Collection Protocol for Human Pancreas

Summary

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.

Abstract

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 β-cells followed by lower proportions of glucagon-containing α-cells and somatostatin-containing δ-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 diabetes^4,5.

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.

Protocol

1. Set-up

1. 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, Appendix 1).
2. Label OCT molds as for cassettes with a permanent marker. Cut out 5-10 cm rectangles of aluminum foil.
3. Print or manually label with permanent marker all vials and tubes. Include enough vials for minced snap frozen samples and tubes with RPMI media for fresh shipments. For the snap frozen vials with RN....

Discussion

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.......

Materials

List of materials used in this article

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