(F-217) Agarose-based 3D cell confinement assay to study nuclear mechanobiology

Introduction::
During many physiological processes and diseases, cells sense and respond to mechanical forces and spatial constraints from the physical environment. In the context of cancer, cells migrate through interstitial spaces as small as 1-2 microns in diameter during the process of metastasis, while cells at the primary tumor experience spatial confinement in the tumor microenvironment. In these physiological processes, cells experience substantial nuclear deformation, as the nucleus is the largest and stiffest organelle. The nucleus has recently gained spotlight as a cellular “mechanosensor” in which nuclear deformation has been shown to regulate changes in chromatin organization, cell contractility, and DNA damage signaling. However, many molecular mechanisms connecting mechanical cues to cellular responses remain undiscovered, and it is still unclear how 3D spatial confinement regulates cellular outputs in many cell types. To address these questions, we fabricated a low-cost, easily customizable cell confinement device capable of inducing precise physical confinement to cells using agarose micropillars. Unlike previously developed devices that use PDMS micropillars, our agarose-based device provides a suitable nutrient supply to cells for long-term confinement. Additionally, confinement is induced with a simple 3D-printed weight holder and disk weights (Figure 1A) without the need for costly external hardware such as syringe pumps or pressurized air systems. The device is compatible with imaging of both live and fixed cells for further analysis to elucidate cellular mechanisms of mechanotransduction.




Materials and Methods::
To form the agarose confinement device, agarose is cast onto a silicon wafer containing the negative tone of the micropillar design. The silicon wafer is fabricated using SU8 photoresist with two distinct layers. The first layer of photoresist contains the negative tone of the array of micropillars fabricated to be the height of cell confinement. We recommend a confinement height of 2-5 µm, with a control height of 10-15 µm, depending on the initial height of the cell nuclei. The second layer of SU8 is fabricated to be a 500 µm tall concentric circle around the micropillar pattern to form a circular undercut in the agarose when casting the device to ensure the micropillars are not damaged. After the wafer is fabricated, agarose is cast onto the wafer, allowed to set, and individual agarose devices are cut out. The device is then placed onto cells seeded on a glass bottom dish. The weight holder and weight are subsequently added on top of the agarose device to induce confinement. Confocal imaging was performed after confinement to assess nuclear height and area. As proof of principle, confined cells were fixed and stained with phalloidin to visualize F-actin. Live/dead cell staining was performed using propidium iodide to confirm cell viability after 24 hours of confinement.




Results, Conclusions, and Discussions::
We successfully developed a 3D agarose-based compression device (Figure 1A) capable of precise and uniform cellular confinement. The device can be fabricated to induce confinement at any height desired, and the amount of weight added can be adjusted, allowing a versatile device that can be applied to many cell types. We validated this device by confining MDA-MB-231 cells expressing fluorescently labeled Histone-2B to either 3 µm height, or a control height of 10 µm that does not induce nuclear deformation. Unconfined cells were used as additional controls. The nuclei of cells confined to 3 µm tall clearly deformed to adjust to the available height, whereas the nuclei of cells in the 10 µm tall control devices were more rounded and similar to the unconfined conditions (Figure 1B). Measurements of nuclear height confirmed these observations, further demonstrating that the 10 µm tall ‘confinement’ serves as an appropriate control height, as the nuclear height of cells confined to 10 µm does not change compared to unconfined cells (Figure 1C). Similarly, the nuclear cross-sectional area in the x-y plane increased significantly during confinement to 3 µm height, whereas the cross-sectional area of nuclei confined to 10 µm did not change compared to unconfined cells. Phalloidin staining was performed to visualize F-actin localization in cells after confinement, confirming this device allows for the fixation of cells for downstream analysis, such as assessing intracellular protein localization or DNA-damage signaling pathways. Live/dead cell staining showed confined cells remained viable for at least 24 hours, demonstrating that the device is suitable for both short-and long-term confinement without impacting cell viability and that it is well suited for live cell imaging. In conclusion, the confinement system provides a cost-effective and robust in vitro assay to probe the consequences of cell confinement and nuclear deformation with precise control over the degree of confinement. This device is ideally suited for studies of nuclear mechanotransduction and the effect of cellular confinement in a broad range of applications, such as immune or cancer cells and many other physiological processes.




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