The demonstrated pancreatic tissue dissociation protocol is simple and provides a tool to isolate viable single cells from pancreatic tissue at different stages during the malignancy process, including solid tumors. Recovering intact and viable acinar cells is a major challenge. The protocol can recover viable single cells from normal pancreata, pancreata having pre-malignant lesions, or pancreatic tumors containing numerous stromal and immune cells.
The use of this protocol to dissect human pancreatic tumors or inflamed pancreas could assist in investigating these diseases to find new biomarkers and treatments. The method is easy to use and can provide the desired results with minimal practice. This video will help to view the small details of the protocol.
Before beginning the dissection, keep all instruments and equipment ready on ice. After fixing the euthanized mouse, spray its abdomen with 70%ethanol. Using scissors and forceps, make a V-shaped incision of 2.5 centimeters in the genital area of the animal, and proceed upward to fully open the abdominal cavity.
Locate the stomach on the left side of the mouse and the pancreas, which is near the spleen. Using two forceps, separate the pancreas from the stomach and duodenum without tearing. Continue and separate the pancreas from the small intestine, jejunum and ileum.
Move the pancreas to the right side and separate the remaining connections between the pancreas and the thoracic cavity with forceps to completely detach the pancreas and attached spleen. Carefully, remove the pancreas without mesenteric fat or other adjacent tissue, and spread it out for examination in a Petri dish on ice. Following the composition mentioned in the text, prepare the dissociation buffers 1 and 2, wash buffer and enzyme activity stop solution in advance.
Place the pancreas in a 50 milliliter tube on ice and rinse it in 10%fetal bovine serum, or FBS, and Hank's Balanced Salt Solution, or HBSS. The fat will float and the pancreas will sink. This is an easy way to visualize and quickly remove the contaminating white adipose tissue still attached to the pancreas.
Next, transfer and keep the mouse pancreatic tissue in a sterile Petri dish containing five milliliters of HBSS on ice till cutting. Using Noyes scissors and a scalpel, cut the pancreas into small pieces of one to three cubic millimeters. After transferring the tissues to a centrifuge tube, centrifuge it at 350G and four degrees Celsius for five minutes.
Aspirate and discard the supernatant to remove cell fragments and blood cells. Resuspend suspend the pieces in dissociation buffer 1 containing 0.02%trypsin-C and 0.05%EDTA for 10 minutes at 37 degrees Celsius with agitation. Immediately wash with 10%FBS and Dulbecco's Modified Eagle's Medium, or DMEM, and centrifuge for five minutes at 350G and four degrees Celsius.
Wash again by resuspending the pellet in 10 milliliters of wash buffer, and centrifuging as previously for five minutes before the next dissociation step. Incubate the pancreas in dissociation buffer 2 for 15 minutes at 37 degrees Celsius with agitation at 180 rotations per minute. After 15 minutes, perform mechanical dissociation by vigorously pipetting the pancreatic fragments up and down 10 times using sterile serological pipettes.
Repeat using pipettes of decreasing size, starting from 25, 10, to 5 milliliters. Incubate the sample to bring it to 37 degrees Celsius. Use light microscopy to monitor the dissociation according to the amount of single cell in the suspension.
Monitor cell viability using trypan blue. Continue incubation and check the dissociation by taking samples every five minutes. Incubate till 90%of the cells are separated into single cells.
After the pancreatic tissue is well dissociated, indicated by the disappearance of pancreatic fragments and the increasing turbidity of the solution, stop the enzymatic reaction by washing twice with enzyme activity stop solution for five minutes at four degrees Celsius. From this step, keep the cell suspension on ice. Pass the cell suspension through a 70 micron nylon mesh.
A smaller nylon mesh size may reduce cell viability. Check the cell viability under the microscope. Resuspend and wash the pellet with 5 to 10 milliliters of ice-cold buffered wash solution before counting the cells.
If several red blood cells are observed, treat the cells with a red blood cell lysis buffer for two minutes at room temperature. In cases where clumps are observed, the samples should be treated again with trypsin, as described previously. If viability is less than 80%live cells should be isolated using the magnetic-activated cell sorting, or MACS dead cells removal kit with MACS MS columns.
The red color of the isolated pancreatic tissue resulted from the expression of tdTomato in the acinar cells. At an early stage of the dissociation, there were low numbers of isolated cells and large numbers of clumps. Later, isolated viable cells were obtained while avoiding reducing the number of viable cells.
Longer incubation reduced the viability of the cells. The tdTomato positive cells were estimated using fluorescence-activated cell sorting. The Gentle Dissociation Protocol supported the recovery of multiple cell types, with high viability that allowed following up with cell sorting of desired cell types.
The live-cell-to-dead-cell counting was done using a hemacytometer. The fluorescence images of frozen pancreatic sections showed the tdTomato positive acinar cells. The single cell RNA sequencing confirmed the detection of all the cell types detected in fluorescence-activated cell sorting in each resected tissue from all the time points.
The carboxypeptidase1, or CPA1 expression in acinar cells was studied, indicating minimal contamination with the transcriptome of other cell types. It is important to note that longer incubation times reduced the viability of the cells, so it is important to monitor the sample and observe the dissociation of the tissue and cell viability every few minutes under the microscope. The cell isolation protocol can be used for single cell RNA sequencing, culturing cells, or as a starting material for organoids.
This technique allows for exploring how pancreatic cancer develops, and investigate the interactions between cells that infiltrate inflamed or cancerous tissue.
