Development, establishment and validation of in vitro and ex vivo assays of vascular calcification

Jana Holmar, Heidi Noels, Michael Boehm, Shruti Bhargava, Joachim Jankowski*, Setareh Orth-Alampour

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

10 Citations (Web of Science)


Objective: Vascular calcification (VC) is one major complication in patients with chronic kidney disease, with a misbalance in calcium and phosphate metabolism playing crucial role. The mechanisms underlying VC have not been entirely revealed to date. As studies aiming at the identification and characterization of the involved mediators are highly relevant, we developed a standardized operating protocol for in vitro and ex vivo approaches in this study to aiming at the comparability of these studies.

Approach and results: We analyzed in vitro and ex vivo experimental conditions to study VC. Therefore, vascular smooth muscle cells were used for in vitro experiments and rat aorta for ex vivo experiments. The degree of calcification was estimated by quantification of calcium concentrations and by von Kossa staining. As a result, a step-by-step protocol for performing experiments on VC was established. We were able to demonstrate that the degree and the location of VC in vascular smooth muscle cells and aortic rings was highly dependent on the phosphate and CaCl2 concentration in the medium as well as the incubation time. Furthermore, the VC was reduced upon increasing fetal calf serum concentration in the medium.

Conclusion: In the current study, we developed and validated a standardized operating protocol for systematic in vitro and ex vivo analyses of medial calcification, which is essential for the comparability of the results of future studies. (C) 2020 The Authors. Published by Elsevier Inc.

Original languageEnglish
Pages (from-to)462-470
Number of pages9
JournalBiochemical and Biophysical Research Communications
Issue number2
Publication statusPublished - 17 Sept 2020


  • Vascular calcification
  • Vascular smooth muscle cells
  • Rat aorta
  • CaCl2
  • Phosphate

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