Kaori Kuribayashi-Shigetomi

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.

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.

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.

High-throughput Measurements of Single Cell Rheology by Atomic Force Microscopy

The compliant mechanical properties of single cells have been extensively investigated and these properties are known to exhibit a strong dependence on the surrounding environments and also cell types, functions and conditions. An understanding of the cell behavior is important for applications of tissue engineering. Accurate rheological measurements are essential to elucidate the mechanisms of cell integrity and fluidity and are also key to mechanically identifying and separating single cells for cellular and tissue engineering. Of the various existing nano- and micro-rheology techniques, atomic force microscopy (AFM) shows great potential as a minimally invasive method. AFM allows mechanical measurements to be performed without the need for chemical modifications, via nano-scale contact between the AFM probe and the cell surface. In this chapter, we describe a recent advance in which micro-fabricated substrates are used for high-speed, automated AFM rheological measurements on size- and position-controlled cells.

Temporal change in complex shear modulus of cells: An atomic force microscopy study

To sort living cells according to our needs, it is important to understand how degree cell property measured for cell sorting fluctuates in time. Mechanical property of cells is one of essential indicators for cell sorting. Thus, in this study, we attempted to measure a time evolution of viscoelastic property such as complex shear modulus, G* of single cells adhered on substrates using atomic force microscopy (AFM). We observed that the G* largely fluctuated in time even the cells are placed on substrates in a confined condition. This indicates that in mechanical cell sorting, mechanical fluctuations of cells should be carefully estimated so that cells are precisely separated by taking the measured data involving cell fluctuations into account.

Quantitative rheological measurements of confluent cell using atomic force microscopy

Rheological properties of cells are associated with various cell functions and thus are considered to be an indicator for diagnosing cell disease. Atomic force microscopy (AFM) is a powerful tool for quantifying the mechanical properties of isolated single cells. For example, our AFM technique reported previously revealed that the complex shear modulus of single cells exhibited a large cell-to-cell variation which depended on frequency. By contrast, rheological properties of cell population such as cells in confluent condition have been less understood. Thus, it is valuable to investigate how quantitatively the AFM technique can be applied to cell population such as cells in confluent condition. As a result, rheological properties of cells in confluent conditions can be relatively rapidly measured by our AFM setup modifying a force modulation AFM mode. This suggests that the AFM technique is useful for diagnosing not only single cells but also cell population and cell assembly.

Atomic force microscopy for mapping mechanical property of the whole cell assembly

Cells rapidly undergo cell division during the embryogenesis. Such dynamic behaviors of cells during the embryogenesis are considered to be strongly associated with their mechanical properties such as cell-cell mechanical interactions and cell stiffness. However, the interplay between the morphogenesis and the mechanical property of whole cell assembly during the developmental process has not been well understood. To exploring the mechanism of forming the whole cell assembly, we proposed atomic force microscopy (AFM) combined with a microarray technique, which allows us to map mechanical property of the whole cell assembly. In this AFM setup, the cell assembly is randomly directed in the microarray well, and thus the average mechanical property of the whole cell assembly can be reconstructed from mapping images obtained from different cell assemblies.

High-throughput measurements of cell mechanics using atomic force microscopy with micro-patterned substrates

Cell mechanics is strongly related to various cell functions such as migration and proliferation. Since the mechanical properties of cells exhibited a large cell-to-cell variation, statistical evaluation of cell mechanics is crucial for understanding cell behavior. In this study, we investigated the deviation of cell complex shear modulus storage modulus, which was measured by atomic force microscopy (AFM) with micro-patterned substrates. The result showed that the cell number more than 50 was required to obtain the converged deviation in the case of NIH3T3 cells cultured in isolated substrates.