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

Published: April 30, 2023

Abstract

Source: Lonardo, E., et al. Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies. J. Vis. Exp. (2015).

This article describes the technique for isolating pancreatic cancer stem cells (CSCs) from human pancreatic ductal adenocarcinoma (PDAC) tissue. These CSCs are used to study the functional effects of drug treatment on the in vitro self-renewal capacity of CSCs for developing more efficient cancer treatments.

Protocol

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.

Representative Results

Figure 1
Figure 1. Metformin Selectively Targets the CSCs.
(A) Metformin decreased the size of spheres. Representative images of spheres obtained after the treatment with the indicated doses of metformin for 7 days (right panel). Quantification of sphere size (n≥6) (left panel), the indicated numbers in abscissas axis represent individual tumor. (B) Sphere formation capacity in the presence or absence of metformin for 7 days (n≥6), the indicated numbers in abscissas axis represent individual tumor. (C) Representative graph of CSC self-renewal capacity in secondary and tertiary spheres of primary pancreatic cancer cells. The spheres were only treated during first generation sphere formation for a total of 7 days (n = 6). Please click here to view a larger version of this figure.

Disclosures

The authors have nothing to disclose.

Materials

Counting Chamber/
Hemocytometer 
Hausser Scientific Co 3200
CASY Cell Counter and Analyzer for proliferation and viability measurement Roche Innovatis AG CASY Model TTC 45,60,150 μm
GentleMACS Dissociator Miltenyi Co 130-093-235
GentleMACS C Tubes Miltenyi Co 130-093-237
100-mm tissue culture dishes BD Falcon 353803
Cell strainer BD Falcon 352350
15-ml polypropylene conical tube BD Falcon 352097
50-ml polypropylene conical tube BD Falcon 352070
1.5 ml sterile tubes Eppendorf 0030 120.086
50 ml centrifuge tubes Corning 430828
Sterile petri dishes, 10 cm dishes Corning 353003
Collagenase type IV Stem Cell Technologies 7909
RPMI Medium 1640 Life technologies 11875-085
Penicillin/Streptomycin solution, 100X Life technologies SV30010
L-glutamine, 200 mM, 100X Life technologies 25030-081
B27 Supplement 50x Life technologies 17504-044
Basic Fibroblast Growth Factor Sigma F0291
Dulbecco’s Modified Eagle Medium/F12 Sigma D8437
Sterile 1x Dulbecco’s Phosphate Buffered Saline Sigma D8537
Trypan Blue Life technologies 15250-061
Incubator with CO2 input
Micro scissors
Curved forceps
Splinter forceps

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Cite This Article
PDAC Stem Cell Isolation: A Method to Isolate Cancer Stem Cells from Pancreatic Tumors. J. Vis. Exp. (Pending Publication), e20319, doi: (2023).

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