Cell lines and media
Cell lines HeLa (carcinoma cells), HCT116 (colon cancer cells), HepG2
(liver cancer cells), A549 (lung cancer cells), MCF-7 (breast cancer
cells), 5637 (bladder cancer cell) and L-02 (human hepatocytes) were
purchased from American Type Culture Collection (ATCC, Manassas, VA,
USA). UCF (Umbilical cord fibroblast) was obtained from National Center
of Bio-Engineering & Technology (Shanghai, CN). PBMC (Peripheral blood
mononuclear cells) was obtained from Otwo Biotech (Shenzhen, CN).
All cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM,
11995-06, Gibco, Grand Island, NY, USA) containing 10% fetal bovine
serum (FBS, 10099-141, Gibco, Grand Island, NY, USA), 100 IU/mL
penicillin and 100 mg/mL streptomycin (B540732, Sangon Biotech,
Shanghai, CN). All cultures were maintained in an incubator (Thermo
Fisher Scientific, Waltham, MA,
USA) at 37 °C with 5% CO2 and 95% humidity, and
passaged when the confluence of cells reached 80% ~
90%.
Screen for optimized
conditions to generate single tumor spheroid
To establish a protocol for rapid spheroid generation in a
high-throughput manner, the formation of single tumor spheroid per well
in a 96-well plate was fully optimized (Figure. 1 ). Cells grown
as a monolayer were detached with trypsin to generate a single-cell
suspension. The cell number was determined
using Countstar (ALIT Life
Science, Shanghai, CN) with the automatic cell counter software. To
prevent cell attachment, Ultra-low attachment 96-well plates in both
“Flat bottom” (3474, Corning, NY, USA) and “U-shaped” (7007,
Corning, NY, USA) were utilized.
For optimal spheroid formation,
the cell seeding density (500~20000 cells/well), serum
concentrations (5% ~ 20%), external forces (gravity
and centrifugal forces), and medium additives (2.5% of Geltrex™
(A15696-01, Gibco, Grand Island, NY, USA) and Matrigel™ (356237,
Corning, NY, USA) were tested. For external forces, the experiments were
carried out as follows: 1. Control: 200 μL cell suspension was directly
seeded without treatment; 2.Hanging drop method: seeded 20 μL cell
suspension at the bottom of the well plate, turned the culture plate
upside down and incubated for 48 h, then put the well plate upright and
supplemented with 180 μL medium; 3. Centrifugation of the whole plate:
seeded 200 μL cell suspension, and the whole culture plate was
centrifuged at 1000 g for 10 min; 4. EP Tube centrifugation: added 200
μL cell suspension to a 1.5 mL EP tube, centrifuged at 1000 g for 10
min, and then transferred the cell clusters to a 96-well plate; 5.
Rotary shaker: seeded 200 μL cell suspension, and placed the whole plate
on a shaker at 60 RPM.