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://www.jove.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. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+tissue+and+whole+organ+decellularization+processes.">An overview of tissue and whole organ decellularization processes.</a> Biomaterials. 32 (12), 3233-3243 (2011).</li><li>Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+metazoan+extracellular+matrix.">The evolution of metazoan extracellular matrix.</a> Journal of Cell Biology. 196 (6), 671-679 (2012).</li><li>Scarritt, M. E., Pashos, N. C., Bunnell, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+cellularization+strategies+for+tissue+engineering+of+whole+organs.">A review of cellularization strategies for tissue engineering of whole organs.</a> Frontiers in Bioengineering and Biotechnol. 3, 43 (2015).</li><li>Goh, S. 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. Biomed Research International. 2015, 432645 (2015).</li><li>Peloso, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Human+Pancreas+as+a+Source+of+Protolerogenic+Extracellular+Matrix+Scaffold+for+a+New-generation+Bioartificial+Endocrine+Pancreas.">The Human Pancreas as a Source of Protolerogenic Extracellular Matrix Scaffold for a New-generation Bioartificial Endocrine Pancreas.</a> Annals of Surgery. 264 (1), 169-179 (2016).</li><li>Mirmalek-Sani, S. H., 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=Porcine+pancreas+extracellular+matrix+as+a+platform+for+endocrine+pancreas+bioengineering.">Porcine pancreas extracellular matrix as a platform for endocrine pancreas bioengineering.</a> Biomaterials. 34 (22), 5488-5495 (2013).</li><li>Taylor, M. J., Baicu, S., Greene, E., Vazquez, A., Brassil, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+isolation+from+juvenile+porcine+pancreas+after+24-h+hypothermic+machine+perfusion+preservation.">Islet isolation from juvenile porcine pancreas after 24-h hypothermic machine perfusion preservation.</a> Cell 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. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+the+protease+inhibitor+phenylmethylsulfonyl+fluoride+in+buffers.">Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers.</a> Analytical Biochemistry. 86 (2), 574-579 (1978).</li><li>Lichstein, H. C., Soule, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+the+Effect+of+Sodium+Azide+on+Microbic+Growth+and+Respiration:+I.+The+Action+of+Sodium+Azide+on+Microbic+Growth.">Studies of the Effect of Sodium Azide on Microbic Growth and Respiration: I. The Action of Sodium Azide on Microbic Growth.</a> Journal of Bacteriology. 47 (3), 221-230 (1944).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
		<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&#39;s PBS with CaCl2 and MgCl2</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&#34; 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&#246;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://www.jove.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://www.jove.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 ...

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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

Decellularization and Recellularization of Whole Livers

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to recreate or mimic the liver microstructure with grounds up approach using tissue engineering and microfabrication techniques have not been successful so far due to this design challenge. In addition, synthetic biomaterials used to create scaffolds for liver tissue engineering applications have been limited in inducing tissue regeneration and repair in large part due to the lack of specific cell binding motifs that would induce the proper cell functions2. Decellularized native tissues such blood vessels3and skin4on the other hand have found many applications in tissue engineering, and have provided a practical solution to some of the challenges. The advantage of decellularized native matrix is that it retains, to an extent, the original composition, and the microstructure, hence enhancing cell attachment and reorganization5.
In this work we describe the methods to perform perfusion-decellularization of the liver, such that an intact liver bioscaffold that retains the structure of major blood vessels is obtained. Further, we describe methods to recellularize these bioscaffolds with adult primary hepatocytes, creating a liver graft that is functional in vitro, and has the vessel access necessary for transplantation in vivo.

Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.

The liver is a complex organ which requires constant perfusion for delivery of nutrients and oxygen and removal of waste in order to survive1. Efforts to ...

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

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 ...

A Decellularization Methodology for the Production of a Natural Acellular Intestinal Matrix

Successful tissue engineering involves the combination of scaffolds with appropriate cells in vitro or in vivo. Scaffolds may be synthetic, naturally-derived or derived from tissues/organs. The latter are obtained using a technique called decellularization. Decellularization may involve a combination of physical, chemical, and enzymatic methods. The goal of this technique is to remove all cellular traces whilst maintaining the macro- and micro-architecture of the original tissue.
Intestinal tissue engineering has thus far used relatively simple scaffolds that do not replicate the complex architecture of the native organ. The focus of this paper is to describe an efficient decellularization technique for rat small intestine. The isolation of the small intestine so as to ensure the maintenance of a vascular connection is described. The combination of chemical and enzymatic solutions to remove the cells whilst preserving the villus-crypt axis in the luminal aspect of the scaffold is also set out. Finally, assessment of produced scaffolds for appropriate characteristics is discussed.

