Bach Christian, Glasow Annegret, Baran-Schmidt Rainer, Oppermann Henry, Bach Christoph, Meixensberger Jürgen, Güresir Erdem, Gaunitz Frank
Department of Neurosurgery, University Hospital Leipzig, Leipzig, Germany.
Department of Radiotherapy and Radiooncology, University of Leipzig, Leipzig, Germany.
Front Bioeng Biotechnol. 2024 Dec 18;12:1471012. doi: 10.3389/fbioe.2024.1471012. eCollection 2024.
Identifying new substances that could potentially be used for tumor therapy and the precise analysis of their spectrum of action requires models that are as similar as possible to the tumor present in the patient. Traditionally, two-dimensional (2D) cell cultures are used. However, these only resemble solid tumors to a limited extent. More realistic models, such as tissue cultures, which are invaluable for the final analysis of the effect of new substances, are unsuitable for high-throughput screening (HTS), such as substance library screening. Therefore, we addressed which parameters need to be optimized to produce 3D cultures suitable for HTS using established tumor cell lines and ultra-low attachment plates, and we tested which experimental parameters need to be considered. In our studies, we have focused on cell lines from gliomas. Gliomas are incurable tumors of the central nervous system and are the subject of intensive research. Our studies used ten glioma cell lines from which we generated spheroids using ultra-low attachment plates. We then determined the spheroid size as a function of the initial cell number and the culture time. We analyzed cell viability using propidium iodide staining, evaluated the effects of temozolomide and radiation on spheroids, and compared the effect to that on 2D cultures. We found that spheroid size correlated linearly with the initial cell number. Fewer cells (250-500) generally resulted in better growth than a higher number. However, not all cell lines produced growing spheroids at all. The spheroids had an outer layer of living cells and an inner core of dead cells. The size of the inner core of dead cells was different in the various cell lines and developed differently during the incubation period. Radiation affected spheroids more than 2D cultures, especially at higher cell densities. Our results provide insight into using glioma cell lines to form spheroids as model systems. We have identified initial cell numbers as a critical parameter for their effective use in research, offering a hopeful outlook for tumor therapy research and drug development.
识别可能用于肿瘤治疗的新物质并精确分析其作用谱，需要尽可能与患者体内肿瘤相似的模型。传统上，使用二维（2D）细胞培养。然而，这些仅在有限程度上类似于实体瘤。更现实的模型，如组织培养，虽然对新物质效果的最终分析非常有价值，但不适用于高通量筛选（HTS），如物质库筛选。因此，我们探讨了使用已建立的肿瘤细胞系和超低附着板产生适合HTS的3D培养物需要优化哪些参数，并测试了需要考虑哪些实验参数。在我们的研究中，我们专注于胶质瘤细胞系。胶质瘤是中枢神经系统的不治之症肿瘤，是深入研究的对象。我们的研究使用了十种胶质瘤细胞系，我们使用超低附着板从中生成球体。然后，我们确定球体大小作为初始细胞数量和培养时间的函数。我们使用碘化丙啶染色分析细胞活力，评估替莫唑胺和辐射对球体的影响，并将其与对2D培养物的影响进行比较。我们发现球体大小与初始细胞数量呈线性相关。细胞数量较少（250 - 500个）通常比数量较多时生长得更好。然而，并非所有细胞系都能产生生长的球体。球体有一层活细胞外层和一层死细胞内核。不同细胞系中死细胞内核的大小不同，并且在孵育期内发育方式也不同。辐射对球体的影响比对2D培养物的影响更大，尤其是在较高细胞密度时。我们的结果为使用胶质瘤细胞系形成球体作为模型系统提供了见解。我们已经确定初始细胞数量是其在研究中有效使用的关键参数，为肿瘤治疗研究和药物开发提供了充满希望的前景。
