Observing the Mechanical Response of Biological Tissues at the Single-Cell Level

While we may not be very conscious of it in our daily lives, our bodies are subjected to various forces from Earth's gravity and daily movements. These forces acting on our bodies are what allow them to maintain their normal functions. You may have seen on the news how astronauts returning from the International Space Station, their muscles weakened by long periods in a zero-gravity environment, need to be supported by others. Tissues like muscles and bones are maintained by being used—that is, by receiving mechanical stimuli. The example of the astronauts is a clear case of a rapid decline in muscle strength due to a reduction in such mechanical stimuli. This relationship between living organisms and mechanical stimuli is just as profound at the cellular level, the microscopic unit of life. Cells sense external mechanical stimuli such as tensile and shear forces, which in turn influence cellular behaviors like proliferation, differentiation, and apoptosis.

This cellular response to mechanical stimuli has also garnered attention in the recent fields of tissue engineering and regenerative medicine. Many studies have attempted to form mature tissues by applying mechanical stimuli based on empirical rules to artificially constructed tissues such as cardiac muscle, blood vessels, and tendons. However, the detailed mechanisms of what kind of mechanical stimuli best promote tissue maturation are not yet fully understood. One reason for this is that biological tissue is a complex composite material with a heterogeneous structure, where numerous cells are densely arranged within an extracellular matrix of collagen and other substances. This means that even when external mechanical stimuli like tension or compression are applied, the stimulus received by each individual cell differs. Furthermore, it has recently been noted that even individual cells (single cells) have their own unique characteristics, and it is expected that the behavior of each single cell will differ even under the same mechanical stimulus. Thus, the mechanical response of cells within three-dimensional biological tissues involves a complex interplay of factors, and our current understanding remains limited.

Therefore, our group has developed a system that enables tracking analysis of single cells within three-dimensional tissues under mechanical stimulation by combining an optical live-imaging system with a mechanical stretching device [1] (Figure). The system consists of a polydimethylsiloxane (PDMS) stretch culture chamber, a three-dimensional C2C12 skeletal muscle tissue, a high-precision stage and its control unit for applying mechanical stretch stimuli, and a laser confocal microscope. The C2C12 cells are triple-fluorescently labeled for the nucleus, cell membrane, and Golgi apparatus through genetic modification, allowing for the acquisition of the cell's position (nucleus), shape (cell membrane), and direction of movement (Golgi apparatus) within the three-dimensional tissue via live imaging. Using this system, we have successfully observed how single C2C12 cells behave in response to mechanical stimuli. We also discovered that under conditions of applied mechanical stimuli, individual cells exhibit different behaviors (cellular individuality). By further developing this system, we hope to contribute to understanding the mechanisms of the hierarchical mechanical responses that occur daily between single cells and tissues within our bodies.

[1] Keitaro Kasahara, Jumpei Muramatsu, Yuta Kurashina, Shigenori Miura, Shogo Miyata, Hiroaki Onoe, “Spatiotemporal single-cell tracking analysis in 3D tissues to reveal heterogeneous cellular response to mechanical stimuli,” Science Advances, Vol. 9, eadf9917, 2023.