TITLE: Human Pancreas Processing
AUTHORS: Jing  Chen, Edward      J Butfiloski, Clayton E Mathews, Martha  Campbell-Thompson

[DESCRIPTION]
The pancreas is a combined endocrine and exocrine organ and is uniquely positioned in the upper abdomen between the stomach (posterior and superior margins), duodenum (proximal or medial), spleen (distal or lateral) and bowel (anterior and inferior). The normal pancreas is shaped as for a pistol with the proximal head region attached to the duodenum and comprising the handle while the distal head/neck, body and tail regions form the barrel. The fluid tone of the soft pancreas makes it difficult to maintain its in vivo 3D shape once trimmed of attached duodenum, spleen and peripancreatic soft tissues. 

The purpose of this protocol is to describe pancreas collection and processing procedures suitable for single cell  mapping. We describe the process of dividing the entire pancreas into sequential cross sections that are subsequently photographed, weighed and processed for the wide range of technologies used in HuBMAP and other programs. Steps describe processing samples to fixed samples for paraffin embedding, optical clearing, or fixed frozen blocks as well as unfixed samples for fresh frozen blocks. Methods are based on those established for the Network for Pancreatic Organ donors with Diabetes program as previously published with minor modifications.

[BEFORE_START]
Set up dissection area in bsl2 hood and 4 stations for sample weighing and photography, freezing, and fixation. Autoclave cutting boards, metal trays, and dissection instruments and supplies and arrange stations. Prepare several liters of 1xPBS, autoclave and refrigerate to cool. Freeze 1xPBS as ice blocks. Prepare 4% paraformaldehyde in 1xPBS as described in materials and cool on ice right before use.

[STEPS]
SECTION: Pancreas processing
1. Unpack the shipping container and verify the organ container is at an ice-cold receiving temperature. Open the organ container in a BSL2 biosafety cabinet. Verify contents include pancreas with attached fat, duodenum, and spleen. Record any deviations for shipping using the   

SECTION: Pancreas processing
3. Photograph the pancreas in the anterior-posterior, superior-inferior, and head-tail views using 1cm grid.   
 
  
SECTION: Pancreas processing
4. Weigh the whole pancreas and record the maximum length and width of each region.  
SECTION: Processing pancreas regions
5. Transect the pancreas into the three major regions (head/neck, body, and tail).
The boundary for the junction between the neck and body is located along the groove from the superior mesenteric artery (SMA) and vein with transection medial to vessels or just distal to the inferior uncinate process. 

 

Divide remaining pancreas sample in half for body and tail (distal half).
 

SECTION: Processing pancreas regions
6. Photograph, weigh, and measure maximum length/widths of each region and record findings. 
SECTION: Processing pancreas regions
7. Place the subdivided pancreas into the opened ProCUT tissue forceps with the inferior surface toward the hinge and proximal pancreas towards operator. Partially close the forceps then place metal end plates into slots to secure the pancreas. Close the forceps and while holding closed, divide the pancreas into ~  sections using long-bladed knives and a sawing action. Optional: After one pancreas cut, the knife can be left in the ProCUT forceps to aid in quickly positioning the next knife and to provide additional rigidity to the pancreas for cutting with a second knife (not shown).
 
Video shows slicing of pancreas tail region with the ProCUT forceps. 

SECTION: Processing pancreas regions
9. Transfer a slice to #5 Whatman filter paper, labeled with 4 orientations (A = anterior, P = posterior,  S = superior, and I = inferior) and the slice number. Trim the filter paper as needed. Note: slices on filter paper can be held on ice until photographed and weighed before further processing.  

   
SECTION: Fresh frozen block preparation
13. After freezing for several minutes, close the Cryotray lid and wrap tray in labeled tin foil  before placing in cardboard box on dry ice. Regular cryomolds are also wrapped in labeled tin foil. 
SECTION: Fresh frozen block preparation
12. Samples to be embedded in CMC or OCT media should be rapidly frozen to avoid freezing artefact.  Samples for fresh frozen blocks will be subdivided to avoid the sample touching the walls of the Cryotray mold or regular cryomold. Cryotray molds are generally used for samples with 4 subdivisions.   

SECTION: Fixed paraffin blocks
10. Place the slices in regular or large format plastic cassettes, depending on sample size and intended use, for fixation before paraffin processing and embedding. Slices, including filter paper, can be transferred into the large format cassettes. Photographs of the cassette labels provides quality control for identity and orientation. Another trimmed filter paper can be placed on top of the tissue in the cassette to ensure smooth surfaces during fixation. 
SECTION: Fixed samples for optical clearing
11.  Place samples for optical clearing in cassettes and fix with 4% PF for 48 hours at 4 °C on a rocker then, wash as in section 10.

SECTION: Pancreas processing
1.1. Maintain cold conditions by placing the container on ice.   
SECTION: Processing pancreas regions
8. When the slicing is completed, open the forceps with the hinge to the right such that the pancreas continues to be viewed as if proximal to distal (medial to lateral). Remove the slices sequentially while maintaining the orientation between slices. 
SECTION: Fresh frozen block preparation
12.1. Prepare dry ice/isopentane slurry in an insulated tray or box with lid. Layer dry ice blocks across bottom of container, cover the dry ice with isopentane, and allow isopentane bath to cool ~ 10 min until boiling stops. Close the container when not in use. 
 

