Pancreatic Tissue Dissection to Isolate Viable Single Cells
May 26th, 2023
Pancreatic metaplastic cells are precursors of malignant cells that give rise to pancreatic tumors. However, isolating intact viable pancreatic cells is challenging. Here, we present an efficient method for pancreatic tissue dissociation. The cells can then be used for single-cell RNA sequencing (scRNA-seq) or for two- or three-dimensional co-culturing.
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.
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Overview
This article presents a simple and efficient protocol for dissociating pancreatic tissue to isolate viable single cells. The method is applicable to various stages of pancreatic malignancy, aiding in the study of pancreatic tumors and pre-malignant lesions.
Key Study Components
Area of Science
- Neuroscience
- Cell Biology
- Oncology
Background
- Pancreatic metaplastic cells are precursors to malignant cells.
- Isolating intact viable pancreatic cells is a significant challenge.
- The protocol can recover cells from normal and diseased pancreatic tissues.
- Understanding these cells can lead to new biomarkers and treatments.
Purpose of Study
- To provide a reliable method for isolating viable pancreatic cells.
- To facilitate research on pancreatic tumors and related diseases.
- To assist in the identification of new therapeutic targets.
Methods Used
- Pancreatic tissue dissociation protocol.
- Isolation of single cells for downstream applications.
- Application to various stages of malignancy.
- Use of the protocol for human pancreatic tumors.
Main Results
- Successful recovery of viable acinar cells from pancreatic tissues.
- Protocol applicable to tissues with pre-malignant lesions and tumors.
- Minimal practice required to achieve desired results.
- Potential to enhance the understanding of pancreatic diseases.
Conclusions
- The protocol is a valuable tool for pancreatic research.
- It aids in the investigation of pancreatic tumors and inflammation.
- Can support the discovery of new biomarkers and treatments.
Frequently Asked Questions
Executive Industry Relevance
Efficient isolation of viable single cells from pancreatic tissue is critical for early discovery and target validation in pancreatic disease research. This protocol addresses the challenge of recovering fragile acinar and other pancreatic cell types, enabling high-quality inputs for downstream molecular and cellular analyses. Its applicability to normal, pre-malignant, and tumor tissues supports translational continuity and risk-adjusted portfolio advancement.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of cell-type-specific pathways in pancreatic disease progression.
- Supports functional validation of targets in both healthy and diseased tissue contexts.
- Facilitates mechanistic de-risking by providing high-viability single-cell suspensions for molecular profiling.
Screening & Assay Development
- Provides validated single-cell preparations for quantitative downstream assays such as flow cytometry and single-cell RNA sequencing.
- Improves assay reproducibility by minimizing cell death and contamination from proteases and RNases.
- Enables scalable preparation of primary cells for compound screening and phenotypic assays.
Translational & Preclinical Research
- Supports disease-relevant model development by isolating cells from pre-malignant and tumor tissues.
- Enables biomarker discovery and validation through high-quality single-cell transcriptomic analysis.
- Facilitates investigation of tumor microenvironment interactions by recovering stromal and immune cells alongside acinar cells.
Pipeline & Workflow Integration
This protocol integrates at the interface of tissue procurement and single-cell analytics, bridging early discovery with translational research in pancreatic disease.
- Discovery Biology: Provides high-integrity single cells for hypothesis testing and pathway analysis.
- Screening: Delivers reproducible, viable cell suspensions for assay development and compound evaluation.
- Analytics: Enables quantitative readouts such as cell viability, cell-type composition, and transcriptomic profiling.
- Translational Research: Aligns with biomarker and target validation efforts in disease-relevant systems.
- Enterprise Reuse: Establishes a standardized workflow adaptable to various pancreatic tissue states and research objectives.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target and biomarker studies by ensuring cell viability and integrity.
- Operational Value: Streamlines tissue processing with reproducible, scalable steps suitable for cross-study standardization.
- Strategic Value: Reduces biological risk and supports informed go/no-go decisions in early-stage pancreatic research.
- Portfolio Impact: Enables risk-adjusted prioritization of targets and models for downstream development.
Implementation Considerations
- Requires expertise in tissue handling and single-cell dissociation techniques.
- Needs access to cell viability assessment tools and flow cytometry or single-cell sequencing platforms.
- Demands strict adherence to timing and temperature controls to preserve cell viability.
- Adaptable to various pancreatic tissue types, including normal, pre-malignant, and tumor samples.
- Viability thresholds and cell-type recovery should be routinely monitored to ensure protocol consistency.
