Pedraza Claudio E, Chien Yung-Ching, McKee Marc D
Faculty of Dentistry, McGill University, Montreal, Quebec, Canada H3A 2B2.
J Cell Biochem. 2008 Apr 1;103(5):1379-93. doi: 10.1002/jcb.21515.
Cell culture methods and models are key investigative tools for cell and molecular biology studies. Fetal bovine serum (FBS) is commonly used as an additive during cell culture since its constituents promote cell survival, proliferation and differentiation. Here we report that commercially available FBS from different major suppliers consistently contain precipitated, calcium oxalate crystals-either in the monohydrate (COM) or dihydrate (COD) form. Mineral structure and phase identification of the crystals were determined by X-ray diffraction, chemical composition by energy-dispersive X-ray microanalysis, and imaging and measurement of crystal growth steps by atomic force microscopy-all identified and confirmed crystallographic parameters for COM and COD. Proteins binding to the crystals were identified by immunoblotting, revealing the presence of osteopontin and fetuin-A (alpha(2)HS-glycoprotein)--known regulators of crystal growth found in serum. Macrophage cell cultures exposed to calcium oxalate crystals showed internalization of the crystals by phagocytosis in a process that induced disruption of cell-cell adhesion, release of reactive oxygen species and membrane damage, events that may be linked to the release of inflammatory cytokines by these cells into the culture media. In conclusion, calcium oxalate crystals found in commercially available FBS are toxic to cells, and their presence may confound results from in vitro studies where, amongst others, phagocytosis, biomineralization, renal cell and molecular biology, and drug and biomaterial testing are being examined.
细胞培养方法和模型是细胞与分子生物学研究的关键研究工具。胎牛血清（FBS）在细胞培养过程中常用作添加剂，因为其成分可促进细胞存活、增殖和分化。在此我们报告，来自不同主要供应商的市售FBS始终含有沉淀的草酸钙晶体——一水合物（COM）或二水合物（COD）形式。通过X射线衍射确定晶体的矿物结构和相鉴定，通过能量色散X射线微分析确定化学成分，并通过原子力显微镜对晶体生长步骤进行成像和测量——所有这些都确定并确认了COM和COD的晶体学参数。通过免疫印迹鉴定与晶体结合的蛋白质，揭示了骨桥蛋白和胎球蛋白-A（α2HS-糖蛋白）的存在——血清中已知的晶体生长调节剂。暴露于草酸钙晶体的巨噬细胞培养物显示，晶体通过吞噬作用被内化，这一过程会导致细胞间粘附破坏、活性氧释放和膜损伤，这些事件可能与这些细胞向培养基中释放炎性细胞因子有关。总之，市售FBS中发现的草酸钙晶体对细胞有毒，其存在可能会混淆体外研究的结果，其中包括吞噬作用研究、生物矿化研究、肾细胞与分子生物学研究以及药物和生物材料测试研究。
