Tumor processing for CAF isolation

Tumor processing for CAF isolation

Tumors were mechanically minced and digested using the tumor dissociation kit (130-096-730; Miltenyi Biotec) in gentleMACS C-Tubes (130-093-237; Miltenyi Biotec) according to the manufacturer's instructions. Tumor samples were minced and incubated in the digestion media at 37°C for 30 min in a gentleMACS Octo dissociator (130-096- 427; Miltenyi Biotec). After the digestion period, cells were suspended in a cold FACS buffer (0.5% BSA and 2 mM EDTA in PBS), filtered through 70-μm filters, and centrifuged to collect the cells.

Then, CAFs were magnetically labelled and isolated using the tumor-associated fibroblast isolation kit (130-116-474; Miltenyi Biotec) according to the manufacturer's instructions. The detailed protocol was described below.

・Reagents

Digestion media A (per tumor): 2.35 mL of serum-free RPMI1640 + 100, 50, or 12.5 µL of Enzyme D, R, or A, respectively

Buffer B: PBS (pH 7.2) containing 0.5% BSA and 2 mM EDTA

・Tumor processing

1. Add 2.5 mL of Digestion media A into each C-tube.
2. Put tumors into C-tubes and mince them (until each tumor becomes 3-mm square in size).
3. Securely screw the caps.
4. Incubate them in a gentleMACS Octo dissociator with the pre-installed program “37C_h_TDK_4”.
5. Add 10 mL of pre-cooled Buffer B into each C-tube and mix well.
6. Collect the cell suspension and filter them through 70-µm filters.
7. Centrifuge them at 300 g, 4°C for 10 min.
8. Discard the supernatant and re-suspend the pellets with Buffer B.

・Magnetic labeling and depletion of non-tumor-associated fibroblasts

*work fast, keep cold, and use pre-cooled solutions.   *up to 10⁸ cells

1. Count cell number and collect the required number of cells. (e.g. 10⁷ cells)
2. Centrifuge the cell suspension at 300 g for 10 min, and aspirate the supernatant completely.
3. Resuspend the cell pellets in 80 µL of buffer per 10⁷ cells.
4. Add 20 μL of Non-Tumor-Associated Fibroblast Depletion Cocktail.
5. Mix well and incubate for 15 min at 4°C (be punctual).
6. Adjust volume to 500 µL using Buffer B.
7. Place LD Column in the magnetic field of MidiMACS.
8. Prepare the column by rinsing 2 mL of Buffer B.
9. Apply the cell suspension onto the column.
10. Collect unlabeled cells that pass through and wash column with 1 mL Buffer B twice.
11. Collect total effluent.

・Magnetic labelling and selection of tumor-associated fibroblasts

1. Centrifuge the cell suspension at 300 g for 10 min, and aspirate the supernatant completely.
2. Resuspend the cell pellet in 80 µL of Buffer B.
3. Add 20 μL of CD90.2 MicroBeads.
4. Mix well and incubate for 15 min at 4°C (must be punctual).
5. Add buffer to a final volume of 500 µL.
6. Place MS Column in the magnetic fields of miniMACS or OctoMACS.
7. Prepare column by rinsing 500 µL of Buffer B.
8. Apply the cell suspension onto the column.
9. Wash the column with 500 µL of Buffer B 3 times.
10. Remove the column from the separator and place it on a suitable collection tube.
11. Pipette 1 mL of Buffer B on the column and immediately flush out the magnetically labelled cells by firmly pushing the plunger into the column.
12. Centrifuge the cell suspension at 300 g for 10 min, and discard the supernatant.
13. Resuspend the cell pellet with appropriate medium. (if the purity was low, repeat this section)