Cell migration, shape maintenance, and intracellular signaling are closely linked to dynamic changes in cell morphology and the cytoskeleton. These processes involve the reorganization of the cytoskeleton within the cytoplasm, affecting all its key components: intermediate filaments, microtubules, and microfilaments. A promising strategy for remotely controlling cellular functions is the use of magnetic nanoparticles, which can influence cellular physiology. This approach, known as magnetogenetics, has been applied in various areas of cell and molecular biology. Applying a magnetic field allows for the non-invasive modulation of biochemical processes, cell migration, and morphological changes in cells containing magnetic nanoparticles. In our study, magnetic nanoparticles were conjugated with antibodies targeting cytoskeletal components, enabling the magnetically induced manipulation and deformation of the cell cytoskeleton. Our research introduces a novel approach to manipulating specific cytoskeletal components and altering cell polarity with spatial precision in vitro using magnetic nanoparticles associated with the cytoskeleton.

1. Cell motility, shape supporting, and intracellular signaling are followed by changes in cell morphology and cytoskeleton. The cell reaction and the reorganization of the cytoskeleton occurs in a single volume of the cytoplasm and affects all components of the cytoskeleton: intermediate filaments, microtubules and microfilaments. A promising way to manipulate cells is magnetic nanoparticles that control cellular physiology. This approach is called magnetogenetics and has found application in various fields of cell and molecular biology. Using a magnetic field, it is possible to non-invasively regulate biochemical processes, migration and changes in the morphology of cells with magnetic nanoparticles. Our work opens up new possibilities for spatial manipulation of individual cytoskeletal components in vitro and operates biochemical pathways associated with individual cytoskeletal components.

Interactions with the extracellular environment and biological responses are based on biochemical pathways. Cytoskeletal reorganization is a dynamic process accompanied by filament polymerization and depolymerization, that allows the cell to effectively perceive and respond to external mechanical stimuli by altering their biomechanical properties. The contribution of mechanical deformations of individual cytoskeletal elements to changes in intracellular signaling has not been covered in the existing literature. In our article, we investigated changes in gene expression after regulated cytoskeletal deformation using a constant magnetic field and magnetic nanoparticles associated with antibodies to cytoskeletal proteins (vimentin, beta-actin and acetylated tubulin). For the first time, we have identified and described the biological pathways involved in the regulation of mechanical deformations of cytoskeletal elements.

3D cell cultures, such as cell spheroids, are actively used in biology for modeling biological processes, studying intercellular interactions and pharmacological compounds screening and are becoming indispensable objects in cell culture laboratories. There are many methods for producing spheroids, varying in cost and convenience. One of the most handy and affordable is the use of agarose microwells. We have developed approaches to fabricate agarose microwells in standard culture plastic with the assistance of a hobby-grade MSLA 3D printer. The use of 3D printing allows you to customize microwells in a wide range of shapes and sizes and scale the production process from a few spheroids to tens of thousands. We have shown that it is possible to create gel microwells in a dish with a glass bottom, which allows us to easily realize time-lapse confocal microscopy of spheroids, and we have also performed in situ optical clearing in the same dishes to study the spheroid structure. We demonstrated the ability to study the cytotoxicity of various substances and nanoparticles in commonly used 96-well plates. And finally, in this article we describe the difficulties and limitations of our approach and suggest ways for solving them, allowing the reader not only to reproduce it, but also to adapt it to the specific needs of a certain laboratory, using provided 3D models and instructions.