Spheroid Generation from Cell Lines: A Three-Dimensional (3D) Cell Culture Method

Overview

Spheroids are a type of 3D cell culture model used to more accurately represent a cell’s environment compared to 2D cell culture monolayer models. In the example protocol, we will see scientists generate spheroids from a colorectal cancer cell line and apply live-cell imaging to track the spheroids’ growth.

Protocol

1. Generation of Spheroids from Colorectal Cancer Cell Line

1. Wash HCT 116 (stably transduced to express Cluster of Differentiation 19 (CD19) and Green Fluorescence Protein (GFP)) cell monolayers with phosphate buffered saline (PBS; 5 mL for a 25 cm² or 10 mL for a 75 cm² flask). Add trypsin (0.5 mL for a 25 cm² or 1 mL for a 75 cm² flask) and incubate cells at 37 °C for 5 min.
2. Check cell detachment under a microscope and neutralize cell dissociation enzyme with complete Roswell Park Memorial Institute 160 medium (RPMI 1640) (RPMI 1640 + 10% FCS + Gentamycin; 10 mL for a 25 cm² or 20 mL for a 75 cm² flask).
3. Centrifuge cell suspension at 500 x g for 5 min. Remove supernatant using a pipette and resuspend by pipetting up and down several times with 5 mL of complete RPM1 1640 medium.
4. Count cells using Trypan blue exclusion on a compatible cell counter.
5. Centrifuge cell suspension at 500 x g for 5 min. Remove supernatant using a pipette and resuspend in RPMI medium to obtain 5 x 10³cells/mL.
6. Coat a 96-well round bottom plate with 100 µL/well of 100 µg/mL of poly-L-lysine (PLL) in PBS during 1 hour at room temperature. Wash twice with PBS and let the plate dry.
7. Transfer the cell suspension to a sterile reservoir and dispense 200 µL/well into the PLL-coated 96-well round bottom plates using a multichannel pipette.
8. Transfer the plate to the automated imaging apparatus inside an incubator (37 °C, 5% CO₂, 95% humidity).
9. Log into the acquisition software, select Schedule To Acquire | Launch Add Vessel | Scan On Schedule | Create Vessel: New.
10. Select Scan Type: Spheroid. Select the channels of interest: Phase + Brightfield (to follow spheroids growth), Green (to follow tumor signal, acquisition time 300 ms) and Red (to follow apoptosis, acquisition time 400 ms).
    1. Select the desired magnification: 10x.
    2. Pick the plate model and its position in the drawer. Select the position of wells to image. Enter the description of the experiment: name, type of cells, number of cells.
11. For the analysis setup, select Defer Analysis Until Later. Right click on the timeline and select Set Selected Scan Group Intervaloption and set Add scans every to 4 h and For a total of to 24 h. Set the desired starting time (at least 1 h after incubation in the automated imaging apparatus).
12. Check every 2 days for the growth of spheroids by logging into the imaging software.
    1. Pick the View Recent Scans option and double-click on the desired experiment. Select Brightfield in the image channels panel and then use the Measure image features tool to measure the diameter of the spheroids. It takes 6 days for a spheroid to reach the desired size: 0.5 mm of diameter. Add 50 µL of complete RPMI medium per well at day 4 to limit medium evaporation effect.

Materials

Name	Company	Catalog Number	Comments
Dulbecco’s Phosphate Buffered Saline	SIGMA-ALDRICH	D8537-500ML	Lot Number: RNBG7037
75 cm² growth area flasks	VWR	430639	Lot Number: 2218002
75 cm² growth area flasks	VWR	734-2705	Lot Number: 3718006
Trypsin-EDTA	SIGM-ALDRICH	T3924-100ml	Lot Number: SLBTO777
RPMI 1640 med L-glutamin, 10 x 500 mL	Life Technology (Gibco)	21875-091	Lot Number: 1926384
Gentamicin	Thermo Fischer	15750060	Lot Number: 1904924A
Trypan Blue Solution, 0.4%	Thermo Fischer	15250061	Lot Number: 1886513
96 well plate, round bottom	VWR	734-1797	Lot Number: 33117036
X-VIVO 15 with Gentamicin L-Gln, Phenol Red, 1 L	BioNordika	BE02-060Q	Lot Number: 8MB036
Reagent Reservoir	VWR	89094-672	Lot Number: 89094-672
15 mL tubes	VWR	734-1867	Lot Number: 19317044
Fetal Bovine Serum	Gibco	10500064	Lot Number: 08Q3066K
HCT 116 Colorectal Carcinoma Line	ATCC	CCL-247	-
Incucyte S3	Essen Bioscience