Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Culture condition

RPE-1 hTERT cell line was cultured in Dulbecco's Modified Eagle Medium (Table of Materials) supplemented with 10% Fetal Bovine Serum, 2 mM L-glutamine, and 100 U/ml penicillin/streptomycin. Cells were incubated at 37 °C with 5% CO₂ in a humidified environment. The protocol described below has been developed by using 10-cm dishes and all technical details reported refer to those dishes. If using different dishes, scale up or down accordingly.

1. Synchronization of RPE-1 cells

1. Use freshly thawed cells that have not undergone more than 15 passages for all experiments. On day 1, thaw and dispense ~2.5 – 5.0 x 10⁵ RPE-1 hTERT cells into a 10-cm tissue culture dish. Add 8 mL of standard growth medium and incubate overnight. Determine cell number using a standard cell counter (such as a hemocytometer).
   NOTE: Cell density refers to growth conditions of RPE-1 cells. Growth conditions might vary among different cell lines; consider adjusting them accordingly.
2. On day 2, synchronize cells at the G1/S border by aspirating regular growth medium and adding 8 mL medium containing 5 mM thymidine.
   NOTE: Thymidine concentration and length of treatment might vary across different cell lines. It is advisable to perform pilot experiments to determine the best conditions for the cell line(s) to be tested.
3. On day 3, 24 h after adding thymidine medium (step 1.2), release from thymidine block by aspirating medium and washing cells three times with 1x PBS. Add 8 mL of regular growth medium and return plates to the incubator.

2. Generation of Aneuploid Cells by Interference with the Activity of the Mitotic Kinase Mps1

1. On day 3, 6 h after release from thymidine block (step 1.3; this is about 3 – 6 h before cells enter mitosis), aspirate medium, wash once with 1x PBS, and replace with medium containing 500 nM reversine (the Mps1 inhibitor).
   NOTE: RPE-1 cells enter mitosis 9 – 12 h after thymidine wash-out^10,11. However, cell cycle kinetics vary among different cell lines. Therefore, it is crucial to determine those kinetics in pilot experiments by performing cell cycle analysis by established methods (e.g., by measuring DNA content via FACS analysis). Additionally, the optimal working concentration of Mps1 inhibitor may vary between cell lines. Previous experiments using RPE1 cells have been successful using reversine at a working concentration of 500 nM^10,11,12. It is advisable to perform pilot experiments to determine the optimal concentration for the cell line to be tested.
2. On day 4, 12 h after reversine treatment (about 6 h after cells were in mitosis), aspirate reversine medium and wash cells three times with 1x PBS. Add 8 mL regular growth medium to the plate and return cells to the incubator.

3. Removal of Aneuploid Cycling Cells and Enrichment of ArCK Population

1. On day 6, about 72 h after cells enter the first mitosis (step 2.2; this is about 66 h after reversine wash-out, which corresponds to about 2 – 3 cell cycles), aspirate medium, wash once with 1x PBS and replace with medium containing 300 nM nocodazole.
   NOTE: Recent work has shown that RPE-1 cells harboring aneuploid karyotypes are genomically unstable and arrest their proliferation about 2 – 3 cell cycles after the first faulty mitosis¹⁰. However, this kinetics might vary among different cell lines. Therefore, it is crucial to perform pilot experiments to determine the proper timing for the removal of aneuploid cycling cells.
2. On day 7, 12 h after nocodazole treatment, aspirate medium and add 3 mL of 1x PBS to the plate. Shake off the mitotic cells by tapping the side of the plate. Rotate the plate between each tap to allow even removal of mitotic cells.
3. To ensure complete removal of mitotic cells, repeat the shake-off process for multiple rounds (3 – 5 times recommended), aspirating and adding 3 mL of 1x PBS between each round.
4. After shake-off, gently aspirate any liquid that adhered to the lid or the edge of the plate. Briefly, check the plate under a microscope at 10X magnification. Ensure that few mitotic cells are observed. If more than five mitotic cells are seen under 10X objective, repeat another round of shake-off.
   NOTE: Mitotic cells would appear rounded and may be partially detached after shake-off. It is crucial to ensure the complete removal of mitotic cells before exposing the cells to the next round of nocodazole treatment. Failure to do so may lead to mitotic cell death and/or result in the generation of tetraploid cells.
5. After final shake-off, wash cells once with 5 mL of 1x PBS. Add 8 mL medium containing 330 nM nocodazole into the plate and incubate for 12 h.
6. Repeat nocodazole treatment/shake-off (steps 3.1 – 3.5) every 12 h until no mitotic cells are observed after incubation. Usually, 4-5 rounds of treatment/shake-off are recommended.
   NOTE: The information provided here refer to RPE-1 cells. The number of rounds of treatment/shake-off might vary among different cell lines. To avoid prolonged and unnecessary exposure to nocodazole it is crucial to carefully determine, in a pilot experiment, the number of rounds required for efficient and complete removal of aneuploid cycling cells.
7. After 4 – 5 rounds of nocodazole treatment/shake-off (steps 3.1 – 3.6), add 10 mL of regular growth medium and incubate. Allow cells to rest for 12 – 24 h before future manipulation or assay use.
   NOTE: After final shake-off, cells remaining on the plate are cell cycle arrested and, as shown recently¹⁰, harbor complex karyotypes. These cells are referred to as ArCK (Arrested with Complex Karyotype) cells. It is advisable to perform assays on ArCK populations within a week of isolation.
8. Optionally, to assess the percentage of arrested cells with complex karyotypes, collect and count cells from each shake-off using a standard cell counter.

Figure 1: Overall schematic of the method used to generate and isolate aneuploid cells with complex karyotype (ArCK) and representative images. (A) Schematic of ArCK cells generation and isolation protocol. (B) Representative images of RPE-1 hTERT cells at each stage of the treatment. The last panel (outlined in red) represents the isolated ArCK cells. Scale bar 100 µm. Please click here to view a larger version of this figure.

4. Characterization of ArCK Population

1. Confirm successful isolation of ArCK population by performing the following assays.
   1. Asses beta-galactosidase staining (the marker of senescence) using a commercially available kit (see Table of Materials).
   2. Measure secretion of pro-inflammatory cytokines, such as CCL2, by employing commercially available kits (see Table of Materials).
   3. Confirm increased levels of the cell cycle inhibitors p53, p21, p16 by Western Blot.
      NOTE: To properly control these assays, ArCK populations should be compared to both euploid and aneuploid cycling cells. The former is low passage, wild-type RPE-1 hTERT cells exponentially growing. The latter can be obtained by inducing chromosome mis-segregation in RPE-1 hTERT and harvesting cells 72 h after the first aberrant mitosis. To do so, follow the protocol described above from step 1.1 and harvest cells right before the first nocodazole treatment (step 3.1).
2. Perform single-cell sequencing to assess the karyotype of the arrested cells.
   NOTE: Single-cell sequencing is a well-established method to determine chromosomal and sub-chromosomal changes across a cell population at the single cell level¹³. Detailed procedures for how to perform single-cell sequencing can be found elsewhere¹⁰.