Three-dimensional (3D) cell and tissue cultures more closely mimic biological environments than two-dimensional (2D) cultures and are therefore highly desirable in culture experiments. However, 3D cultures often fail to yield repeatable experimental results because of variation in the initial culture conditions, such as cell density and distribution in the extracellular matrix, and therefore reducing such variation is a paramount concern. Here, we present a 3D culture platform that demonstrates highly repeatable experimental results, obtained by controlling the initial cell cluster shape in the gel cube culture device. A micro-mould with the desired shape was fabricated by photolithography or machining, creating a 3D pocket in the extracellular matrix contained in the device. Highly concentrated human bronchial epithelial cells were then injected in the pocket so that the cell cluster shape matched the fabricated mould shape. Subsequently, the cubic device supplied multi-directional scanning, enabling high-resolution capture of the whole tissue structure with only a low-magnification lens. The proposed device significantly improved the repeatability of the developed branch pattern, and multi-directional scanning enabled quantitative analysis of the developed branch pattern formations. A mathematical simulation was also conducted to reveal the mechanisms of branch pattern formation. The proposed platform offers the potential to accelerate any research field that conducts 3D culture experiments, including tissue regeneration and drug development.

An in vitro 3D culturing platform enabling multidirectional observations of 3D biosamples is presented. The 3D structure of biosamples can be recognized without fluorescence. The cubic platform employs two types of hydrogels that are compatible with conventional culture dishes or well plates, facilitating growth in culture, ease of handling, and viewing at multiple angles.

Abstract:In vitro three-dimensional (3D) culturing is considered essential in many biological fields. However, the imaging of developed 3D formations is often difficult, especially if the size of the sample is relatively large. The z-resolution of fluorescent imaging is low using low magnification lenses (4× and 10×) due to large focal depths. This paper describes 3D culture platform enabling large scale 3D imaging by fine spatial alignment of the image dataset obtained from multiple directions. A gel cube device was employed to conduct the multi-scanning and then a self-fluorescent microstructure in a cubic frame allows us spatially align image dataset within a few pixels. By synthesizing data from multiple scans, the platform enables us to visualize millimeter-sized 3D sample structure and individual cellular actin filaments at the same time. Millimeter depth imaging of a developed bronchial tree was achieved with high z-resolution. The device, which is applicable to most microscopy systems, can enhance the image quality without modifying current systems.

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An in vitro 3D culturing platform enabling multidirectional observations of 3D biosamples is presented by M. Hagiwara and co-workers on page 1566. 3D recognition of a sample structure can be achieved by facilitating multi-directional views using a standard microscope without a laser system. The cubic platform has the potential to promote 3D culture studies, offering easy handling and compatibility with commercial culture plates at a low price tag.