Microstructures Printed via 2-Photon Polymerization (2PP) to Mechanically Stimulate 3D Multicellular Systems

In nature, cells live immersed in an extracellular matrix with other cells, forming tissues and organs. These three-dimensional environments are essential for a wide array of vital biological processes. As a result, many recent studies are now exploring the utilization of 3D multicellular systems such as spheroids, organoids, and organotypic cell cultures. These alternatives, when compared to conventional 2D cell culture methods, hold a significant advantage in closely resembling physiological conditions. Indeed, the mechanical forces acting upon cells can yield differing outcomes depending on whether the cells are in a 2D or 3D environment. Traditionally, most studies on mechanotransduction, especially in terms of the effects induced by mechanical stretching, have been conducted on two-dimensional cultures. This approach, however, has provided only a limited understanding of the effects within a 3D culture.<br/>To address this limitation, we have developed a device for the mechanical stimulation of 3D multicellular systems using Two-Photon Polymerization (2PP). This device can be used to study the mechanical forces in the biological complexities of tissues at an exceptional micrometer-scale resolution. The design of the device allows the application of forces capable of stretching the encapsulated 3D multicellular systems with extreme precision, using a cantilever. Simultaneously, this system enables high-resolution imaging to assess the effects of stretching in vivo. Our findings have revealed that cyclic stretching over 30 minutes at a frequency of 0.5 Hz, and an average displacement of 0.22um, leads to noteworthy alterations in the morphology and orientation of actin within the 3D multicellular system. Furthermore, this device can be easily customized to mechanically stimulate various types of multicellular systems, accommodating differences in shape and cell types.