The overall goal of this procedure is to isolate pancreatic mesenchymal cells for their gene and surface marker expression analysis. This method can help answer key questions in the pancreas development field such as what cues are expressed by mesenchymal cells and how do they influence epithelial development. The main advantages of this technique are that it is relatively simple and fast, and that it allows a good yield of sorted cells.
Begin by depositing the internal organs in a culture dish containing HBSS and placing the dish under a stereo microscope. Detach the liver and kidneys to expose the pancreas using fine forceps to remove the pancreas from the stomach, duodenum, and spleen. As each pancreas is collected, transfer the tissue into a conical tube containing freshly prepared digestive buffer and place the tube on ice.
When all the pancreata have been harvested, transfer the tissues to a 37 degree celsius heating block for 30 minutes with agitation at 700 RPM, manually inverting the tubes three to four times after 15 minutes. If large pieces of pancreas are still present after the first half of the digestion, invert the tube every five minutes for the last 15 minutes and incubate the tissue for an additional five to 10 minutes as necessary. At the end of the digestion, place the tubes on ice and add 10 milliliters of cold HBSS to each sample.
Collect the cells by centrifugation and resuspend the pellets in two milliliters of fresh PBS. Next, filter the cells through 35 micron strainers into five milliliter round-bottom polystyrene collection tubes and wash the digestion tubes with two milliliters of fresh PBS straining the washes into their corresponding collection tubes. Then centrifuge the cells again and carefully aspirate the supernatants without disturbing the pellets.
Resuspend the cells in sorting buffer according to the age of the animals and filter them through new 35 micron cell strainers into new five milliliter collection tubes. Aliquot 50 to 100 microliter volumes of cells into new FACS tubes for the staining controls including a tube for each fluorophore, the viability dye, the fluorescent proteins, and an unstained control. Wash each tube with up to four milliliters of sorting buffer and then centrifuge.
Next, resuspend the pellets in fresh sorting buffer according to the age of the animals. Then stain the cells with the viability dye. For cell sorting prior to RNA extraction, immediately before the beginning of the sort, coat a sterile 1.5 milliliter collection tube with one milliliter of sorting buffer containing RNAse inhibitor.
After five minutes, vortex the collection tube and remove the liquid. Vortex the first staining control and load the tube into the flow cytometer to begin determining the sorting parameters. When all of the sorting parameters have been set, load the samples and sort the cells into 1.5 milliliter collection tubes.
Then collect the sorted cells by centrifugation. Aspirate the supernatant down to the pellet and extract the RNA according to the standard RNA extraction protocols. Here, the isolated whole gastrointestinal tract including the stomach, spleen, intestine, and pancreas of an embryonic day 15.5 embryo and a postnatal day four pup are shown.
Flow cytometric analysis of single cells from embryonic, neonatal, and adult pancreatic tissues isolated as just demonstrated reveals that transgenic pancreatic tissues from all analyzed ages contains distinct YFP-labeled cell populations. After sorting, these mesenchymal cells can be cultured to establish a cell line for at least five passages. Note the fibrotic morphology of the cultured cells typical of mesenchymal cells.
The sorted cells express the panmesenchymal marker vementin, as analyzed by qPCR after RNA extraction and cDNA synthesis. Further, isolated pancreatic cell surface marker expression indicates that all of the fluorescently labeled cells in the transgenic YFP pancreas express CD9, a cell surface glycoprotein reportedly expressed by fibroblasts. Following this procedure, other methods like RNA sequencing can be performed to answer additional questions about mesenchymal cell gene expression.
