Temporal Variation in Single-Cell Power-Law Rheology Spans the Ensemble Variation of Cell Population.

Changes in the cytoskeletal organization within cells can be characterized by large spatial and temporal variations in rheological properties of the cell (e.g., the complex shear modulus G∗). Although the ensemble variation in G∗ of single cells has been elucidated, the detailed temporal variation of G∗ remains unknown. In this study, we investigated how the rheological properties of individual fibroblast cells change under a spatially confined environment in which the cell translational motion is highly restricted and the whole cell shape remains unchanged. The temporal evolution of single-cell rheology was probed at the same measurement location within the cell, using atomic force microscopy-based oscillatory deformation. The measurements reveal that the temporal variation in the power-law rheology of cells is quantitatively consistent with the ensemble variation, indicating that the cell system satisfies an ergodic hypothesis in which the temporal statistics are identical to the ensemble statistics. The autocorrelation of G∗ implies that the cell mechanical state evolves in the ensemble of possible states with a characteristic timescale.

Parylene mobile microplates integrated with an enzymatic release for handling of single adherent cells.

An approach for manipulating single adherent cells is developed that is integrated with an enzymatic batch release. This strategy uses an array of releasable microfabricated mobile substrates, termed microplates, formed from a biocompatible polymer, parylene. A parylene microplate array of 10-70 µm in diameter can be formed on an alginate hydrogel sacrificial layer by using a standard photolithographic process. The parylene surfaces are modified with fibronectin to enhance cell attachment, growth, and stretching. To load single cells onto these microplates, cells are initially placed in suspension at an optimized seeding density and are allowed to settle, stretch, and grow on individual microplates. The sacrificial layer underneath the microplate array can be dissolved on a time-scale of several seconds without cytotoxicity. This system allows the inspection of selected single adherent cells. The ability to assess single cells while maintaining their adhesive properties will broaden the examination of a variety of attributes, such as cell shape and cytoskeletal properties.

Metre-long cell-laden microfibres exhibit tissue morphologies and functions.

Artificial reconstruction of fibre-shaped cellular constructs could greatly contribute to tissue assembly in vitro. Here we show that, by using a microfluidic device with double-coaxial laminar flow, metre-long core-shell hydrogel microfibres encapsulating ECM proteins and differentiated cells or somatic stem cells can be fabricated, and that the microfibres reconstitute intrinsic morphologies and functions of living tissues. We also show that these functional fibres can be assembled, by weaving and reeling, into macroscopic cellular structures with various spatial patterns. Moreover, fibres encapsulating primary pancreatic islet cells and transplanted through a microcatheter into the subrenal capsular space of diabetic mice normalized blood glucose concentrations for about two weeks. These microfibres may find use as templates for the reconstruction of fibre-shaped functional tissues that mimic muscle fibres, blood vessels or nerve networks in vivo.

Mechanical properties of epithelial cells in domes investigated using atomic force microscopy.

As epithelial cells in vitro reach a highly confluent state, the cells often form a microscale dome-like architecture that encloses a fluid-filled lumen. The domes are stabilized by mechanical stress and luminal pressure. However, the mechanical properties of cells that form epithelial domes remain poorly characterized at the single-cell level. In this study, we used atomic force microscopy (AFM) to measure the mechanical properties of cells forming epithelial domes. AFM showed that the apparent Young's modulus of cells in domes was significantly higher when compared with that in the surrounding monolayer. AFM also showed that the stiffness and tension of cells in domes were positively correlated with the apical cell area, depending on the degree of cell stretching. This correlation disappeared when actin filaments were depolymerized or when the ATPase activity of myosin II was inhibited, which often led to a large fluctuation in dome formation. The results indicated that heterogeneous actomyosin structures organized by stretching single cells played a crucial role in stabilizing dome formation. Our findings provide new insights into the mechanical properties of three-dimensional deformable tissue explored using AFM at the single-cell level.

Circadian rhythms in Per1, PER2 and Ca2+ of a solitary SCN neuron cultured on a microisland.

