cold.</li>
</ol>
4. Preparation of the Porcine Pancreas for Decellularization
<ol>
	<li>Keep the pancreas at 4 &#176;C or on ice throughout the process.</li>
	<li>Cut the sutures of the duodenum using scissors, clean it of food by flushing 50 - 150 mL of ultrapure water using a 25-mL pipette, and ligate it again with sutures.</li>
	<li>Ligate one end of the aorta and all branches besides the SMA and CT with sutures to prevent leakage.</li>
	<li>Insert from the other end of the aorta a 4-mm arteriotomy cannula and ligate it with sutures.</li>
	<li>Perfuse the pancreas with Solution 3 for 1 h at 20 mL/min using the decellularization set-up. 
	NOTE: Prefill the tubes of pump with solution 3 such that no bubbles enter the pancreas. Look for any leakages from all sides of the organ and ligate all open vascular branches with sutures, except the portal vein.</li>
	<li>Freeze the pancreas at -20 &#176;C in Solution 4 until the start of the decellularization.</li>
</ol>
5. Decellularization of the Porcine Pancreas
<ol>
	<li>Thaw the pancreas at 4 &#176;C.</li>
	<li>Run the peristaltic pump in the decellularization set-up with Solution 3 at 20 mL/min until no air bubbles are seen in the detergent inlet tube.</li>
	<li>Place the pancreas in the decellularization container and connect the detergent inlet tube to the aorta of the pancreas. Wash the organ by perfusion with Solution 3 overnight at 20 mL/min at 4 &#176;C.</li>
	<li>Pour out the solution left in the organ chamber.</li>
	<li>Replace Solution 3 with Solution 5 and perfuse the pancreas for 30 min at 20 mL/min at 4 &#176;C. Pour out the solution in the organ chamber.</li>
	<li>Add Solution 6 and perfuse the pancreas for 8 h at 20 mL/min at 4 &#176;C. Pour out the solution in the organ chamber.</li>
	<li>Wash the organ by perfusion with Solution 5 for 96 h at 20 mL/min at 4 &#176;C. Pour out the solution in the organ chamber.</li>
	<li>Prepare the pancreas for DNase treatment by recirculating 500 mL of Dulbecco&#39;s PBS containing CaCl2 and MgCl2 for 30 min at 37 &#176;C. Pour out the solution in the organ chamber.</li>
	<li>Add 250 mL of Solution 7 and perfuse the pancreas for 4 h at 20 mL/min at 37 &#176;C. Pour out the solution in the organ chamber.</li>
	<li>Wash the organ by perfusion with Solution 5 for 120 h at 20 mL/min at 4 &#176;C.</li>
	<li>Store the organ in Solution 8 at 4 &#176;C for short periods or at -20 &#176;C for long periods.</li>
</ol>
6. Verification of Decellularization
<ol>
	<li>Using scissors, cut 3- to 10-mm biopsies from all lobes of the pancreas and fix them in formaldehyde for 48 h at room temperature.</li>
	<li>Wash the pieces in ultrapure water for 15 min, process them in a tissue processor following standard protocols, and embed them in paraffin.</li>
	<li>Cut 5-&#181;m sections using microtome and stain them by Meyer&#39;s hematoxylin and 0.2% alcoholic eosin (HE) following standard protocols.</li>
	<li>View the slides under a light microscope to check for the loss of nuclei. 
	NOTE: A piece from a fresh tissue processed in the same way can be used as a control to check for the presence of nuclei.</li>
</ol>

