Intercellular flow-mediated force relaxation measurement on the three-dimensional multicellular tissue

Abstract

Three-dimensional (3D) multicellular tissues are prevailing over 2D monolayer or single cells; their mechanical properties like stiffness, surface tension, and viscosity have been shown to relate to diseases like fibrosis or tumor metastasis. Multicellular tissues have been traditionally modeled as a viscoelastic material due to their apparent shape rearrangement, which hardly considers the internal structure, including the extracellular matrix (ECM) and resulting intercellular water flow. These intercellular communications usually provide significant information on diseases such as tumor invasion, but immediate supporting evidence of this behavior is lacking. In this work, we investigate the bulk response of 3D multicellular tissues due to such intercellular flows and explore the related mechanism through a tailored micro-mechanics platform. Firstly, we design and establish a micro-mechanics platform based on the parallel plate compression (PPC) method. We adopt a precise micro-balance as the sensor to detect the force variation of the sample during compression. A piezo linear stage is incorporated to exert such tiny vertical displacement. Besides, a lateral microscope is designed to monitor the compression process instantaneously. This platform has proved to be applicable to various samples, including hydrogels, cell spheroids, and natural tissues or organs. Then, we propose the critical criterion, the size dependency of force relaxation time, to distinguish a material's properties, i.e., viscoelasticity and poroelasticity. For poroelastic material, the force relaxation is due to water redistribution; hence, the speed highly depends on the sample sizes. In contrast, for viscoelastic material, it is determined by the bulk material properties, thus independent of the size. We theoretically verify this criterion via Abaqus simulation and experimentally on classic poro-/visco-elastic materials with various dimensions. Next, we apply the size-dependency criterion on the 3D multicellular tissues to distinguish the poro-/visco-elasticity in this biomaterial. We take the PPC on multiple cell spheroids with different sizes through the platform. It is observed that the force relaxation times are linearly proportional to the size of all tested cell lines, demonstrating poroelasticity in our experimental time range. Intriguingly, we take tests on the natural organs of the mouse islets and find such linear correlation as well. Hence, both cultured spheroids and natural tissues are poroelastic. Finally, we explore the mechanism determining the poroelasticity inside the 3D multicellular tissues. By inhibiting the cell-cell junctions, we demonstrate the intercellular water flow through the extracellular gaps dominates this poroelastic force relaxation in the biomaterial. Further experiments show that the stiffness of the structure and the extracellular gaps inside the 3D multicellular tissues couple to contribute to the intercellular water flow, i.e., the stiffer the structure and/or the larger the gaps, the faster the water flows, thus quicker the force decays after compression. These findings highlight the fundamental role of intercellular water flow in the mechanical properties of 3D multicellular tissues. The designed micro-mechanics platform is also beneficial to research at the tissue level with micro-newton forces owing to the development of artificial organoids for early disease diagnosis and treatment.