Name: collection protocol for human pancreas
Uploaded: 2012-05-23T12:00:00
Description: Watch this Scientific Journal Video about Collection Protocol for Human Pancreas at JoVE.com

The authors thank the donors' families and the Organ Procurement Organizations involved in this research and Maria Martino and Irina Kusmartseva for their expert assistance. This work was funded by the Juvenile Diabetes Research Foundation (M.C-T., M.A.) in support of the Network for Pancreatic Organ Donors with Diabetes.

1. Set-up
<ol>
	<li>Label cassettes for paraffin blocks manually with a pencil, or automatically with a cassette printer. Include the donor identifier (CaseID), sample type, aliquot number, and any additional unique identification (Case worksheet, <a href="https://www.jove.com/files/ftp_upload/4039/4039appendix1.doc" target="_blank">Appendix 1</a>).</li>
	<li>Label OCT molds as for cassettes with a permanent marker. Cut out 5-10 cm rectangles of aluminum foil.</li>
	<li>Print or manually label with permanent marker all...

<ol>
	<li>Brissova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+human+pancreatic+islet+architecture+and+composition+by+laser+scanning+confocal+microscopy.">Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy.</a> J. Histochem. Cytochem. 53, 1087-1097 (2005).</li><li>Rahier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pancreatic+polypeptide+cells+in+the+human+pancreas:+the+effects+of+age+and+diabetes.">The pancreatic polypeptide cells in the human pancreas: the effects of age and diabetes.</a> J. Clin. Endocrinol. Metab. 56, 441-444 (1983).</li><li>Uchida, T., Takada, T., Ammori, B. J., Suda, K., Takahashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+the+ventral+and+dorsal+pancreas:+a+new+insight+into+anatomy+and+embryonic+development.">Three-dimensional reconstruction of the ventral and dorsal pancreas: a new insight into anatomy and embryonic development.</a> J. Hepatobiliary Pancreat. Surg. 6, 176-180 (1999).</li><li>Wittingen, J., Frey, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+concentration+in+the+head,+body,+tail+and+uncinate+process+of+the+pancreas.">Islet concentration in the head, body, tail and uncinate process of the pancreas.</a> Ann. Surg. 179, 412-414 (1974).</li><li>Rahier, J., Goebbels, R., Henquin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+composition+of+the+human+diabetic+pancreas.">Cellular composition of the human diabetic pancreas.</a> Diabetologia. 24, 366-371 (1983).</li><li>Vaught, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+NCI+perspective+on+creating+sustainable+biospecimen+resources.">An NCI perspective on creating sustainable biospecimen resources.</a> J. Natl. Cancer Inst. Monogr. 42, 1-7 (2011).</li><li>Gianani, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+results+of+screening+of+nondiabetic+organ+donors+for+expression+of+islet+autoantibodies.">Initial results of screening of nondiabetic organ donors for expression of islet autoantibodies.</a> J. Clin. Endocrinol. Metab. 91, 1855-1861 (2006).</li><li>Tauriainen, S., Salmela, K., Rantala, I., Knip, M., Hy&#246;ty, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collecting+high-quality+pancreatic+tissue+for+experimental+study+from+organ+donors+with+signs+of+&#946;-cell+autoimmunity.">Collecting high-quality pancreatic tissue for experimental study from organ donors with signs of &#946;-cell autoimmunity.</a> Diabetes Metab. Res. Rev. 26, 585-592 (2010).</li><li>In't Veld, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+insulitis+in+adult+autoantibody-positive+organ+donors.">Screening for insulitis in adult autoantibody-positive organ donors.</a> Diabetes. 56, 2400-2404 (2007).</li><li>T&#246;ns, H. A., Terpstra, O. T., Bouwman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+human+pancreata+in+perspective+of+the+isolation+of+the+islets+of+langerhans.">Heterogeneity of human pancreata in perspective of the isolation of the islets of langerhans.</a> Transplant Proc. 40, 367-369 (2008).</li></ol>

The goal of this procedure is to define a standard pancreas processing that can be harmonized across multiple collection sites and thus allow comparisons of results obtained by different groups. The dissection method is based on standard surgical pathology practices with additional steps for annotation of major pancreas regions followed by intra-regional subdivisions. Standardization of biospecimen processing methods is required to facilitate collection of high quality samples exemplified by the publication of best pract...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>Dissection instruments including scalpels with 22 blades</td>
			<td>Various</td>
			<td>Various</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge tubes (15 and 50 ml)</td>
			<td>Fisher Scientific</td>
			<td>05-539-5, 430828</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Cryotubes(1.8 ml)</td>
			<td>VWR North America</td>
			<td>89004-300</td>
			<td>Sterile, bar codes optional</td>
		</tr>
		<tr>
			<td>RNAlater</td>
			<td>Ambion</td>
			<td>AM7021</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical Pathology Ink</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cassettes</td>
			<td>Mercedes Medical</td>
			<td>CAS ECO109</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10% Neutral Buffered Formalin</td>
			<td>Fisher Scientific</td>
			<td>23-245-685</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>OCT Freezing Media</td>
			<td>TissueTek</td>
			<td>4583</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Molds</td>
			<td>ThermoScientific</td>
			<td>58952</td>
			<td>Various sizes</td>
		</tr>
		<tr>
			<td>Isopentane</td>
			<td>Fisher Scientific</td>
			<td>O3551-4</td>
			<td>OCT chilling bath</td>
		</tr>
		<tr>
			<td>Dry ice</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>OCT chilling bath</td>
		</tr>
		<tr>
			<td>Liquid nitrogen in Dewar flask</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Snap freezing cryovials</td>
		</tr>
		<tr>
			<td>Cell culture dish (100mm)</td>
			<td>Corning</td>
			<td>430167</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Hyclone DMEF</td>
			<td>ThermoScientific</td>
			<td>SH30023.01</td>
			<td>Sterile, store refrigerated, glutamine included</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Cellgro</td>
			<td>35-010-CV</td>
			<td>Sterile, store frozen aliquots</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin/Antifungal</td>
			<td>GIBCO</td>
			<td>15240-062</td>
			<td>Sterile, store frozen aliquots</td>
		</tr>
		<tr>
			<td>Type 9 Paraffin</td>
			<td>Richard-Allan Scientific</td>
			<td>8337</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1X D-PBS</td>
			<td>GIBCO</td>
			<td>14190-144</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>EM Sciences</td>
			<td>15710</td>
			<td>Make fresh, store refrigerated</td>
		</tr>
		<tr>
			<td>8% Glutaraldehyde</td>
			<td>EM Sciences</td>
			<td>16019</td>
			<td>Make fresh, store refrigerated</td>
		</tr>
		<tr>
			<td>Biosafety Level 2 hood</td>
			<td>Various</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ultra-low freezer</td>
			<td>Various</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sakura Cassette Printer</td>
			<td>Sakura</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sakura VIP300</td>
			<td>Sakura</td>
			<td>&nbsp;</td>
			<td>Automatic paraffin processor</td>
		</tr>
		<tr>
			<td>Paraffin Embedding Station</td>
			<td>ThermoFisher</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table 1. Specific reagents and equipment.

collection protocol for human pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

Watch this Scientific Journal Video about Collection Protocol for Human Pancreas at JoVE.com

Collection Protocol for Human Pancreas

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

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