Circadian rhythms in Per1, PER2 expression and intracellular Ca2+ were measured from a solitary SCN neuron or glial cell which was physically isolated from other cells. Dispersed cells were cultured on a platform of microisland (100-200 µm in diameter) in a culture dish. Significant circadian rhythms were detected in 57.1% for Per1 and 70.0% for PER2 expression. When two neurons were located on the same island, the circadian rhythms showed desynchronization, indicating a lack of oscillatory coupling. Circadian rhythms were also detected in intracellular Ca2+ of solitary SCN neurons. The ratio of circadian positive neurons was significantly larger without co-habitant of glial cells (84.4%) than with it (25.0%). A relatively large fraction of SCN neurons generates the intrinsic circadian oscillation without neural or humoral networks. In addition, glial cells seem to interrupt the expression of the circadian rhythmicity of intracellular Ca2+ under these conditions.

Origami-based self-folding of co-cultured NIH/3T3 and HepG2 cells into 3D microstructures.

This paper describes an origami-inspired self-folding method to form three-dimensional (3D) microstructures of co-cultured cells. After a confluent monolayer of fibroblasts (NIH/3T3 cells) with loaded hepatocytes (HepG2 cells) was cultured onto two-dimensional (2D) microplates, degradation of the alginate sacrificial layer in the system by addition of alginate lyase triggered NIH/3T3 cells to self-fold the microplates around HepG2 cells, and then 3D cell co-culture microstructures were spontaneously formed. Using this method, we can create a large number of 3D cell co-culture microstructures swiftly with ease in the same time. We find that HepG2 cells confined in the 3D cell co-culture microstructures have an ability to enhance the secreted albumin compared to 2D system in a long culture period. The result indicates that the origami-based cell self-folding technique presented here is useful in regenerative medicine and the preclinical stage of drug development.

Visualising the dynamics of live pancreatic microtumours self-organised through cell-in-cell invasion.

Pancreatic ductal adenocarcinoma (PDAC) reportedly progresses very rapidly through the initial carcinogenesis stages including DNA damage and disordered cell death. However, such oncogenic mechanisms are largely studied through observational diagnostic methods, partly because of a lack of live in vitro tumour imaging techniques. Here we demonstrate a simple live-tumour in vitro imaging technique using micro-patterned plates (micro/nanoplates) that allows dynamic visualisation of PDAC microtumours. When PDAC cells were cultured on a micro/nanoplate overnight, the cells self-organised into non-spheroidal microtumours that were anchored to the micro/nanoplate through cell-in-cell invasion. This self-organisation was only efficiently induced in small-diameter rough microislands. Using a time-lapse imaging system, we found that PDAC microtumours actively stretched to catch dead cell debris via filo/lamellipoedia and suction, suggesting that they have a sophisticated survival strategy (analogous to that of starving animals), which implies a context for the development of possible therapies for PDACs. The simple tumour imaging system visualises a potential of PDAC cells, in which the aggressive tumour dynamics reminds us of the need to review traditional PDAC pathogenesis.

Mobile Microplates for Morphological Control and Assembly of Individual Neural Cells.

A microfabricated device that enables morphological control and assembly of cultured single neural cells is described. Assembly of morphologically controlled single neural cells allows neuroengineers to design in vitro neural circuits with a single-cell resolution. Compared to conventional cell-patterning techniques, the device allows for the highly precise positioning of neural somas and neurites in a reproducible fashion.

Magnetically responsive microflaps reveal cell membrane boundaries from multiple angles.

A microflap system to incline adherent cells in the desired orientation is described. Inclination angles of cell-laden microflaps are precisely controlled by the applied magnetic field, enabling us to observe cell-membrane boundaries from multiple angles. This system is equipped with conventional microscopes, allowing clear focused images of cell-membrane boundaries to be obtained with high magnification.

Cell origami: self-folding of three-dimensional cell-laden microstructures driven by cell traction force.

This paper describes a method of generating three-dimensional (3D) cell-laden microstructures by applying the principle of origami folding technique and cell traction force (CTF). We harness the CTF as a biological driving force to fold the microstructures. Cells stretch and adhere across multiple microplates. Upon detaching the microplates from a substrate, CTF causes the plates to lift and fold according to a prescribed pattern. This self-folding technique using cells is highly biocompatible and does not involve special material requirements for the microplates and hinges to induce folding. We successfully produced various 3D cell-laden microstructures by just changing the geometry of the patterned 2D plates. We also achieved mass-production of the 3D cell-laden microstructures without causing damage to the cells. We believe that our methods will be useful for biotechnology applications that require analysis of cells in 3D configurations and for self-assembly of cell-based micro-medical devices.