3D Co-culture of Lung Cancer Cells with CAFs: An In Vitro Model System to Study Tumor Progression

1.Passaging and Culturing TUM622 Cells and CAFs in 2D Cultures

1. Passaging and culturing TUM622 cells
   1. Warm 3D culture medium and cell dissociation reagents (see Table of Materials) for TUM622 cells at 37 °C.
   2. Passage TUM622 cells at 80% confluency in 2D flasks. Usually, this occurs 1 week after passaging.
   3. Discard old medium from a T75 flask and wash once with 6 mL of HEPES buffer. Avoid pipetting directly onto the cells.
   4. Aspirate the HEPES buffer. Add 4 mL of trypsin/EDTA (0.25 mg/mL, see Table of Materials) for a quick rinse and discard the trypsin/EDTA.
   5. Add 2 mL of trypsin/EDTA and incubate at 37 °C for 5 min. Remove flasks from the incubator and tap the flasks to loosen the cells without creating air bubbles and return flasks to the incubator for an additional 5 min.
      NOTE: Prolonged exposure to trypsin will irreversibly damage the cells and alter their phenotype, thus it is recommended to limit the time cells are exposed to trypsin.
   6. Confirm cells have detached and dissociated under a light microscope (4x or 10x). Add 4 mL of neutralization buffer (TNS buffer) (see subculture reagent information in the Table of Materials) followed by 10 mL of 3D culture medium (see Table of Materials).
   7. Pipette up-and-down gently to further dissociate the cells using a 10 mL pipette. Transfer the suspension through a 40 µm cell strainer into a 50 mL conical tube.
   8. Count cell numbers using a hemocytometer or automated cell counter.
   9. Seed 0.8 x 10⁶ cells/T75 flask in 20 mL of 3D culture medium (see Table of Materials).
   10. Feed the cells every other day by replacing half of the spent medium with fresh medium.
2. Passaging and culturing CAFs
   1. Passage CAFs when cells reach confluency. Usually, this occurs after 5 days of culturing from a 1:2 split.
   2. Prepare CAF medium using RPMI basal medium with 20% heat-inactivated fetal bovine serum, 1% L-Glutamine and 1% Penicillin/Streptomycin. Warm the medium to 37 °C.
   3. In a T75 flask, rinse CAFs with phosphate-buffered saline (PBS) once then add 2 mL of trypsin/EDTA and incubate at 37 °C for 5 min.
   4. Observe under a light microscope to ensure cells have dissociated in the flask (4x or 10x). If not, extend the incubation for another 2-3 min.
   5. Once cells have detached and dissociated, add 10 mL of 3D culture medium to neutralize the trypsin/EDTA and pipette up and down several times to further dissociate the CAFs.
   6. Transfer the cell suspension into a 50 mL conical tube and spin down at 300 x g for 5 min at room temperature.
   7. Discard the supernatant and resuspend the pellet in an appropriate volume of 3D culture medium (see Table of Materials) and passage into two new T75 flasks.