SECTION: Fixed paraffin blocks
10.2. Label the cassettes with a #2 pencil for HuBMAP pancreas ID (sequential number 1, 2, etc) followed by slice number with subdivision when used (e.g. P1-3A).  
SECTION: Pancreas processing
2.1. Collect regional pancreatic lymph nodes (PLN) and process them as fixed and frozen samples in pancreas cassettes or molds, respectively, or as individual samples, as needed. 
SECTION: Pancreas processing
2.  
Remove spleen and duodenum from the pancreas by blunt dissection using scissors and forceps. Remove peripancreatic tissues containing lymph nodes, fat, and fascia with major vessels. Remove any sutures or staples from the pancreas samples before processing. (Samples for fixation, treat as in steps 10.3-10.5; samples to be frozen, treat as in steps 12-14.)
SECTION: Pancreas processing
1.2. Inspect the pancreas and other organs for gross abnormalities and record any findings in the case worksheet. Abnormalities may include hemorrhage/hematoma, mass/cyst, calcifications (fat necrosis), edema and/or stiffness due to fibrosis.  
SECTION: Fresh frozen block preparation
12.2. Label all cryomolds using a cryo-safe pen with the case, slice, and subdivision identifiers. 
Place a small amount of CMC or OCT media in the bottom of each cryomold and pre-chill on regular wet ice. 
SECTION: Fixed frozen block preparation
15. Transfer washed, fixed samples to 30% sucrose for 2880 min at 4 °C to equilibrate. Samples may be in cassettes or in other containers. 
SECTION: Fresh frozen block preparation
14. Seal frozen blocks in boxes that are sealed in plastic zip-lock bags at -80 °C until use.
SECTION: Fixed frozen block preparation
16. Optional: Transfer samples from 30% sucrose to 1:1 30% sucrose:OCT and equilibrate 1440 min at 4 °C  followed by  equilibration in 100% OCT for several hours to 960 min at 4 °C. 
17. Transfer samples to a petri dish or weight boat and cover them with 100% OCT for 5 min if not previously equilibrated in 100% OCT. 
   
 
18. Place a small amount of OCT media in the bottom of a cryomold then add the sample, pushing sample flat against the bottom of the cryomold as for fresh frozen samples. Fill the cryomold with OCT media while avoiding introduction of any air bubbles. 
19. Freeze samples using dry ice/isopentane bath as in 12.4. 
  
20. Wrap the frozen blocks in labelled tin foil and place in a cardboard freezer box sealed with a zip-lock bag for storage at -80 °C  until use.

SECTION: Fixed paraffin blocks
10.3. Place the cassettes into fresh cold 4% paraformaldehyde (PF) in 1xPBS in a 1 liter Nalgene plastic container. The ratio of sample to fixative should be at least 1:20 (vol/vol). The Nalgene container is kept on ice until all the cassettes have been added. Then, move the container to a rocker plate in a cold room to complete fixation. 
 


Fixation times are 24 to 48 hours depending on sample thickness and intended use. Document all sample start and finish times on CaseWorkSheet.   
SECTION: Fixed paraffin blocks
10.4. End the fixation by washing the samples with copious cold 1xPBS. Wash samples on a rocker plate in a cold room three times. The first two washes should be >1 hour, followed by a third wash overnight. Discard the first two PBS washes in PF waste container.  
SECTION: Fixed paraffin blocks
10.5. Store washed, fixed samples in 1x PBS at 4 °C prior to paraffin processing.   

   
SECTION: Fresh frozen block preparation
12.3. Add the sample to the cryomold and gently push it flat to the bottom. Cover sample with additional CMC or OCT media while avoiding the introduction of any air bubbles. 
SECTION: Pancreas processing
2.2. Process 0.5  thick spleen samples by inclusion with pancreas cassettes and molds, or individually, as needed.
SECTION: Pancreas processing
2.3. Samples from duodenum may be prepared as individual cassettes or molds, as for pancreas samples. 
SECTION: Fresh frozen block preparation
12.4. Immediately freeze by placing the cryomold on top of a dry ice/isopentane bath. 
SECTION: Pancreas processing
3.1. Optional: Photograph the whole pancreas, pancreatic regions, and pancreatic slices using a LED light box with 1cm grid. 3D imaging of the pancreas may also be performed (LiDAR, iPhone).
SECTION: Fresh frozen block preparation
13.1. Discard isopentane to chemical waste container after dry ice evaporates. 
SECTION: Fixed paraffin blocks
10.1. If a slice is needs subdivision, photograph the subdivisions on the filter paper and label starting with A for the superior/anterior quadrant or anterior pieces. Continue lettering in clockwise manner usually up to 4 subdivisions for the largest slices. Samples should not touch the sides of the cassette to prevent compression artefacts. Transfer subdivisions into cassettes.
SECTION: Pancreas processing
2.4. Place any tissues not to be saved into Nalgene container with fixative for disposal according to UF biological waste regulations. Inactivate the shipping media and/or PBS in contact with tissues with fresh 10% bleach for at least 30 minutes before disposal.
SECTION: Processing pancreas regions
6.1. Return the body and tail samples to cold shipping media or PBS to hold for later dissection. 
SECTION: Fixed samples for optical clearing
11.1. Stored fixed tissues for optical clearing in 1xPBS with 0.1% PF at 4 °C  until use.