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J Vis Exp. 2018 Aug 28;(138). doi: 10.3791/58144.

A Proximal Culture Method to Study Paracrine Signaling Between Cells.

Author information

1
Indiana University School of Medicine.
2
Urology, Indiana University Health Southern Indiana Physicians.
3
Indiana University School of Medicine; Indiana University Melvin and Bren Simon Cancer Center; Department of Medical and Molecular Genetics, Indiana University School of Medicine; anmitra@indiana.edu.

Abstract

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Paracrine or juxtacrine signaling mediates such interactions. The use of a conditioned medium and coculture studies are the most common methods to discriminate between these two types of interactions. However, the effect of localized high concentrations of secreted factors in the microenvironment during the paracrine interactions is not accurately recapitulated by conditioned medium and, thus, may lead to imprecise conclusions. To overcome this problem, we have devised a proximal culture method to study paracrine signaling. The two cell types are grown on either surface of a 10 µm-thick polycarbonate membrane with 0.4 µm pores. The pores allow the exchange of secreted factors and, at the same time, inhibit juxtacrine signaling. The cells can be collected and lysed at the endpoint to determine the effects of the paracrine signaling. In addition to allowing for localized concentration gradients of secreted factors, this method is amenable to experiments involving prolonged periods of culture, as well as the use of inhibitors. While we use this method to study the interactions between ovarian cancer cells and the mesothelial cells they encounter at the site of metastasis, it can be adapted to any two adherent cell types for researchers to study paracrine signaling in various fields, including tumor microenvironment, immunology, and development.

PMID:
30222146
PMCID:
PMC6231914
[Available on 2020-08-28]
DOI:
10.3791/58144
[Indexed for MEDLINE]

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