Mar 28, 2025
Standard Protocol for Magnetic Spheroid Formation
- Marine Simonneau1
- 1LabTAU
Protocol Citation: Marine Simonneau 2025. Standard Protocol for Magnetic Spheroid Formation . protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4z772vo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 28, 2025
Last Modified: March 28, 2025
Protocol Integer ID: 125617
Abstract
Our study introduces three-dimensional (3D)
pancreatic adenocarcinoma spheroid models using magnetic aggregation of
pancreatic cancer cells and immortalized fibroblasts
Materials
●
KPC A219 Cells
●
iMEF Cells
●
Nanoshuttles (Greiner
Bio-One 657846)
●
Complete DMEM/F-12 Culture Medium (10% FBS, 1%
L-Glutamine, 1% Penicillin/Streptomycin) (Gibco 21331-020)
●
DPBS (Gibco
14190-086)
●
Trypsin (Gibco
25300-054)
●
TGFβ Solution
(Transforming Growth Factor) (0.01 mg/ml) (PEPROTECH 100-21)
●
96-Magnet Plate (Greiner Bio-One 130188)
●
24-Magnet Plate
●
Medium Aspirator
●
Tube Holder
●
Malassez/Thomas Hemocytometer
●
Blue Trypan
●
Micropipettes
20µL and 100µL (STAR LAB)
●
Pipetboy
●
T75 Culture Flask (Corning 430641U) / T25 Culture
Flask (Thermo Fisher Scientific 156367)
●
96-Well Transparent Plate with Flat Bottom (Greiner
Bio-One 655970) / 96-Well Transparent Plate with Round Bottom (Greiner Bio-One
650970)
● 15ml Centrifuge Tubes (Fisher Scientific 431885) / 50ml
Centrifuge Tubes (Fisher Scientific 431176)
Day -3 Cell Passage
Day -3 Cell Passage
●
Warm the DMEM culture medium, DPBS, and trypsin in a
water bath.
●
Aspirate the medium from the flasks.
●
Add 5 mL of DPBS to the T75 flask and 3 mL of DPBS to
the T25 flask to rinse the cells.
● Aspirate the DPBS.
●
Add 2 mL of trypsin to the T75 flask and 1 mL of
trypsin to the T25 flask.
●
Incubate KPC A219 cells for 6 minutes and iMEF cells
for 5 minutes in the incubator (37°C, 5% CO2).
●
Check if the cells detach properly from the flask
walls.
●
Add 10 mL of DMEM to the T75 flask and 5 mL of DMEM to
the T25 flask to inhibit the trypsin reaction.
●
Take 20 µL of cell suspension and mix it with 20 µL of
Blue Trypan.
●
Distribute the mixed solution on the Malassez/Thomas
cell counter, then count the cells.
●
Centrifuge the cell suspensions for 5 minutes at 300g
in a centrifuge at room temperature.
Preparation of Cell Suspensions at 2x10⁶ Cells/mL
Preparation of Cell Suspensions at 2x10⁶ Cells/mL
●
Calculate the volume of DMEM needed to dilute or
concentrate the cell suspensions to a concentration of 2x10⁶ cells/mL:
Volume of DMEM to add = Total number of cells / 2x10⁶
●
Aspirate the supernatant and add the calculated volume
of DMEM to achieve a cell suspension at 2x10⁶ cells/mL.
●
Homogenize the cell suspension by pipetting.
Preparation of New T75 or T25 Flasks for KPC A219/iMEF Cell Culture
Preparation of New T75 or T25 Flasks for KPC A219/iMEF Cell Culture
●
To form 0-40 spheroids: use 1 T25 flask of KPC and 1
T25 flask of iMEF.
●
To form 40-100 spheroids: use 1 T25 flask of KPC and 1
T75 flask of iMEF / 2 T25 flasks of iMEF.
●
For the T75 flask: add 15 mL of DMEM + 300 µL of cell
suspension at 2x10⁶ cells/mL.
●
For the T25 flask: add 5 mL of DMEM + 100 µL of cell
suspension at 2x10⁶ cells/mL.
Cell Incubation
Cell Incubation
●
Observe the plate under the optical microscope.
●
Incubate all flasks in the incubator (37°C, 5% CO2).
Day -1: Addition of TGF Beta and Nanoshuttles
Day -1: Addition of TGF Beta and Nanoshuttles
Addition of TGFβ to iMEF Flask 24 Hours Before Co-culture●
For the T75 iMEF flask, add 75 µL of TGFβ to achieve 50 ng/mL of TGFβ in the culture medium.
●
For the T25 iMEF flask, add 25 µL of TGFβ to achieve 50 ng/mL of TGFβ in the culture medium.
Addition of
Nanoshuttles to KPC A219 and iMEF Flasks the Day Before Co-culture
●
For KPC A219: add 100 µL of Nanoshuttles to the T25
flask.
●
For iMEF: add 50 µL of Nanoshuttles to the T25 flask
and 150 µL of Nanoshuttles to the T75 flask.
Day 0: Co-culture and Spheroid Formation
Day 0: Co-culture and Spheroid Formation
Cell Passage and
Preparation of Cell Suspensions at 2x10⁶ Cells/mL Concentration
●
Perform the cell passage and record the concentrations
and cell counts.
Cell Count●
Prepare the cell suspensions of KPC and iMEF at 2x10⁶
cells/mL concentration and record the volumes of the cell solution.
Preparation of KPC and iMEF Mixing Solution●
Calculate the volume of KPC suspension, iMEF
suspension, and DMEM culture medium needed to form spheroids:
1 spheroid = 10,000 KPC + 20,000 iMEF
●
Mix the KPC suspension, iMEF suspension, and DMEM
after the calculations.
●
Dispense 150 µL of the mixed solution into each well
of the 96-well plate.
Incubate
the cells in the 96-magnet plate in the incubator (37°C, 5% CO2) for 6 hours,
then remove the magnet plate.