In order to find out cancer markers in human breath, in vitro cell culture is often used to study the characteristic volatile organic compounds (VOCs). In the cell culture process, disposable vessels are frequently adopted. However, these vessels are normally made of plastic, and they have the possibility to release some VOCs, which may interfere with the cell-specific volatiles and even can result in an incorrect conclusion. In this study, by using glass cell culture flasks as control, the headspace solid-phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS) analyses of the VOCs in plastic cell culture flasks were systematically carried out for the first time. A total of 35 VOCs were detected in five brands of flasks. In each flask, there were between 13 and 25 volatile compounds. Furthermore, the components and packaging bag of each flask were also sampled and analyzed by HS-SPME-GC-MS. The results show that the flask cap, septum, flask body, and packaging bag exhibit respectively different volatile behaviors. The former two parts release the most volatiles which have obvious contributions to the headspace gases in the flasks, while the flask body mainly liberates styrene. For different flasks packed within the same bag, the headspace analyses show that their residual VOCs are inconsistent with each other. Moreover, the residual VOCs in the same flask are variable in three consecutive days. These results indicate that the multiple flasks in parallel cell culture experiments, or the same flask with different cell culture durations, will produce an indelible disturbance to the cell-specific VOCs. In addition, among the 35 VOCs detectable in five brands of empty plastic flasks, 15 VOCs were previously reported as characteristic VOCs from lung cancer, melanoma, cervical cancer cells, or normal cells. This is an alert that, when using plastic flasks, it must be careful to treat the possible interference from the background VOCs in the flasks. This study demonstrates that the cell culture tool needs to be standardized, and the clean glass or metal vessels are strongly recommended for usage when studying cell volatile biomarkers.

Graphical abstract

为了找出人类呼吸中的癌症标志物，经常使用体外细胞培养来研究特征性挥发性有机化合物（VOC）。在细胞培养过程中，经常使用一次性容器。但是，这些容器通常由塑料制成，并且有可能释放一些VOC，这可能会干扰特定于细胞的挥发物，甚至可能导致错误的结论。在这项研究中，以玻璃细胞培养瓶为对照，首次系统地进行了顶空固相微萃取气相色谱质谱（HS-SPME-GC-MS）分析塑料细胞培养瓶中的VOC。在五个品牌的烧瓶中共检测到35种VOC。在每个烧瓶中，有13至25种挥发性化合物。此外，还对每个烧瓶的成分和包装袋取样并通过HS-SPME-GC-MS分析。结果表明，瓶盖，隔垫，瓶体和包装袋分别表现出不同的挥发性。前两个部分释放的挥发性最大，对烧瓶中的顶空气体有明显的贡献，而烧瓶主体主要释放出苯乙烯。对于装在同一袋中的不同烧瓶，顶空分析表明它们的残留VOC彼此不一致。此外，同一烧瓶中的残留VOC在连续三天内变化。这些结果表明，平行细胞培养实验中的多个烧瓶，或具有不同细胞培养持续时间的同一烧瓶，将对细胞特异性VOC产生不可磨灭的干扰。此外，在五个品牌的空塑料瓶中可检测到的35种VOC中，先前有15种VOC被报道为来自肺癌，黑素瘤，宫颈癌细胞或正常细胞的特征性VOC。这是一个警告，当使用塑料瓶时，必须小心处理瓶中背景VOC可能产生的干扰。这项研究表明，细胞培养工具需要标准化，在研究细胞挥发性生物标记物时，强烈建议使用干净的玻璃或金属容器。

图形概要