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

Abstract

Source: Dasari, S. et al. A Proximal Culture Method to Study Paracrine Signaling Between Cells. J. Vis. Exp. (2018)

In this video, we develop a proximal co-culture model of ovarian cancer cells and primary human mesothelial cells. This physiologically relevant model allows the study of paracrine signaling, where the localized concentrations of the secreted factors are maintained while preventing direct cellular contact.

Protocol

A. Cell Preparation Isolation and culture of human primary mesothelial cells 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 …

Representative Results

Figure 1: Assembly of the proximal culture system. (A) This panel shows human primary mesothelial cells (HPMCs). (B) This panel shows HeyA8 ovarian cancer cells. Scale bars = 200 µm (A-B). (C) HeyA8 cells were seeded on the lower surface of a transwell insert with 0.4 µm pores. Once the cells were attached, the…

Materials

24 mm Transwell permeable support with 0.4 µm Pore Polycarbonate Membrane Insert	Corning (Costar)	3412	• 10 µm thick translucent polycarbonate membrane. • Treated for optimal cell attachment. • Packaged 6 inserts in a 6 well plate, 4 plates per case. • Membrane must be stained for cell visibility. • Sterilized by gamma radiation.
6 well plate	Corning (Falcon)	353046	Flat Bottom, TC-treated, sterile, with Lid
15 cm culture dish	Corning (Falcon)	353025	Sterile, TC-treated Cell Culture Dish
DMEM	Corning (Cellgro)	10-013-CV
Penicillin Streptomycin	Corning	30-002-CI
MEM Nonessential amino acids	Corning (Cellgro)	25-025-CI
Pipets			Any make is fine
CO2 Incubator			Any make is fine
Biosafety level II cabinet			Any make is fine