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      Oxidative degradation of polyamines by serum supplement causes cytotoxicity on cultured cells

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          Abstract

          Serum is a common supplement for cell culture due to it containing the essential active components for the growth and maintenance of cells. However, the knowledges of the active components in serum are incomplete. Apart from the direct influence of serum components on cultured cells, the reaction of serum components with tested drugs cannot be ignored, which usually results in the false conclusion on the activity of the tested drugs. Here we report the toxicity effect of polyamines (spermidine and spermine) on cultured cells, especially on drug-resistant cancer cell lines, which resulted from the oxidative degradation of polyamines by amine oxidases in serum supplement. Upon adding spermidine or spermine, high concentration of H 2O 2, an enzyme oxidation product of polyamines, was generated in culture media containing ruminant serum, such as fetal bovine serum (FBS), calf serum, bovine serum, goat serum or horse serum, but not in the media containing human serum. Drug-resistant cancer cell lines showed much higher sensitivity to the oxidation products of polyamines (H 2O 2 and acrolein) than their wild cell lines, which was due to their low antioxidative capacity.

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          Optimization of chemically defined cell culture media--replacing fetal bovine serum in mammalian in vitro methods.

          Quality assurance is becoming increasingly important. Good laboratory practice (GLP) and good manufacturing practice (GMP) are now established standards. The biomedical field aims at an increasing reliance on the use of in vitro methods. Cell and tissue culture methods are generally fast, cheap, reproducible and reduce the use of experimental animals. Good cell culture practice (GCCP) is an attempt to develop a common standard for in vitro methods. The implementation of the use of chemically defined media is part of the GCCP. This will decrease the dependence on animal serum, a supplement with an undefined and variable composition. Defined media supplements are commercially available for some cell types. However, information on the formulation by the companies is often limited and such supplements can therefore not be regarded as completely defined. The development of defined media is difficult and often takes place in isolation. A workshop was organised in 2009 in Copenhagen to discuss strategies to improve the development and use of serum-free defined media. In this report, the results from the meeting are discussed and the formulation of a basic serum-free medium is suggested. Furthermore, recommendations are provided to improve information exchange on newly developed serum-free media. Copyright 2010 Elsevier Ltd. All rights reserved.
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            The natural polyamine spermine functions directly as a free radical scavenger.

            The polyamines are small organic cations that are absolutely required for eukaryotic cell growth. Although their growth requirements are well established, the molecular functions of the polyamines are ill-defined. Oxidative damage to DNA by reactive oxygen species is a continual problem that cells must guard against to survive. The polyamine spermine, which is normally found in millimolar concentrations in the nucleus, is shown here to function directly as a free radical scavenger, and adducts formed as a result of this function are identified. These data suggest that spermine is a major natural intracellular compound capable of protecting DNA from free radical attack.
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              Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells.

              Culture media for therapeutic cell preparations-such as mesenchymal stromal cells (MSCs)-usually comprise serum additives. Traditionally, fetal bovine serum is supplemented in basic research and in most clinical trials. Within the past years, many laboratories adapted their culture conditions to human platelet lysate (hPL), which further stimulates proliferation and expansion of MSCs. Particularly with regard to clinical application, human alternatives for fetal bovine serum are clearly to be preferred. hPL is generated from human platelet units by disruption of the platelet membrane, which is commonly performed by repeated freeze and thaw cycles. Such culture supplements are notoriously ill-defined, and many parameters contribute to batch-to-batch variation in hPL such as different amounts of plasma, a broad range of growth factors and donor-specific effects. The plasma components of hPL necessitate addition of anticoagulants such as heparins to prevent gelatinization of hPL medium, and their concentration must be standardized. Labels for description of hPL-such as "xenogen-free," "animal-free" and "serum free"-are not used consistently in the literature and may be misleading if not critically assessed. Further analysis of the precise composition of relevant growth factors, attachment factors, microRNAs and exosomes will pave the way for optimized and defined culture conditions. The use of hPL has several advantages and disadvantages: they must be taken into account because the choice of cell culture additive has major impact on cell preparations.
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                Author and article information

                Contributors
                hszhang@iccas.ac.cn
                sgdh@iccas.ac.cn
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                10 July 2018
                10 July 2018
                2018
                : 8
                : 10384
                Affiliations
                [1 ]ISNI 0000 0004 0596 3295, GRID grid.418929.f, Department Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, CAS Research/Education Center for Excellence in Molecular Sciences, , Institute of Chemistry, Chinese Academy of Sciences, ; Beijing, 100190 China
                [2 ]ISNI 0000 0004 1797 8419, GRID grid.410726.6, University of the Chinese Academy of Sciences, ; Beijing, 100049 China
                [3 ]ISNI 0000 0004 1806 6075, GRID grid.419265.d, CAS Center for Excellence in Nanoscience, , National Center for Nanoscience and Technology, ; Beijing, 100190 China
                Author information
                http://orcid.org/0000-0002-5121-053X
                http://orcid.org/0000-0002-5746-803X
                Article
                28648
                10.1038/s41598-018-28648-8
                6039494
                176edcc8-26dc-45ec-8e7e-de20c9e6a5f6
                © The Author(s) 2018

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 16 February 2018
                : 10 May 2018
                Funding
                Funded by: We gratefully acknowledge the financial support from NSF of China (21575147, 21535009, 21635008, 21705153 and 21621062).
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