<ol>
	<li>Yang, H. K., Yoon, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+encapsulated+islet+transplantation.">Current status of encapsulated islet transplantation.</a> Journal of Diabetes and its Complications. 29 (5), 737-743 (2015).</li><li>Badylak, S. F., Taylor, D., Uygun, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-organ+tissue+engineering:+Decellularization+and+recellularization+of+three-dimensional+matrix+scaffolds.">Whole-organ tissue engineering: Decellularization and recellularization of three-dimensional matrix scaffolds.</a> Annual Review of Biomedical Engineering. 13, 27-53 (2011).</li><li>Crapo, P. M., Gilbert, T. W., Badylak, S. 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K., 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=Perfusion-decellularized+pancreas+as+a+natural+3D+scaffold+for+pancreatic+tissue+and+whole+organ+engineering.">Perfusion-decellularized pancreas as a natural 3D scaffold for pancreatic tissue and whole organ engineering.</a> Biomaterials. 34 (28), 6760-6772 (2013).</li><li>Wu, 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=3D+Culture+of+MIN-6+Cells+on+Decellularized+Pancreatic+Scaffold.">3D Culture of MIN-6 Cells on Decellularized Pancreatic Scaffold.</a> In Vitro and In Vivo Study. 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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 Transplantation. 19 (5), 613-628 (2010).</li><li>Elebring, E., Kuna, V. K., Kvarnstrom, N., Sumitran-Holgersson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold-perfusion+decellularization+of+whole-organ+porcine+pancreas+supports+human+fetal+pancreatic+cell+attachment+and+expression+of+endocrine+and+exocrine+markers.">Cold-perfusion decellularization of whole-organ porcine pancreas supports human fetal pancreatic cell attachment and expression of endocrine and exocrine markers.</a> Journal of Tissue Engineering. 8, 2041731417738145 (2017).</li><li>Gilpin, A., Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+Strategies+for+Regenerative+Medicine:+From+Processing+Techniques+to+Applications.">Decellularization Strategies for Regenerative Medicine: From Processing Techniques to Applications.</a> Biomed Research International. 2017, 9831534 (2017).</li><li>James, G. 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S.S.H. holds shares in Verigraft, a company that has licensed the technology of blood vessel tissue engineering. The other authors have nothing to disclose.

The proposed protocol, using perfusion of SDC and Triton X-100 at 4 &#176;C, will decellularize whole porcine pancreas successfully. The challenge in this technique is the dissection of the intact pancreas containing all the three lobes without damaging the parenchyma and its supplying vessels, as well as the ligation of the other vascular branches of the specimen in order to perfuse the organ without leakage. The porcine pancreas has a different anatomy compared to the human pancreas. It consists of three lobes and stays in close contact with the small intestine by partly surrounding it. We dissected the duodenum together with the pancreas, as several small blood vessels from pancreas connect the intestine. During the optimization studies, blunt cutting of these blood vessels has shown leakage and an incomplete perfusion of solutions.
Since the aorta connects to the pancreas through the coeliac and superior mesenteric arteries, we chose the aorta as an inlet to keep the perfusion simple by only using one cannula and, therefore, one inlet. In our experience, ligation of the aorta above the CT and below the SMA will decrease the time of dissection and reduces the risk of damaging any of the two vessels. In addition to a heparinized pig, we also noticed that a perfusion of cold heparin via the aorta immediately after the dissection helps in achieving perfusion of solutions throughout the organ. We speculate if this occurs by preventing the formation of blood clots in the blood vessels. The initial perfusion of a pancreas with ultrapure water after dissection will lyse red blood cells and remove the blood remnants in the organ, thereby preventing the formation of blood clots. This period can also be used to find any unligated small branches of veins and arteries, as blood flow can be easily noticed above the background.
We chose to keep the whole decellularization procedure at cold temperatures (4 &#176;C), as this will hinder the action of exocrine enzymes that release from exocrine cells of the pancreas. The exocrine enzymes, when not inhibited, can cause a deleterious effect on cells and the ECM, as they can digest cell membranes and proteins12. As freezing and thawing can effectively burst the cells, we included a freeze/thaw step, initially even before the perfusion of detergents4,13. The initial wash after thawing will remove the remnants of cell bursts. The detergent treatment we used is a mix of SDC and Triton X-100 at unusually high concentrations and at a high perfusion speed. We chose this approach to achieve faster decellularization by removing the exocrine cells that damage the ECM. We speculate that a hard and fast protocol is beneficial for pancreas decellularization, as less time will be available for pancreatic enzymes to interact with the ECM, thus preserving good ECM components. To preserve the ECM components, we also added serine protease inhibitor (PMSF) to the detergent solutions, as that will inhibit the activation of enzymes released from exocrine cells14. Sodium azide is added to all decellularization solutions, as it acts as a bacteriostatic agent, thereby inhibiting the chance of bacterial contamination15.
The pancreas decellularized following this protocol showed a preservation of ECM structures and the ECM proteins collagen and elastin. However, a significant loss of glycosaminoglycans was noticed in the decellularized pancreas. The pancreas decellularized in this fashion also showed promise for the attachment of human fetal pancreatic stem cells and the expression of exocrine and endocrine markers in pieces recellularized for 14 days12. However, to generate an intact and functional pancreas, further research is required in evaluating correct cell sources, cell types, cell seeding strategies, and bioreactor culture.

