Apr 01, 2025
Standard Protocol for Magnetics Spheroids in hydrogel Formation
- Marine Simonneau1,
- magali perier1,
- Jacqueline Ngo-Reymond1
- 1INSERM U1032
Protocol Citation: Marine Simonneau, magali perier, Jacqueline Ngo-Reymond 2025. Standard Protocol for Magnetics Spheroids in hydrogel Formation. protocols.io https://dx.doi.org/10.17504/protocols.io.261geeoxyg47/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: April 01, 2025
Last Modified: April 01, 2025
Protocol Integer ID: 125854
Abstract
This protocol is used to grow spheroids in hydrogel.
Materials
● KPC A219 Cells
● iMEF Cells
● Nanoshuttles (GreinerBio-One 657846)
● Complete DMEM/F-12 Culture Medium (10% FBS, 1%L-Glutamine, 1% Penicillin/Streptomycin) (Gibco 21331-020)
● DPBS (Gibco14190-086)
● Trypsin (Gibco25300-054)
● 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
● Pipetboy
● T75 Culture Flask (Corning 430641U) / T25 Culture Flask (Thermo Fisher Scientific 156367)
● Growdex-T (UPM Biomedicals ref 100 103 005)
● Growdase (UPM Biomedicals ref 900 102 002)
● 96-Well Transparent Plate with Flat Bottom (GreinerBio-One 655970) / 96-Well Transparent Plate with Round Bottom (Greiner Bio-One650970)
● 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.
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.
Take new culture flasks.
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.
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.
Incubate the flasks in the incubator (37°C, 5% CO2).
Day 0: Co-culture and Spheroid Formation
Day 0: Co-culture and Spheroid Formation
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.
Dispensing the Mixed Solution into the 96-Well Plate
Dispense 150 µL of the mixed solution into each well of the 96-well plate.
Cell Incubation
Incubate the cells in the 96-magnet plate in the incubator (37°C, 5% CO2) for 6 hours, then remove the magnet plate.
Note: If the spheroids are for elastography, the spheroids should be transferred to a 4-well plate (use P1000 to transfer the spheroids to avoid breaking them); otherwise, leave the spheroids incubating in the incubator for 4 days for chemotherapy or cavitation testing.
Day 4: Hydrogel addition
Day 4: Hydrogel addition
Aspirate the medium gently, keeping the spheroid in the medium. It is possible to remove only 50 to 80µl of medium.
Preparation of 0.4% hydrogel solution for a final volume of 10ml: 4ml Growdex 1% + 6ml medium
La solution mère est à 1% de cellulose.
Est-ce qu’il faut le mentionner quelque part ?
Add Growdex to the medium with a low retention cone, swirling with the pipette.
Then pipette back and forth for 90sec to mix well without bubbling!
Add 100 µl the prepared hydrogels to the wells
Add 100µl of medium on top of the gel or 100µl of medium with 0.5mM Gemcitabine on top of the hydrogel.
Day 10 : Growdex hydrogel degradation
Day 10 : Growdex hydrogel degradation
I have 0.4mg of cellulose in a well, so I need 180µg of Growdase for one well.
The concentration of Growdase is 10µg/µl, i.e. 18µl to obtain 180µg.
I have 96 wells, so 18x96 = 1728µl Growdase + 7872µl medium
I aspirate the medium and add 100µl of this solution/well
Let incubate 8h at 37°C