Magnetically Bioprinted 3D Spheroid Co-cultures: A Method for Co-culturing Cancerous Cells and Fibroblasts

– In magnetic bioprinted 3D co-cultures, multiple cell types form 3D structures or spheroids under magnetic fields' influence. To set up the culture, first, prepare single-cell suspensions of cancer cells and fibroblasts in their appropriate medium. Next, add biocompatible magnetic nanoparticles to both suspensions. These nanomaterials contain positively charged poly L-lysine residues that facilitate the nonspecific binding of magnetic particles to the cell membrane via electrostatic interactions.

Incubate to achieve maximum magnetization of cancer cells and fibroblasts. Subsequently, mix both cell suspensions in the desired ratio. Transfer this cell-nanoparticle suspension to a cell-repellent culture plate, specifically optimized to prevent cell attachment. Mount this setup on a magnetic drive and incubate. Magnetic forces guide the aggregation or bioprinting of magnetized cancer cells and fibroblasts into 3D spheroid structures at the bottom of the well.

Once spheroids form, remove the magnet and allow the 3D co-cultures to grow to the desired volume. In this protocol, we will demonstrate the generation of three-dimensional co-culture spheroids from pancreatic cancer cells and PS1-activated pancreatic fibroblast cells using magnetic bioprinting technique.

Using standard aseptic tissue culture technique, culture the cancer cells and fibroblasts of interest in T75 flasks in the appropriate growth medium. When the cells reach 70% to 80% confluency, wash the cultures one time in 5 milliliters of PBS and detach the cells from the flask surfaces with 2 milliliters of 0.05% Trypsin-EDTA per flask for five minutes at 37 degrees Celsius.

After confirming detachment under a microscope, deactivate the Trypsin with 8 milliliters of growth medium per flask. And pipette the cells a few times to generate single-cell suspensions for counting. Collect the cells by centrifugation and resuspend the pellets in 1 times 10 to the sixth cells per milliliter concentrations in fresh growth medium.

To magnetize the cells, add 10 microliters of NanoShuttle in growth medium per 100 microliters of cells and agitate the solutions gently. Gently invert the tubes a few times and transfer the cell-NanoShuttle mixes into individual wells of a 24-well plate. Incubate the cells at room temperature for two hours with gentle shaking to facilitate the NanoShuttle binding to the cell surface. At the end of the binding incubation, gently mix each magnetized cell suspension with pipetting and adjust the concentration to 1.5 times 10 to the fourth magnetized cancer cells or fibroblasts per 150 microliters of medium.

Mix the fibroblast and cancer cells in a 2 to 1 ratio for a total of 150 microliters of seeded cells per well. Place the cell-repellent, 96-well plate on top of a 96-well magnetic spheroid drive with thinner magnets and seed 150 microliters of cell mixture into each well. When all of the cells have been plated, incubate the plate overnight at 37 degrees Celsius and 5% CO2 with the magnetic spheroid drive still attached. The next morning, remove the magnetic drive and return the cells to the incubator for up to seven more days.