Diabetes mellitus is characterized by the presence of increased levels of glucose in blood. It is recognized as a major public health challenge in most countries1. High levels of blood glucose affect the blood vessels and nervous system, causing damage to the eyes, the heart, and the kidneys, and extremity ischemia. Traditional methods of treatment include injections of exogenous insulin, drugs, and lifestyle changes. Putting aside a cure for the disease, in some cases, available treatments fail to maintain the insulin at therapeutic levels, resulting in hyperglycemia. Although the transplantation of islets or whole pancreas eliminates the disease, it is not commonly done because of a shortage of suitable donor organs and because of the risks and difficulties involved from immunosuppression and encapsulation2.
Current improvements in the field of tissue engineering and regenerative medicine possess the capacity for providing a solution for these issues. With the technique of decellularization, the cellular material from a human or animal donor can be removed while the important extracellular matrix (ECM) proteins, growth factors, and signaling molecules are preserved in the scaffold. Such scaffolds can potentially be transplanted without the need for immunosuppression, to restore the organ function after recellularization with the recipient&#39;s own non-immunogenic stem cells3,4. The tissue-engineered organs from allogeneic or xenogeneic sources can be used in clinical transplantation, as the major extracellular matrix proteins are conserved among species and might not be rejected after transplantation5.
Decellularization is a well-explored method involving the optimal use of physical forces, chemical detergents, and enzymes in a physiological setting to remove cells and nuclear material from a tissue or organ. Recellularization is a procedure of seeding cells back into the acellular organ. It is an intellectually tough procedure, requiring a large number of cells, an optimum cell-seeding strategy, and a bioreactor system for the culture of the organ at physiologically acceptable conditions like temperature, pressure, and gases6.
The pancreas can be considered a challenging tissue for tissue engineering because of its exocrine and endocrine capacities. The exocrine tissue secretes several digestive enzymes, while the endocrine part secretes hormones, including insulin. The decellularization of intact pancreases from mouse7,8, human9, and pig10 has already been reported using enzymes (trypsin, deoxyribonuclease [DNase]) and non-ionic (Triton X-100) and ionic detergents (sodium deoxycholate [SDC] and sodium dodecyl sulfate [SDS]). However, following the published protocols, we struggled with a successful dissection and complete perfusion and decellularization while maintaining an ECM structure. We speculated that the applied detergents during the decellularization cause lysis of the cells, thereby releasing digestive enzymes into the organ. The released enzymes will cause an irreversible damage to the ECM scaffold and make it inefficient for decellularization and recellularization. A design of the method that effectively decellularizes pancreas while inhibiting the action of digestive enzymes may solve the problem. We chose the strategy of Peloso et al., of decellularization of the pancreas at a cold temperature, although they did not report on why cold temperature is used9. At the same time, we designed a dissection strategy with modifications from Taylor et al. by choosing the aorta as a perfusion inlet over the coeliac trunk (CT) and the superior mesenteric artery (SMA)11.
In a recently published article12, we demonstrate a method for the effective isolation and decellularization of porcine pancreas while preserving some ECM components. In this paper, we show a detailed description of how to dissect a whole porcine pancreas containing splenic, duodenal, and connection lobes, and present a stepwise protocol for successful decellularization.

