Riken has made progress in the topologies of femtosecond laser maskless photolithography and the infiltration mechanism of cell spheres

With the development of tissue engineering, the interaction between biomaterial interface and cell and its physical mechanism have become a research hotspot. The topological morphology of biological interface can effectively regulate cell behavior and influence cell function. However, some physiological processes in vivo, such as embryonic development, immune response and tissue renewal and remodeling, often involve multi-cellular collective behavior. The invasion and metastasis of tumor are also related to the coordinated movement of collective cells. As an in vitro three-dimensional cell culture model with strong cell-cell interactions, the cell sphere can better simulate the in vivo environment in terms of cell physiology, signaling pathways, gene and protein expression, and gas/nutrient gradients. Therefore, clarifying the interaction between the topological structure of the material surface and the cell sphere is of great significance for exploring the physiological and pathological mechanisms in vivo. However, at present, it is difficult to quickly prepare the multi-scale micro/nano topologies with both cm scale and micro/nano precision.

Recently, Meiling Zheng, a researcher at the Laboratory of Organic Nanophotonics, Biomimetic Intelligent Interface Science Center, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, and her team have made new progress in the preparation of micro-nano topologies across scales and the regulation of cell sphere infiltration. The team proposes to use femtosecond laser maskless projection lithography (MOPL) to prepare large area microdisk array topologies with high accuracy to study the infiltration of cell spheres. It was found that cell spheres exhibited different infiltration rates on a variety of microdisk array topologies with different cell diameters. By analyzing cell morphology, skeleton distribution and cell adhesion, we analyzed the mechanism of infiltration rate of cell spheres, and found that cell spheres took different infiltration patterns on large and small size microdisk structural units. This study reveals the response mechanism of cell spheres to the micro-nano topologies at different scales and provides a reference for exploring tissue infiltration behavior.

MOPL is an efficient and flexible technology for preparing micro-nano topologies. Taking into account the size of individual cells and their interaction with large area topologies during cell sphere infiltration, the MOPL technique was used to prepare large area (8 mm × 10 mm) microdisk array structures with height less than 1μm and topological unit diameter of 2, 5, 20, and 50 μm, respectively (FIG. 1).

In this study, the uniform size of human kidney clear cell carcinoma cells were prepared by ultra-low adsorption method. Further, the researchers used laser scanning confocal fluorescence microscopy to observe the dynamic infiltration behavior of cell spheres on the microdisk array topology. The cell spheres were completely infiltrated in a series of microdisk array topologies and showed different infiltrated areas. In combination with the cell sphere spreading theory, by quantifying the infiltration area of cell spheres at different time points, it was found that the infiltration rate of cell spheres decreased sequentially on the microdisk unit with a diameter of 2, 5, 50 and 20 μm, and the cell spheres had a small cell-substrate adhesion energy on the microdisk unit with a diameter of 20 μm (Figure 2).

Further, we analyzed cell morphology, actin, and adhesion plaque distribution on a variety of microdisk structures using immunofluorescence staining, and proposed that cell spheres infiltrate in climbing mode on small microdisk structures with diameters of 2μm and 5 μm, and in circling mode on larger microdisk structures with diameters of 20μm and 50 μm (FIG. 3). The infiltration process of the cell ball is a kind of multi-cell collective coordination movement.

This study reveals the infiltration mechanism of cell spheres on the surface of isotropic microdisk array topologies and deepens the understanding of the interaction between cell spheres and interface topologies. This work is an extension of femtosecond laser surface projection nanolithography technology and its application. The research is published in Small. The research work is supported by the National key research and development Program "Nanotechnology", the National Natural Science Foundation and the International Partnership Program of the Chinese Academy of Sciences.

Figure 1. Schematic diagram of femtosecond laser maskless projection lithography and the prepared microdisk array topology

Figure 2. Dynamic infiltration process and analysis of cell spheres on various topologies

Figure 3. Analysis of climbing infiltration and circumferential infiltration patterns of cell spheres

Source: Technical Institute of Physics and Chemistry, Chinese Academy of Sciences