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.
1. Generation of Spheroids from Colorectal Cancer Cell Line
- 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.
- 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).
- 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.
- Count cells using Trypan blue exclusion on a compatible cell counter.
- 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.
- 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.
- 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.
- Transfer the plate to the automated imaging apparatus inside an incubator (37 °C, 5% CO2, 95% humidity).
- Log into the acquisition software, select Schedule To Acquire | Launch Add Vessel | Scan On Schedule | Create Vessel: New.
- 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).
- Select the desired magnification: 10x.
- 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.
- 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).
- Check every 2 days for the growth of spheroids by logging into the imaging software.
- 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.
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|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|