Assembly and Use of a Microfluidic Device to Study Nuclear Mechanobiology During Confined Migration

Agrawal, Rajat; Windsor, Aaron; Lammerding, Jan

doi:10.1007/978-1-0716-2337-4_22

Cited by 4 publications

(3 citation statements)

References 19 publications

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“…However, if desired, a plastic mold can be generated from a PDMS cast of the silicon wafer to provide an even more permanent mold that can be used to cast the agarose devices. We have previously published a detailed protocol for the fabrication of plastic molds from PDMS casts (Agrawal et al, 2022).…”

Section: Su-8 Fabrication Of 500 μM Upper Layer Features and Final Pr...mentioning

confidence: 99%

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Agarose‐based 3D Cell Confinement Assay to Study Nuclear Mechanobiology

et al. 2023

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Cells in living tissues are exposed to substantial mechanical forces and constraints imposed by neighboring cells, the extracellular matrix, and external factors. Mechanical forces and physical confinement can drive various cellular responses, including changes in gene expression, cell growth, differentiation, and migration, all of which have important implications in physiological and pathological processes, such as immune cell migration or cancer metastasis. Previous studies have shown that nuclear deformation induced by 3D confinement promotes cell contractility but can also cause DNA damage and changes in chromatin organization, thereby motivating further studies in nuclear mechanobiology. In this protocol, we present a custom‐developed, easy‐to‐use, robust, and low‐cost approach to induce precisely defined physical confinement on cells using agarose pads with micropillars and externally applied weights. We validated the device by confirming nuclear deformation, changes in nuclear area, and cell viability after confinement. The device is suitable for short‐ and long‐term confinement studies and compatible with imaging of both live and fixed samples, thus presenting a versatile approach to studying the impact of 3D cell confinement and nuclear deformation on cellular function. This article contains detailed protocols for the fabrication and use of the confinement device, including live cell imaging and labeling of fixed cells for subsequent analysis. These protocols can be amended for specific applications. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Design and fabrication of the confinement device wafer Basic Protocol 2: Cell confinement assay Support Protocol 1: Fixation and staining of cells after confinement Support protocol 2: Live/dead staining of cells during confinement

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Section: Su-8 Fabrication Of 500 μM Upper Layer Features and Final Pr...mentioning

confidence: 99%

“…2. 18-24 hr before the cell confinement assay, coat the glass-bottom dishes with the appropriate extracellular matrix molecules (e.g., 5 μg/ml fibronectin or 0.05 mg/ml type I collagen) for 2 hr at 37°C or overnight at 4°C (Agrawal et al, 2022).…”

Section: Extracellular Matrix Coating and Cell Seedingmentioning

confidence: 99%

Agarose‐based 3D Cell Confinement Assay to Study Nuclear Mechanobiology

et al. 2023

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“…To address this issue, modern microfabrication technologies have been employed in a number of studies to investigate the movement of individual cells, using microfluidics. 37–58 Recently, we have developed a high-throughput microfluidic migration platform that utilizes robotic liquid handling and computer vision to rapidly quantify individual cellular motility. 59…”

Section: Introductionmentioning

confidence: 99%

Microfluidic single-cell migration chip reveals insights into the impact of extracellular matrices on cell movement

Zhou,

Ma,

Rock

et al. 2023

Lab Chip

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Chromatin compaction during confined cell migration induces and reshapes nuclear condensates

Zhao,

Xia,

Brangwynne

2024

Nat Commun

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Cell migration through small constrictions during cancer metastasis requires significant deformation of the nucleus, with associated mechanical stress on the nuclear lamina and chromatin. However, how mechanical deformation impacts various subnuclear structures, including protein and nucleic acid-rich biomolecular condensates, is largely unknown. Here, we find that cell migration through confined spaces gives rise to mechanical deformations of the chromatin network, which cause embedded nuclear condensates, including nucleoli and nuclear speckles, to deform and coalesce. Chromatin deformations exhibit differential behavior in the advancing vs. trailing region of the nucleus, with the trailing half being more permissive for de novo condensate formation. We show that this results from increased chromatin heterogeneity, which gives rise to a shift in the binodal phase boundary. Taken together, our findings show how chromatin deformation impacts condensate assembly and properties, which can potentially contribute to cellular mechanosensing.

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Assembly and Use of a Microfluidic Device to Study Nuclear Mechanobiology During Confined Migration

Cited by 4 publications

References 19 publications

Agarose‐based 3D Cell Confinement Assay to Study Nuclear Mechanobiology

Agarose‐based 3D Cell Confinement Assay to Study Nuclear Mechanobiology

Microfluidic single-cell migration chip reveals insights into the impact of extracellular matrices on cell movement

Chromatin compaction during confined cell migration induces and reshapes nuclear condensates

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