Defining the most potent osteoinductive culture conditions for MC3T3-E1 cells reveals no implication of oxidative stress or energy metabolism

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Semicheva, A, Ersoy, U, Vasilaki, A, Myrtziou, I and Kanakis, I (2024) Defining the most potent osteoinductive culture conditions for MC3T3-E1 cells reveals no implication of oxidative stress or energy metabolism. International Journal of Molecular Sciences, 25 (8). ISSN 1661-6596

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Abstract

The MC3T3-E1 preosteoblastic cell line is widely utilised as a reliable in vitro system to assess bone formation. However, the experimental growth conditions for these cells hugely diverge, and, particularly, the osteogenic medium (OSM)’s composition varies in research studies. Therefore, we aimed to define the ideal culture conditions for MC3T3-E1 subclone 4 cells with regard to their mineralization capacity and explore if oxidative stress or the cellular metabolism processes are implicated. Cells were treated with nine different combinations of long-lasting ascorbate (Asc) and β-glycerophosphate (βGP), and osteogenesis/calcification was evaluated at three different time-points by qPCR, Western blotting, and bone nodule staining. Key molecules of the oxidative and metabolic pathways were also assessed. It was found that sufficient mineral deposition was achieved only in the 150 μg.mL<sup>−1</sup>/2 mM Asc/βGP combination on day 21 in OSM, and this was supported by Runx2, Alpl, Bglap, and Col1a1 expression level increases. NOX2 and SOD2 as well as PGC1α and Tfam were also monitored as indicators of redox and metabolic processes, respectively, where no differences were observed. Elevation in OCN protein levels and ALP activity showed that mineralisation comes as a result of these differences. This work defines the most appropriate culture conditions for MC3T3-E1 cells and could be used by other research laboratories in this field.

Item Type: Article
Uncontrolled Keywords: mineralisation; oxidative stress; energy metabolism; Cell Line; Osteoblasts; Animals; Mice; Ascorbic Acid; Glycerophosphates; Culture Media; Cell Culture Techniques; Cell Differentiation; Calcification, Physiologic; Energy Metabolism; Oxidative Stress; Osteogenesis; MC3T3-E1 subclone 4; energy metabolism; mineralisation; oxidative stress; Animals; Mice; Oxidative Stress; Osteogenesis; Energy Metabolism; Osteoblasts; Cell Line; Glycerophosphates; Calcification, Physiologic; Cell Differentiation; Cell Culture Techniques; Ascorbic Acid; Culture Media; 31 Biological Sciences; 3106 Industrial Biotechnology; Animals; Mice; Oxidative Stress; Osteogenesis; Energy Metabolism; Osteoblasts; Cell Line; Glycerophosphates; Calcification, Physiologic; Cell Differentiation; Cell Culture Techniques; Ascorbic Acid; Culture Media; 0399 Other Chemical Sciences; 0604 Genetics; 0699 Other Biological Sciences; Chemical Physics; 3101 Biochemistry and cell biology; 3107 Microbiology; 3404 Medicinal and biomolecular chemistry
Subjects: R Medicine > RM Therapeutics. Pharmacology
R Medicine > RS Pharmacy and materia medica
Divisions: Pharmacy and Biomolecular Sciences
Publisher: MDPI AG
Date of acceptance: 8 April 2024
Date of first compliant Open Access: 9 October 2026
Date Deposited: 09 Oct 2026 15:02
Last Modified: 09 Oct 2026 15:02
DOI or ID number: 10.3390/ijms25084180
URI: https://researchonline.ljmu.ac.uk/id/eprint/29644
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