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

Protokol

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 5x10⁶ viable cells.

Sonuçlar

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 fo...

Malzemeler

Name	Company	Catalog Number	Comments
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