The article describes a methodology for the production of an acellular matrix from rat intestine. The derivation of intestinal scaffolds is important for future applications in tissue engineering, stem cell biology and drug testing.

Successful tissue engineering involves the  combination of scaffolds with appropriate cells in vitro or in vivo.  Scaffolds may be synthetic, ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent ...

Quantification of Strain in a Porcine Model of Skin Expansion Using Multi-View Stereo and Isogeometric Kinematics

Tissue expansion is a popular technique in plastic and reconstructive surgery that grows skin in vivo for correction of large defects such as burns and giant congenital nevi. Despite its widespread use, planning and executing an expansion protocol is challenging due to the difficulty in measuring the deformation imposed at each inflation step and over the length of the procedure. Quantifying the deformation fields is crucial, as the distribution of stretch over time determines the rate and amount of skin grown at the end of the treatment. In this manuscript, we present a method to study tissue expansion in order to gain quantitative knowledge of the deformations induced during an expansion process. This experimental protocol incorporates multi-view stereo and isogeometric kinematic analysis in a porcine model of tissue expansion. Multi-view stereo allows three-dimensional geometric reconstruction from uncalibrated sequences of images. The isogeometric kinematic analysis uses splines to describe the regional deformations between smooth surfaces with few mesh points. Our protocol has the potential to bridge the gap between basic scientific inquiry regarding the mechanics of skin expansion and the clinical setting. Eventually, we expect that the knowledge gained with our methodology will enable treatment planning using computational simulations of skin deformation in a personalized manner.

This protocol uses multi-view stereo to generate three-dimensional (3D) models out of uncalibrated sequences of photographs, making it affordable and adjustable to a surgical setting. Strain maps between the 3D models are quantified with spline-based isogeometric kinematics, which facilitate representation of smooth surfaces over coarse meshes sharing the same parameterization.

Tissue expansion is a popular technique in plastic and reconstructive surgery that grows skin in vivo for correction of large defects such as burns and ...

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to &#60;20 &#181;m as formalin fixed paraffin section on glass slides or &#60;500 &#181;m for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples &#62;500 &#181;m using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth &#62;150 &#181;m with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Here, we present a protocol to image human pancreas sections in three dimensions (3D) using optimized passive clearing methods. This manuscript demonstrates these procedures for passive optical clearing followed by multiple immunofluorescence staining to identify key elements of the autonomic and sensory neural networks innervating human islets.

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological ...

Comparable Decellularization of Fetal and Adult Cardiac Tissue Explants as 3D-like Platforms for In Vitro Studies

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components are presented as a surface coating. These culture systems constitute a simplification of the complex nature of the tissue ECM that encompasses biochemical composition, structure, and mechanical properties. To better emulate the ECM-cell communication shaping the cardiac microenvironment, we developed a protocol that allows for the decellularization of the whole fetal heart and adult left ventricle tissue explants simultaneously for comparative studies. The protocol combines the use of a hypotonic buffer, a detergent of anionic surfactant properties, and DNase treatment without any requirement for specialized skills or equipment. The application of the same decellularization strategy across tissue samples from subjects of various age is an alternative approach to perform comparative studies. The present protocol allows the identification of unique structural differences across fetal and adult cardiac ECM mesh and biological cellular responses. Furthermore, the herein methodology demonstrates a broader application being successfully applied in other tissues and species with minor adjustments, such as in human intestine biopsies and mouse lung.

The cardiac extracellular matrix (ECM) is a complex network of molecules that orchestrate key processes in tissues and organs while enduring physiological remodeling throughout life. Standardized decellularization of fetal and adult hearts permits comparative experimental studies of both tissues in a 3D context by capturing native architecture and biomechanical properties.

Current knowledge of extracellular matrix (ECM)-cell communication translates to large two-dimensional (2D) in vitro culture studies where ECM components ...