<table><tbody><tr><td>4mm DLP arteriotomy cannula</td><td>Medtronic</td><td>31104</td><td></td></tr><tr><td>2ml Unlabelled pipette</td><td>vWR</td><td>612-3720</td><td></td></tr><tr><td>Degasser</td><td>Biotech AB</td><td>0001-6484</td><td></td></tr><tr><td>DNase-I</td><td>Worthington</td><td>LS0020007</td><td></td></tr><tr><td>Dulbecco&#039;s PBS with CaCl&lt;sub&gt;2&lt;/sub&gt; and MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>D8662</td><td></td></tr><tr><td>EDTA disodium salt dihydrate</td><td>AlfaAesar</td><td>A15161.OB</td><td></td></tr><tr><td>Heparin</td><td>Leo</td><td>387107</td><td></td></tr><tr><td>Luer Male with 1/8&quot; ID Barb</td><td>Oina</td><td>LM-2PP-QC</td><td>For 3X5mm silicon tube</td></tr><tr><td>Peristaltic pump</td><td>Oina</td><td>SP-1X4</td><td></td></tr><tr><td>PMSF</td><td>Roche</td><td>10837091001</td><td>Unstable in aqueous solution. Should be added fresh before perfusion.</td></tr><tr><td>Potassium chloride</td><td>Sigma Aldrich</td><td>P5405</td><td></td></tr><tr><td>Potassium hydrogen phosphate</td><td>Sigma Aldrich</td><td>P9791</td><td></td></tr><tr><td>SDC</td><td>Sigma Aldrich</td><td>30970</td><td></td></tr><tr><td>Silicon tube 3X5mm</td><td>VWR</td><td>2280706</td><td></td></tr><tr><td>Sodium Azide</td><td>Sigma Aldrich</td><td>71290</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma Aldrich</td><td>13423</td><td></td></tr><tr><td>Sodium hydrogen phosphate</td><td>Merck</td><td>71640-M</td><td></td></tr><tr><td>Suture</td><td>V&amp;ouml;mel</td><td>14817</td><td></td></tr><tr><td>Syrringe 50mL</td><td>Becton Dickinson</td><td>300137</td><td></td></tr><tr><td>Triton-X-100</td><td>AlfaAesar</td><td>A16046.OF</td><td></td></tr></tbody></table>

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.

Representative porcine pancreas dissection pictures, which can help in locating and dissecting the inferior mesenteric artery and vein tree, the portal vein, the hepatic artery, the bile duct, and the aorta branching to CT and SMA, are shown in Figure 2A, 2B, and 2C (yellow arrows), respectively. Figure 3A shows the gross morphology of a normal pancreas, which appears light pink and contains splenic, connection, and duodenal lobes. After decellularization, the pink color is lost and the decellularized pancreas looks pale white in color. The gross morphology picture showing splenic, connection, and duodenal lobes of a decellularized pancreas is shown in Figure 3B. Figure 3C shows the presence of many blue nuclei in a normal pancreas by staining with HE. In a decellularized pancreas, the HE staining showed a loss of nuclei, as no blue nuclei are seen (Figure 3D).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58302/58302fig2v2.jpg" /> 
Figure 2: Pictures of a porcine pancreas dissection. (A) Location of the inferior mesenteric artery and vein tree (yellow arrow). (B) Ligation of the portal vein, the hepatic artery and the bile duct (yellow arrow). (C) Aorta branching to the coeliac trunk and the superior mesenteric artery (yellow arrow). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58302/58302fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58302/58302fig3.jpg" /> 
Figure 3: Gross morphology and HE staining of normal and decellularized pancreases. (A) Gross morphology of a normal pancreas. (B) Gross morphology of a decellularized pancreas. (C) HE staining shows the presence of blue nuclei in a normal pancreas. (D) HE staining shows the absence of blue nuclei in a decellularized pancreas. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/58302/58302fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation and Decellularization of a Whole Porcine Pancreas at JoVE.com

Isolation and Decellularization of a 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 ...

isolation-and-decellularization-of-a-whole-porcine-pancreas

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

9.4K Views.  University of Gothenburg. 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.

Video: Isolation and Decellularization of a Whole Porcine Pancreas

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 ...

