Luo C J, Wightman Raymond, Meyerowitz Elliot, Smoukov Stoyan K
Department of Materials Science and Metallurgy, University of Cambridge, 27 Charles Babbage Road, Cambridge, CB3 0FS, UK.
Sainsbury Laboratory, University of Cambridge, Bateman Street, Cambridge, CB2 1LR, UK.
BMC Plant Biol. 2015 Aug 26;15:211. doi: 10.1186/s12870-015-0581-7.
Cell culture methods allow the detailed observations of individual plant cells and their internal processes. Whereas cultured cells are more amenable to microscopy, they have had limited use when studying the complex interactions between cell populations and responses to external signals associated with tissue and whole plant development. Such interactions result in the diverse range of cell shapes observed in planta compared to the simple polygonal or ovoid shapes in vitro. Microfluidic devices can isolate the dynamics of single plant cells but have restricted use for providing a tissue-like and fibrous extracellular environment for cells to interact. A gap exists, therefore, in the understanding of spatiotemporal interactions of single plant cells interacting with their three-dimensional (3D) environment. A model system is needed to bridge this gap. For this purpose we have borrowed a tool, a 3D nano- and microfibre tissue scaffold, recently used in biomedical engineering of animal and human tissue physiology and pathophysiology in vitro.
We have developed a method of 3D cell culture for plants, which mimics the plant tissue environment, using biocompatible scaffolds similar to those used in mammalian tissue engineering. The scaffolds provide both developmental cues and structural stability to isolated callus-derived cells grown in liquid culture. The protocol is rapid, compared to the growth and preparation of whole plants for microscopy, and provides detailed subcellular information on cells interacting with their local environment. We observe cell shapes never observed for individual cultured cells. Rather than exhibiting only spheroid or ellipsoidal shapes, the cells adapt their shape to fit the local space and are capable of growing past each other, taking on growth and morphological characteristics with greater complexity than observed even in whole plants. Confocal imaging of transgenic Arabidopsis thaliana lines containing fluorescent microtubule and actin reporters enables further study of the effects of interactions and complex morphologies upon cytoskeletal organisation both in 3D and in time (4D).
The 3D culture within the fibre scaffolds permits cells to grow freely within a matrix containing both large and small spaces, a technique that is expected to add to current lithographic technologies, where growth is carefully controlled and constricted. The cells, once seeded in the scaffolds, can adopt a variety of morphologies, demonstrating that they do not need to be part of a tightly packed tissue to form complex shapes. This points to a role of the immediate nano- and micro-topography in plant cell morphogenesis. This work defines a new suite of techniques for exploring cell-environment interactions.
细胞培养方法能够对单个植物细胞及其内部过程进行详细观察。虽然培养的细胞更便于显微镜观察，但在研究细胞群体之间的复杂相互作用以及对与组织和整个植物发育相关的外部信号的反应时，其应用有限。与体外简单的多边形或卵形相比，这些相互作用导致了在植物体内观察到的细胞形状多种多样。微流控装置可以分离单个植物细胞的动态，但在为细胞相互作用提供类似组织和纤维状的细胞外环境方面应用受限。因此，在理解单个植物细胞与其三维（3D）环境的时空相互作用方面存在差距。需要一个模型系统来弥合这一差距。为此，我们借鉴了一种工具，即一种3D纳米和微纤维组织支架，该支架最近用于动物和人体组织生理学及病理生理学的生物医学工程体外研究。
我们开发了一种用于植物的3D细胞培养方法，该方法使用与哺乳动物组织工程中使用的类似的生物相容性支架来模拟植物组织环境。这些支架为在液体培养中生长的分离的愈伤组织来源的细胞提供发育线索和结构稳定性。与为显微镜观察而生长和制备整个植物相比，该方案速度更快，并提供了关于细胞与其局部环境相互作用的详细亚细胞信息。我们观察到了单个培养细胞从未观察到的细胞形状。细胞不是仅呈现球形或椭圆形，而是使其形状适应局部空间，并且能够相互生长，呈现出比甚至在整个植物中观察到的更为复杂的生长和形态特征。对含有荧光微管和肌动蛋白报告基因的转基因拟南芥品系进行共聚焦成像，能够进一步研究相互作用和复杂形态对3D和实时（4D）细胞骨架组织的影响。
纤维支架内的3D培养允许细胞在包含大小空间的基质中自由生长，这项技术有望补充当前光刻技术，在光刻技术中生长受到仔细控制和限制。一旦接种到支架中的细胞可以呈现多种形态，表明它们不需要成为紧密堆积组织的一部分就能形成复杂形状。这表明了即时纳米和微观形貌在植物细胞形态发生中的作用。这项工作定义了一套用于探索细胞 - 环境相互作用的新技术。
