Isolation of Mouse Pancreatic Endothelial Cells

The aim of this research is to deepen our understanding of the role of pancreatic vasculature in both normal pancreatic biology and disease development. By successfully enriching the endothelial fraction, we seek to identify specific pathways within the vasculature that govern pancreatic functions, which can potentially be targeted to restore and enhance pancreatic functions in disease settings. Recently, an increasing emphasis has been placed on the role of the endothelial cell dysfunction in pancreatic pathologies.

Identification of the heterogeneity of endothelial cell populations in the pancreas has allowed for the understanding of how endothelial pancreas interactions strive or protect against diseases. Omics approaches such as single cell or single nucleus RNA sequencing, proteomics and genomics are being used to study the pancreas at a cellular level. To gain insights at the tissue or spatial level, new platforms using unbiased and probe-based approaches such as Visium and Xenium are being implemented.

In this protocol, we describe a gentle dissociation technique that minimizes endogenous pancreatic enzymes from decreasing cell viability and a magnetic cell sorting procedure to selectively enrich endothelial cells. The dissociation and enrichment strategy of our protocol will enable further understanding of both cell surface markers and the transcriptome of pancreatic endothelial cells that was previously under explored due to technical limitations. To begin, position the euthanized mouse on the operating table.

Stretch the mouse limbs and secure them with pins. Using surgical scissors, make a small midline incision through the skin and peritoneum from the lower abdominal area and extend towards the thoracic region. Cut through the diaphragm and lift the rib cage to expose the heart.

Insert a 25 to 30 gauge needle connected to a syringe containing ice cold sterile PBS into the left ventricle of the heart. Perfuse the heart at a rate of five to 10 milliliters per minute and stop after injecting 10 milliliters. Then, using dissecting scissors and forceps, extract the pancreas carefully.

Transfer the pancreas to a 50 milliliter tube containing 10 milliliters of ice cold PBS, along with the trypsin inhibitor and place the tube on ice. To begin, obtain the murine pancreas in a Petri dish containing ice cold PBS. Using surgical scissors and tweezers, carefully remove excess tissue.

Transfer the trimmed pancreas to a five milliliter tube containing one milliliter of dissociation solution. With dissecting scissors, mince the pancreatic tissues into fine pieces, keeping the tube on ice. Transfer the lysate into a 50 milliliter tube placed on ice.

Add two milliliters of collagenase solution to the five milliliter tube to remove any residual pancreatic tissue and transfer it to the 50 milliliter tube. Incubate the tube in a water bath tempered at 37 degrees Celsius for 20 minutes. Place the tube with tissue homogenate on ice and let it settle by gravity.

Then pass the supernatant through a 70 micrometer filter and add five milliliters of dissociation stop solution. Using a 27 gauge needle, triturate the remaining pellet with an additional one milliliter of dissociation solution and pass it through the filter as before. Spin the cell suspension at 300 G for 10 minutes at four degrees Celsius and resuspend the pellet in one milliliter of wash buffer.

Incubate one times 10 to the power of seven cells with one microliter of anti-CD31 biotin antibody on a rotator. Add 20 microliters of anti-biotin MicroBeads and incubate for 40 more minutes. Then set up a magnetic stand with the column separator holder and apply the column.

After equilibrating the column, add the sample to it and wash with a total of nine milliliters of wash buffer. Remove the column from the holder and place it in a 15 milliliter tube. Add five milliliters of wash buffer to the column.

Using a plunger, push down cells in the column to collect the elution containing the endothelial cells. Finally, spin down the flow-through and elution fractions. After resuspending, count the cells and measure viability.

Quantitative PCR analysis to validate the enrichment of endothelial cells showed that the enriched fractions had significantly higher levels of marker genes, Pecam1 and Kdr compared to the flow-through samples. A lower level of NK6 homeobox 1, a transcription factor in the pancreatic endocrine cells, was observed in the enriched fractions. Western blotting showed strong signals of CD31 at the expected size, further confirming the endothelial cell enrichment in the fractions.