2. Plating TUM622 Cells in the Extracellular Matrix for 3D Culturing

1. The day before the experiment, thaw vials of basement membrane matrix in a 4 °C refrigerator overnight. Cooldown plastic pipettes (2 mL) and tips at -20 °C overnight.
   NOTE: Not all lots of basement membrane matrix have the same capacity to support the 3D growth of TUM622 cells. Therefore, it is necessary to acquire and test multiple lots of basement membrane matrix to identify those that support robust acini formation. Usually, this requires a higher protein concentration (16-18 mg/mL) in the matrix.
2. On the day of the experiment, warm 3D culture medium, HEPES buffer, trypsin/EDTA and trypsin neutralization buffer (TNS) in a 37 °C water bath. Immediately before setting up the culture, take the thawed basement membrane matrix out of the fridge and put the vial on ice.
3. Cool down the tissue culture plates on a metal platform cooler placed on ice. Place centrifuge tubes on a metal cooling rack on ice.
4. Using TUM622 cells obtained from step 1.1.7, calculate the desired number of cells needed for plating. Typically, 15,000-30,0000 cells are needed per well of a 24-well plate. Lower density is more suited for imaging and quantification, while higher density is preferred when collecting cells for RNA extraction or western blotting.
5. Transfer cell suspension into a cooled centrifuge tube (each tube containing cells for triplicate plating) and spin down at 300 x g in a hanging bucket centrifuge at 4 °C for 5 min.
6. Aspirate the supernatant carefully with an aspirating pipette attached to an unfiltered tip (20 µL), leaving approximately 100 µL of the medium in the tube (use markings on the tube as a guide).
7. Gently tap on the side of the tube to dislodge and dissociate the pellet before returning it to the cooling rack.
8. Using the 2 mL pre-cooled pipettes, gently mix the matrix by pipetting up and down a few times while keeping the vial in contact with the ice. Pipette at an even and moderate speed so that no bubbles are introduced into the matrix during this procedure.
9. Transfer the appropriate volume of the matrix into each centrifuge tube. For plating triplicates in a 24-well plate, add 1.1 mL of basement membrane matrix to each tube.
10. Using pre-cooled tips, pipette the matrix in each tube up and down about 10 times to make a uniform cell suspension.
11. Transfer 310 µL of cell/matrix suspension into each well of a pre-cooled 24-well plate. The pipette is placed at a 90° angle to the plate surface and the suspension added to the center of the well. The suspension should spread and cover the entire well without needing to tilt the plate.
12. To facilitate downstream immunofluorescence analysis, plate the cell/matrix suspension in parallel into 2-well chamber slides. Transfer 100 µL of cell/matrix suspension into the center of a well of 2-well chamber slide (see Table of Materials). This allows the matrix to form a dome-like structure with much smaller volume.
13. Return the plate and the chamber slide back into a tissue culture incubator and incubate for 30 min to allow the matrix to solidify. Examine the plate/slide under a light microscope to ensure that single cells are evenly distributed within the matrix (4x or 10x).
14. Add 1 mL of pre-warmed 3D culture complete medium into each well and 1.5 mL of 3D culture medium to each well of the chamber slide then return them to the incubator.

3. 3D Coculturing of TUM622 Cells and CAFs in the Extracellular Matrix

1. Prepare cell suspensions of TUM622 and CAFs according to section 2.
2. Count the CAF cell density by taking 10 µL of cell suspension and mixing it with 10 µL of trypan blue.
3. Add 10 µL of the mixture to each of the two chambers on a hemacytometer to count and calculate cell density.
   NOTE: CAFs have irregular shapes and may not be accurately counted on an automatic cell counter.
4. Co-embedding TUM622 cells and CAFs in basement membrane matrix
   1. Based on the cell density information, calculate the desired number of cells used for plating. CAFs are seeded at a 2:1 ratio of TUM622 cells. For example, for 30,000 TUM622 cells seeded, 60,000 CAFs are co-embedded.
   2. Transfer the appropriate volume of TUM622 as well as CAFs cell suspension into the same centrifuge tube and follow steps 2.5-2.11 for plating into 24-well plates. For immunofluorescence, transfer 60 µL of TUM622/CAFs mix to chamber slides as described in step 2.12).
5. Coculturing TUM622 with overlaid CAFs in basement membrane matrix (see Table of Materials)
   1. Set up TUM622 mono-culture according to steps 2.5-2.13.
   2. Transfer twice the number of CAFs suspension (compared to the number of TUM622 cells seeded) into a centrifuge tube and spin down at 300 x g for 5 min at room temperature.
   3. Aspirate the supernatant and resuspend the CAFs in 1 mL of 3D culture medium.
   4. Transfer the 1 mL of CAFs suspension to the well containing the embedded TUM622 cells.