Proximal Culture System: An In Vitro Culture Technique to Study Paracrine Signaling Between Cells

A. Cell Preparation

1. Isolation and culture of human primary mesothelial cells
   1. Isolate human primary mesothelial cells (HPMCs) from the human omentum and grow them in a complete growth medium [Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum, 1% penicillin-streptomycin, 1% non-essential amino acids, and 1% vitamins] at 37 °C and 5% CO₂.
      NOTE: The HPMCs are typically grown to 100% confluence (Figure 1A).
2. ​Growth conditions of ovarian cancer cells
   1. Grow HeyA8 ovarian cancer cells in complete growth medium at 37 °C and 5% CO₂.
      NOTE: Use HeyA8 cells grown to about 80% confluence (Figure 1B).

B. Cell Culture

1. Proximal culture set-up
   1. Use Transwell inserts for 6-well plates with a 10 μm-thick tissue-culture-treated polycarbonate membrane with 0.4 μm pores (10⁸ pores/cm²) and a 4.67 cm² growth area. If needed, optimize for different insert sizes by scaling the number of cells seeded according to the growth surface area. Warm complete growth medium, trypsin, and phosphate-buffered saline (PBS) to 37 °C before use.
2. Preparing the inserts
   1. To seed the HeyA8 cells on the lower surface of the membrane of the insert, remove the insert from its packaging using sterile forceps and place it inverted in a sterile 15 cm culture dish.
   2. Use a 15 cm dish, as it has the depth to accommodate the inverted insert, or substitute it with any appropriate sterile container. Depending on the experiment, place the required number of inserts in the 15 cm dish.
   3. Label the three controls and three experimental conditions accordingly with a marker on the edge of the inserts.
3. Preparing the cancer cells
   1. To trypsinize the HeyA8 cells grown at 80% confluence in a 25 cm² flask (~2 x 10⁶ cells), add 1 mL of trypsin (0.25%) for 40–60 s and neutralize the trypsin with 6 mL of complete growth medium.
   2. Centrifuge the cells at 500 x g at room temperature (RT) for 3 min, discard the supernatant, and resuspend the cell pellet in 5 mL of complete growth medium. Count the live cells using the trypan blue dye exclusion method.
4. Seeding the cancer cells
   1. Suspend 100,000 cells/mL of live HeyA8 cells in complete growth medium. Carefully seed 80,000 HeyA8 cells suspended in 800 μL of complete growth medium on the bottom of the insert (which is now facing up, since it is inverted).
   2. Seed the cells to form a dome-like shape so the cells remain on the insert (Figure 1C). Start from the center of the membrane and move outward in concentric circles while slowly pipetting. Avoid going more than 3 mm from the edge to prevent any spilling of the medium.
      NOTE: The number of cells seeded will depend on the growth rate of the cells and the duration of the experiment. The number of HeyA8 cells seeded for this proximal culture were optimized, and the cells will be 80–90% confluent at the end of the third day.
5. Cancer cell attachment
   1. Cover the 15 cm dish and carefully move the dish containing the inserts to the CO₂ incubator. It is critical not to disrupt the drop of medium and cells on the insert at this step. Leave the cells at 37 °C for 4 h to allow the cancer cells to attach to the insert.
6. Preparing the mesothelial cells
   1. After around 4 h, trypsinize the HPMCs grown at 100% confluence in a 75 cm² flask (~4 x 10⁶ cells) with 2 mL of trypsin (0.25%) for 1–2 min and neutralize the trypsin with 12 mL of complete growth medium.
   2. Centrifuge the cells at 500 x g at RT for 3 min, resuspend the cell pellet in 10 mL of complete growth medium, and count the live cells using the trypan blue dye exclusion method.
7. Seeding the mesothelial cells
   1. Suspend 300,000 cells/mL of live HPMCs in complete growth medium. Add 2.5 mL of fresh complete growth media to each well of a 6-well plate. Carefully bring the 15 cm dish containing the inserts out to the biosafety hood. Using sterile forceps, flip the insert and place it in the well of the 6-well plate so that it is upright, with the HeyA8 cells attached to the lower surface immersed in the complete growth medium (Figure 1C).
   2. Seed 450,000 HPMCs suspended in 1.5 mL of growth medium in the inside of the inserts with the HeyA8 cells attached to the surface facing the bottom of the well (Figure 1C) or to inserts without any HeyA8 cells for the HPMC controls. Add 1.5 mL of growth medium without cells to the HeyA8 controls.
8. Growing the proximal culture
   1. Let the proximal culture grow in the CO₂ incubator at 37 °C and 5% CO₂ for 72 h. If required, the medium can be replenished as follows.
      1. For the inside of the insert, remove 750 µL and add 750 µL of fresh complete growth medium.
      2. For the outside of the insert (the 6-well plate), remove 1 mL and add 1 mL of fresh complete growth medium. The 1 mL was chosen for the convenience of changing the medium in one step. If desired, 1.25 mL can be removed and replaced instead.