Abstract
BackgroundCell 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.ResultsWe 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).ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0581-7) contains supplementary material, which is available to authorized users.
Highlights
Cell culture methods allow the detailed observations of individual plant cells and their internal processes
Cultures are used to seed pre-wetted scaffolds consisting of poly(ethylene terephthalate) (PET) : PLA in a ratio of 70 % : 30 %
The scaffolds are organised as a layered-meshwork of the PET microfibres incorporating the finer PLA nanofibres (Fig. 1a-b)
Summary
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. Single, isolated cells permit easier access to the subcellular dynamics, especially for cell types that are poorly accessible or difficult to orient for imaging It is, difficult to isolate processes on the single cell-scale whilst concurrently maintaining the tissue-scale response to external signals from a 3D environment. Difficult to isolate processes on the single cell-scale whilst concurrently maintaining the tissue-scale response to external signals from a 3D environment This makes a new model system based on cultured cells interacting within a tissuelike scaffold a desirable biological tool
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