initial-3d-cell-cluster-control-hybrid-gel-cube-device-for-repeatable

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its ...

Osaka Prefecture University

A multicellular organization is a complex resulting from the coordinated migration of cells to form a specific pattern. The directionality of migration is governed by the mechanical and molecular dynamics of factors secreted from the cells. The mechanism underlying pattern formation is too complex to unveil by culture experiments alone. A mathematical model could provide a powerful tool for elucidating the mechanism of pattern formation by computing the molecular dynamics, which are difficult to visualize by culture experiments. However, there tends to be a gap between mathematical models and experimental research due to incongruity between the idealized conditions of the model and the experimental results. This paper presents an in vitro-in silico interface platform for elucidating the logic of multicellular pattern formation. Two-dimensional collective cell pattern formation was developed using normal human bronchial epithelial cells. Then, geometrical control of collective cells followed by feedback iteration was used to bridge the gap between the mathematical model and in vitro experiments. The mechanisms underlying the pattern formation of bronchial epithelial cells were evaluated using a reaction-diffusion model. The results indicated that differences in the diffusion rates of the activator and inhibitor determine the direction of collective cell migration to form a specific pattern.

An in vitro-in silico interface platform for spatiotemporal analysis of pattern formation in collective epithelial cells.

Gefitinib is a tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR) and has been approved for the treatment of non-small cell lung cancers (NSCLCs) with EGFR mutations. Here we demonstrated that gefitinib induced a significantly enhanced biological activity of succinate-tetrazolium reductase (STR) in mitochondria and mitochondrial membrane potential in HCC827 cells (EGFR mutation NSCLCs, sensitive to gefitinib) at a high cell density.

Gefitinib Enhances Mitochondrial Biological Functions in NSCLCs with  Mutations at a High Cell Density.

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.

High repeatability from 3D experimental platform for quantitative analysis of cellular branch pattern formations.

Lung branching morphogenesis is a complex system involving many molecular interactions to filling the three dimensional spaces; however, the underlying developmental mechanisms are still not fully understood. In this paper, we have investigated the effect of epidermal growth factor (EGF) on normal human bronchial epithelial cells and their three-dimensional (3D) branching pattern formation by using in vitro experiments and mathematical simulation. The results show that EGF is essential for 3D branch pattern formation and its receptor is highly expressed at the tip of branches to generate the drive force for cells to migrate. Macropinocytosis induced by EGFR expression is firmly contributed to the nutrition uptake at the tip of branches. Our findings for effective branching formation of human lung cells contribute to further understanding molecular mechanisms of organogenesis, and the important mechanisms also possibly participate in related lung disease such as malformation.

Article	Year
An in vitro-in silico interface platform for spatiotemporal analysis of pattern formation in collective epithelial cells. Integrative biology : quantitative biosciences from nano to macro\| PubMed ID: 27452205	2016
Gefitinib Enhances Mitochondrial Biological Functions in NSCLCs with Mutations at a High Cell Density. Anticancer research\| PubMed ID: 28870896	2017
High repeatability from 3D experimental platform for quantitative analysis of cellular branch pattern formations. Integrative biology : quantitative biosciences from nano to macro\| PubMed ID: 29687138	2018
Epidermal growth factor induced macropinocytosis directs branch formation of lung epithelial cells. Biochemical and biophysical research communications\| PubMed ID: 30442367	2018

Masaya Hagiwara

Biography

JoVE Journal Publications

Other Publications