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Concentration-dependent cytotoxicity of copper ions on mouse fibroblasts in vitro: effects of copper ion release from TCu380A vs TCu220C intra-uterine devices

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Abstract

Sustained release of copper (Cu) ions from Cu-containing intrauterine devices (CuIUD) is quite efficient for contraception. However, the tissue surrounding the CuIUD is exposed to toxic Cu ion levels. The objective for this study was to quantify the concentration dependent cytotoxic effects of Cu ions and correlate the toxicity due to Cu ion burst release for two popular T-shaped IUDs - TCu380A and TCu220C on L929 mouse fibroblasts. Fibroblasts were cultured in 98 well tissue culture plates and 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphehyltetrazolium bromide (MTT) assay was used to determine their viability and proliferation as a function of time. For cell seeding numbers ranging from 10,000 to 100,000, a maximum culture time of 48 h was identified for fibroblasts without significant reduction in cell proliferation due to contact inhibition. Thus, for Cu cytotoxicity assays, a cell seeding density of 50,000 and a maximum culture time of 48 h in 96 well plates were used. 24 h after cell seeding, culture media were replaced with Cu ion containing media solutions of different concentrations, including 24 and 72 h extracts from TCuIUDs and incubated for a further 24 h. Cell viability decreased with increasing Cu ion concentration, with 30 % and 100 % reduction for 40 μg/ml and 100 μg/ml respectively at 24 h. The cytotoxic effects were further evaluated using light microscopy, apoptosis and cell cycle analysis assays. Fibroblasts became rounded and eventually detached from TCP surface due to Cu ion toxicity. A linear increase in apoptotic cell population with increasing Cu ion concentration was observed in the tested range of 0 to 50 μg/ml. Cell cycle analysis indicated the arrest of cell division for the tested 25 to 50 μg/ml Cu ion treatments. Among the TCuIUDs, TCu220C having 265 mm2 Cu surface area released 9.08 ± 0.16 and 26.02 ± 0.25 μg/ml, while TCu380A having 400 mm2 released 96.7 ± 0.11 and 159.3 ± 0.15 μg/ml respectively following 24 and 72 h extractions. The effects of TCuIUD extracts on viability, morphology, apoptosis and cell cycle assay on L929 mouse fibroblasts cells, were appropriate for their respective Cu ion concentrations. Thus, a concentration of about 46 μg/ml (~29 μM) was identified as the LD50 dose for L929 mouse fibroblasts when exposed for 24 h based on our MTT cell viability assay. The burst release of lethal concentration of Cu ions from TCu380A, especially at the implant site, is a cause of concern, and it is advisable to use TCuIUD designs that release Cu ions within cytotoxic limits yet therapeutic, similar to TCu220C.

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Acknowledgements

The authors acknowledge the financial support from National Science and Technology Support Project of China (Grant No.2006BAI15B08), National Natural Science Foundation of China Project (Grant No. 51073024) and the Royal Society-NSFC international joint project grant (No. 5111130207).

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Authors and Affiliations

  1. School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing, 100083, People’s Republic of China

    Bianmei Cao, Yudong Zheng, Tingfei Xi, Huai Yang & Yanxuan Ma

  2. National Institute for the Control of Pharmaceutical & Biological Products, Beijing, 100050, People’s Republic of China

    Tingfei Xi & Chuanchuan Zhang

  3. Wolfson Center for Materials Processing, School of Engineering and Design, Brunel University, West London, UB8 3PH, UK

    Wenhui Song

  4. Brunel Institute for Bioengineering, Brunel University, Uxbridge, Middlesex, UB8 3PH, UK

    Krishna Burugapalli

  5. Beijing Institute of Medical Device Testing, Beijing, 100120, People’s Republic of China

    Bianmei Cao

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  1. Bianmei Cao
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Correspondence to Yudong Zheng or Tingfei Xi.

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Cao, B., Zheng, Y., Xi, T. et al. Concentration-dependent cytotoxicity of copper ions on mouse fibroblasts in vitro: effects of copper ion release from TCu380A vs TCu220C intra-uterine devices. Biomed Microdevices 14, 709–720 (2012). https://doi.org/10.1007/s10544-012-9651-x

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