Hormone-deprived serum impairs angiogenic properties in human endothelial cells regardless of estrogens

ABSTRACT

Aims: In vitro studies on hormone biological activities are commonly performed on cells cultured in nominally hormone-free media consisting of phenol-red-free media supplemented with charcoal-stripped (CS) serum. These media are largely used in almost all cell types, including endothelial cells (ECs). Methods: Cell number and metabolic activity were measured with standard methods. Angiogenesis was evaluated in a three-dimensional spheroid sprouting assay. Results: When we compared human umbilical vein ECs (HUVECs) cultured in standard conditions (199 medium supplemented with normal serum) with HUVECs grown in the hormone-free medium (phenol-red-free 199 medium supplemented with CS serum), we found that cells stop to grow in the absence of hormones. Notably, neither 17-β2 estradiol nor dihydrotestosterone reversed this inhibition. Moreover, the presence of the CS serum was sufficient to abrogate the ability of HUVECs to sprout in a three-dimensional spheroid assay, thus affecting a functional property of ECs. Conclusions: Our results suggest that one or possibly more substances removed by stripping procedure from serum and different from sex hormones are crucial for the maintenance of in vitro ECs distinctive properties. Therefore, caution should be used when ECs are studied in media containing the CS serum.

KEYWORDS:

• Cell growth
• endothelial cells
• hormone-deprived serum
• in vitro angiogenesis
• sex steroids

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Acknowledgments

We thank Dr. Donatella Lattuada for FACS analysis and Dr. Silvia Bini for HUVEC isolation, and all the nurses of the Macedonio Melloni Hospital for helping in the umbilical cord collection.

Funding

This work was partly supported by the Department of Medical Biotechnology and Translational Medicine, University of Milano (Piano Sviluppo Linea B, grant numbers 15-6-3003005-14 to Maria Grazia Cattaneo).

Additional information

Funding

This work was partly supported by the Department of Medical Biotechnology and Translational Medicine, University of Milano (Piano Sviluppo Linea B, grant numbers 15-6-3003005-14 to Maria Grazia Cattaneo).