Materials and Reagents

12 well cell culture plate (Thermo Fisher Scientific, catalog number: 150628 )
100 mm cell culture dish (Thermo Fisher Scientific, catalog number: 172931 )
5 ml centrifuge tube (Thermo Fisher Scientific, catalog number: 339650 )
Phosphate buffered saline (PBS) (pH 7.2) (Life Technologies, catalog number: 20012027 )
Note: Currently, it is “Thermo Fisher Scientific, Gibco^TM, catalog number: 20012027”.
100x Antibiotic-Antimycotic (Life Technologies, catalog number: 15240062 )
Note: Currently, it is “Thermo Fisher Scientific, Gibco^TM, catalog number: 15240062”.
Dulbecco’s modified Eagle's medium (DMEM) (Wako Pure Chemical Industries, catalog number: 04330085 )
3,000 U/ml Collagenase from Clostridium histolyticum (Sigma-Aldrich, catalog number: C5138 )
1,000 U/ml Hyaluronidase (Wako Pure Chemical Industries, catalog number: 08006201 )
20 mg/ml Deoxyribonuclease I from bovine pancreas (Sigma-Aldrich, catalog number: DN25 )
Fetal bovine serum (FBS) (Thermo Fisher Scientific, catalog number: SH3091003 )
Note: Currently, it is “GE Healthcare, catalog number: SH3091003”.
Penicillin-Streptomycin Mixed Solution (Nakarai tesque, catalog number: 2625384 )
0.5 g/l-Trypsin/0.53 mmol/l-EDTA Solution, with Phenol Red (Nakarai tesque, catalog number: 3277834 )
7-AAD (BD Pharmingen, catalog number: 5168981E )
CELLBANKER1 (Nippon Zenyaku Kogyo Co., ZENOAQ, catalog number: CB011 )
Wash buffer (see Recipes)
Digestion solution (see Recipes)
Growth medium (see Recipes)

Equipment

Surgical scissors and forceps
Pipettes
Centrifuge
Cell culture hood
37 °C, 5% CO₂ cell culture incubator
Microscope
FACS Aria ll flow cytometer (BD Biosciences)

Procedure

Euthanize the mouse when the thyroid tumor has reached appropriate size, approximately 50-500 mm³ (refer to Video 1 and Figure 1).
Expose and remove the thyroid tumor from trachea using sterile scissors. Remove the surrounding tissues as carefully as possible.
Place the thyroid tumor onto 12 well type plate, and wash it several times with 2 ml of cold wash buffer.
Mince the thyroid tumor into small pieces as small as possible using sterile scissors on ice.
Collect all of the tumor pieces into 15 ml centrifuge tube.
Wash with 5 ml wash buffer by centrifugation at 300 rcf for 5 min at 4 °C.
Resuspend the tumor pieces in 5 ml digestion solution.
Incubate at 37 °C for 60 min. Meanwhile mix gently every 15 min.
Centrifuge at 300 rcf for 5 min at room temperature, and remove the supernatant.
Wash 2 times with 5 ml wash buffer by centrifugation at 300 rcf for 5 min at room temperature.
Resuspend the tumor pieces in 10 ml growth medium, and plate them onto a 100 mm cell culture dish.
Incubate at 37 °C in 5% CO₂ for 4~5 days.
You will see some cells attach to the dish and grow, while the major population of cells is floating.
Collect all culture medium containing floating cells into 15 ml centrifuge tube.
Add 1 ml growth medium to the 100 mm cell culture dish, wash and recollect it into 15 ml centrifuge tube. Repeat this step at least twice in order to recover all of the floating cells.
Centrifuge at 300 rcf for 5 min at room temperature, discard the supernatant, resuspend the cells in 10 ml growth medium, and plate them onto a new 100 mm cell culture dish.
Add 10 ml growth medium to the used 100 mm cell culture dish, and maintain the attached cells in the cell culture incubator as adhesive population.
Repeat steps 13-17 after 3-4 day culture at least 3-4 times until attached cells never appear from floating cells in order to completely separate the non-adhesive population from the adhesive population.
You will detect the markers of C cells such as Calcitonin and Ascl1 positive cells are highly enriched in the non-adhesive population.
Dead cells should be removed by cell sorter using staining reagents such as 7-AAD for some applications.
Non-adhesive and adhesive populations can be stored in liquid nitrogen tank with cell stock solution such as CELLBANKER1.

Video 1. The video for how to remove the thyroid tumor

Figure 1. The picture of mouse thyroid tumor

Figure 2. Phase-contrast image of the non-adhesive cell population

Figure 3. The non-adhesive population highly expressed C cell markers (A. Calcitonin, B. Ascl1) as compared to adhesive population and mouse embryonic fibroblast

Recipes

Wash buffer
2x antibiotic
Antimycotic in PBS (pH 7.2)
Digestion solution
DMEM containing 300 units/ml collagenase, 100 units/ml hyaluronidase and 0.1 mg/ml DNase l
Growth medium
DMEM supplemented 10 % FBS, 100 U/ml Penicillin and 100 μg/ml Streptomycin

Acknowledgments

We thank S. Kohno, H. Muranaka and Y. Nishimoto for helping preparation of this manuscript. Also, we thank A. Berns’s group for stimulating us to create this protocol. This work was supported by Funding Program for Next Generation World-Leading Researchers (NEXT), Grant-in-Aid for Scientific Research (MEXT), Astellas Foundation for Research on Metabolic Disorders, Takeda Science Foundation, Naito Foundation, Daiichi-Sankyo Foundation for Life Science, NOVARTIS Foundation (Japan) for Promotion of Science and Hokkoku Foundation for Cancer Research.

References

Calbo, J., van Montfort, E., Proost, N., van Drunen, E., Beverloo, H. B., Meuwissen, R. and Berns, A. (2011). A functional role for tumor cell heterogeneity in a mouse model of small cell lung cancer. Cancer Cell 19(2): 244-256.
Kitajima, S., Kohno, S., Kondoh, A., Sasaki, N., Nishimoto, Y., Li, F., Abdallah Mohammed, M. S., Muranaka, H., Nagatani, N., Suzuki, M., Kido, Y. and Takahashi, C. (2015). Undifferentiated State induced by Rb-p53 double inactivation in mouse thyroid neuroendocrine cells and embryonic fibroblasts. Stem Cells 33(5): 1657-1669.
Shamma, A., Takegami, Y., Miki, T., Kitajima, S., Noda, M., Obara, T., Okamoto, T. and Takahashi, C. (2009). Rb Regulates DNA damage response and cellular senescence through E2F-dependent suppression of N-ras isoprenylation. Cancer Cell 15(4): 255-269.
Takahashi, C., Contreras, B., Iwanaga, T., Takegami, Y., Bakker, A., Bronson, R. T., Noda, M., Loda, M., Hunt, J. L. and Ewen, M. E. (2006). Nras loss induces metastatic conversion of Rb1-deficient neuroendocrine thyroid tumor. Nat Genet 38(1): 118-123.