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

1.Culture of Pancreatic Cancer 3D Spheroids using Magnetic Bioprinting

1. Using standard aseptic tissue culture technique, culture cells of interest in a T75 flask to a confluency of 70-80% in appropriate growth media.
   NOTE: Typically, 5-7 × 10⁶ cells from a 70-80% confluent T75 flask were harvested. Two different cell types were used in this study – Patu8902 (pancreatic tumor cells) and PS1 cells (activated pancreatic stellate cells). Both cell lines were cultured in Roswell Park Memorial Institute media 1640 (RPMI-1640) supplemented with 10% (v/v) fetal bovine serum (FBS), 1× penicillin-streptomycin (p/s).
2. Wash cells once with 5 mL of Dulbecco's phosphate buffered saline (DPBS).
3. Detach cells from plastic surface by trypsinizing with 2 mL of 0.05% Trypsin-EDTA for 5-10 min at 37 °C. Check for cell detachment under a microscope.
4. Deactivate trypsin by addition of 8 mL growth media to detached cells. Avoid over trypsinization, as this can adversely affect the health/viability of cells.
5. Pipet cells up and down to generate a single cell suspension and count cell density using an automated cell counter or a hemocytometer.
6. In the meantime, equilibrate a vial of NS to ambient temperature.
7. Calculate the amount of cells required for seeding the desired number of spheroids (wells) and aliquot to a new 15 mL conical tube. For example, to seed an entire 96 well plate with 5,000 cells/well, aliquot at least 5.5 × 10⁵ cells.
8. Collect cells by centrifuging at 500 x g for 3 min. Carefully asp irate or decant the supernatant and resuspend the cells at a concentration of 1.0 x 10⁶ cells/mL in growth media. Pipette gently using a wide bored pipet tip to avoid shearing. For example, resuspend 5.5 x 10⁵ cells in 550 µl of growth media.
9. Magnetize the desired amount of cells using the equilibrated NS (step 1.6).
   1. Directly add NS to the cell suspension in growth media and agitate gently. For efficient magnetic labeling of cells, use 10 µL NS per 100 µL cells (resuspended at 1 x 10⁶ cells/mL).
      NOTE: For a typical experiment, label 5.5×10⁵ Patu8902 cells in 550 µL with 55 µL NS and 1.1×10⁶ PS1 cells in 1100 µL, (separately) with 110 µL NS.
   2. Gently invert tube a few times to ensure cell suspension. Temporarily, transfer the cell-NS mix into a single well of a 24-well plate. Do this separately for each cell type to be magnetized. Cover plate and incubate cells at room temperature for 2 h with gentle shaking to allow NS binding to cell surface.
      NOTE: Culture and assay of spheroids starting either from Patu8902 or PS1 alone cells, in addition to co-culture spheroids starting with both cell types premixed before printing on magnetic drives was successfully achieved using this method in independent experiments. A method for generating co-culture spheroids from Patu8902 and PS2 cells is described below in the subsequent steps.
10. Pipet up and down the magnetized cells gently to mix evenly and prepare a master mix containing the desired cell number. For seeding spheroids, use a total of 15,000 cells (5,000 Patu8902 + 10,000 PS1) and adjust to a total volume of 150 µL per well of a 96-well plate.
    NOTE: The final volume dispensed into each well must be the same regardless of the number of cells used.
11. Place a cell repellent 96-well plate atop the 96-well magnetic spheroid drive and dispense 150 µL of cell mix into each well. Observe the cells slowly collect to the center of the well above the magnet. Cover and incubate the plate overnight in a 5% CO₂ incubator set at 37 °C with the magnetic spheroid drive still attached. Remove the magnetic drive and incubate for further growth for up to 7 days.
    NOTE: It is critical to use cell repellent growth plates for spheroid printing using magnetic spheroid drives. Ensure that the spheroid drive with thinner smaller magnets is used, not the holding drive.
12. Monitor the growth of spheroids every day. Replenish with fresh growth media every three days by placing the growth plate mounted on the magnetic holding drive at an angle and using a multichannel pipet to draw out spent media.
    NOTE: Patu8902 and PS1 cells co-seeded at 15,000 cells/well will grow into 3D spheroids with a diameter of 400-600 µm within 5-7 days. At this point spheroids are ready for metabolic characterization, drug evaluation, and other downstream applications.