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Proximal Culture System: An In Vitro Culture Technique to Study Paracrine Signaling Between Cells
Overview
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 .......
- 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.
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.......
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Source: Dasari, S. et al. A Proximal Culture Method to Study Paracrine Signaling Between Cells. J. Vis. Exp. (2018)
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