Proximal Culture System: An In Vitro Culture Technique to Study Paracrine Signaling Between Cells
Published: April 30, 2023
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…
Declarações
The authors have nothing to disclose.
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 |
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Proximal Culture System: An In Vitro Culture Technique to Study Paracrine Signaling Between Cells. J. Vis. Exp. (Pending Publication), e20374, doi: (2023).