PDAC Stem Cell Isolation: A Method to Isolate Cancer Stem Cells from Pancreatic Tumors

1. Isolation of CSCs from Human PDAC (Figure 1)

1. In a sterile biosafety cabinet transfer the human PDAC tissue to a 60 x 15 mm culture dish containing 1 ml of sterile 1X phosphate-buffered saline (PBS). Mince the tumor into small pieces (1 – 5 mm) using a sterile scalpel and forceps.
2. Add 1 ml of sterile 1X PBS to the culture dish and repeat the trituration step until the tissue is completely dissociated, regularly this requires 3-4 rounds. Transfer the tissue suspension (top up to a final volume of 5 ml with 1X PBS) into a sterile tube and mechanically homogenize it with a dissociator such as gentleMACS.
3. Incubate the homogenized tissue with collagenase (use 2.5 mg/ml of collagenase in 1X PBS) for 60 min at 37°C and then centrifuge for 5 min at 900 x g. Decant the supernatant and resuspend the cell pellets in 10 ml of complete medium. Filter the cell suspension through a 40 µm strainer and centrifuge for 5 min at 900 x g.
4. Decant the supernatant and resuspend the pellet with 5 ml of red blood cell lysis buffer (ammonium-chloride-potassium, ACK), and incubate the
5. Resuspend the pellet in CSCs medium, plate on a gelatin-coated dish, and incubate at 37°C for 1 h. This step will remove most of the fibroblast cells that quickly attach to the plate.
6. Remove the plate from the incubator and carefully recover cell suspension and quantify the number of viable (trypan blue-negative) cells using a hemocytometer. For this purpose, gently mix the suspension and pipette 20 µl of the suspension into an Eppendorf tube. Add 20 µl (1:1 ratio) of trypan blue to the cells in the microcentrifuge tube and mix well. Pipette approximately 10 µl of the mixture onto the hemocytometer.
7. Count all clear cells within the four corner quadrants of the counting chamber for viable cell count.
   NOTE: The viability and yield of cells may vary considerably between tumor specimens. For PDAC samples viability regularly ranges between 45-70%, and tissue pieces from a macroscopic tumor sample with a diameter of 3 – 5 mm should yield to approximately 5×10⁶ viable cells.