He Lijuan, Sneider Alexandra, Chen Weitong, Karl Michelle, Prasath Vishnu, Wu Pei-Hsun, Mattson Gunnar, Wirtz Denis
Department of Chemical and Biomolecular Engineering, Johns Hopkins University; Johns Hopkins Physical Sciences - Oncology Center, Johns Hopkins University.
Department of Chemical and Biomolecular Engineering, Johns Hopkins University.
J Vis Exp. 2017 Nov 29(129):56364. doi: 10.3791/56364.
The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.
尽管三维环境中哺乳动物细胞分裂的调控研究具有生理相关性和治疗意义，但其仍在很大程度上未被探索。缺乏探索的可能原因是实验限制和技术挑战，这些使得三维培养中的细胞分裂研究效率低下。在此，我们描述了一种基于成像的方法，用于在三维胶原基质中高效研究哺乳动物细胞分裂和细胞与基质的相互作用。用荧光H2B标记的细胞通过胸腺嘧啶阻断和诺考达唑处理相结合的方法进行同步化，随后采用机械摇脱技术。然后将同步化的细胞嵌入三维胶原基质中。使用活细胞显微镜监测细胞分裂。细胞分裂期间和之后胶原纤维的变形，这是细胞与基质相互作用的一个指标，可以使用定量共聚焦反射显微镜进行监测和量化。该方法为在生理相关的三维环境中研究哺乳动物细胞分裂和细胞与基质的相互作用提供了一种高效且通用的方法。这种方法不仅为正常组织和疾病发展的分子基础提供了新的见解，还允许设计新的诊断和治疗方法。