Medicine

Procedure for Decellularization of Porcine Heart by Retrograde Coronary Perfusion

Perfusion-based whole organ decellularization has recently gained interest in the field of tissue engineering as a means to create site-specific extracellular matrix scaffolds, while largely preserving the native architecture of the scaffold. To date, this approach has been utilized in a variety of organ systems, including the heart, lung, and liver 1-5. Previous decellularization methods for tissues without an easily accessible vascular network have relied upon prolonged exposure of tissue to solutions of detergents, acids, or enzymatic treatments as a means to remove the cellular and nuclear components from the surrounding extracellular environment6-8. However, the effectiveness of these methods hinged upon the ability of the solutions to permeate the tissue via diffusion. In contrast, perfusion of organs through the natural vascular system effectively reduced the diffusion distance and facilitated transport of decellularization agents into the tissue and cellular components out of the tissue. Herein, we describe a method to fully decellularize an intact porcine heart through coronary retrograde perfusion. The protocol yielded a fully decellularized cardiac extracellular matrix (c-ECM) scaffold with the three-dimensional structure of the heart intact. Our method used a series of enzymes, detergents, and acids coupled with hypertonic and hypotonic rinses to aid in the lysis and removal of cells. The protocol used a Trypsin solution to detach cells from the matrix followed by Triton X-100 and sodium deoxycholate solutions to aid in removal of cellular material. The described protocol also uses perfusion speeds of greater than 2 L/min for extended periods of time. The high flow rate, coupled with solution changes allowed transport of agents to the tissue without contamination of cellular debris and ensured effective rinsing of the tissue. The described method removed all nuclear material from native porcine cardiac tissue, creating a site-specific cardiac ECM scaffold that can be used for a variety of applications.

A method to rapidly and completely remove cellular components from an intact porcine heart through retrograde perfusion is described. This method yields a site specific cardiac extracellular matrix scaffold which has the potential for use in multiple clinical applications.

Perfusion-based  whole organ decellularization has recently gained interest in the field of  tissue engineering as a means to create site-specific ...

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

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix (ECM) Production

Islet transplantation (ITx) has the potential to become the standard of care in beta cell replacement medicine but its results remain inferior to those obtained with whole pancreas transplantation. The protocols currently used for human islet isolation are under scrutiny because they are based on the enzymatic digestion of the organ, whereby the pancreas is demolished, its connections to the body are lost and islets are irreversibly damaged. Islet damage is characterized by critical factors such as the destruction of the extracellular matrix (ECM), which represents the 3D framework of the islet niche and whose loss is incompatible with islet euphysiology. Researchers are proposing the use of ECM-based scaffolds derived from the mammalian pancreas to address this problem and ultimately improve islet viability, function, and lifespan. Currently available methods to obtain such scaffolds are harsh because they are largely detergent based. Thus, we propose a new, detergent-free method that creates less ECM damage and can preserve critical components of pancreatic ECM. The results show that the newly developed decellularization protocol allowed the achievement of complete DNA clearance while the ECM components were retained. The ECM obtained was tested for cytotoxicity and encapsulated with human pancreatic islets which showed a positive cellular behavior with insulin secretion when stimulated with glucose challenge. Collectively, we propose a new method for the decellularization of the human pancreas without the use of conventional ionic and non-ionic chemical detergents. This protocol and the ECM obtained with it could be of use for both in vitro and in vivo applications.

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix ECM Production

The protocol described in this manuscript explains the steps for the fabrication of a soluble extracellular matrix (ECM) from the human pancreas. The solubilized ECM powder obtained through this protocol may be used for the recapitulation of pancreatic islets&#8217; microenvironment in vitro and, potentially, in vivo settings.

Islet transplantation (ITx) has the potential to become the standard of care in beta cell replacement medicine but its results remain inferior to those ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

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 ...

Purification of Porcine Islets by Surgical Perfusion of Pig Pancreas

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

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 ...

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 ...

Pancreatic Islets Isolation through Collagenase Perfusion: An Enzymatic Method to Isolate Pancreatic Islet Cells

Source: Villarreal D. et al.&#160;<a href="https://jove.unicartagenaproxy.elogim.com/v/57048/" target="_blank">A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice</a>. J. Vis. Exp. (2019)
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.&#160;This protocol explains a method for isolating pancreatic islets by first perfusing collagenase via Ampulla of Vater to digest the exocrine pancreatic tissue and a simplified density gradient centrifugation to purify the islets from mice.

Izolacja wysp trzustkowych poprzez perfuzję kolagenazy: enzymatyczna metoda izolowania komórek wysp trzustkowych

Source: Villarreal D. et al.&#160;A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice. J. Vis. Exp. (2019)
Pancreatic islets, also ...