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

Area of Science:

• Cell biology
• Biophysics
• Mechanobiology

Background:

• Cells experience mechanical forces and physical confinement in tissues.
• These forces influence cell behavior, impacting physiological and pathological processes like cancer metastasis.
• Previous research indicates 3D confinement causes nuclear deformation, affecting cell contractility, DNA, and chromatin.

Purpose of the Study:

• To present a novel, user-friendly, and cost-effective method for inducing precise 3D physical confinement on cells.
• To enable detailed investigation into the effects of nuclear deformation on cellular functions.
• To provide comprehensive protocols for device fabrication and cell confinement assays.

Main Methods:

• Fabrication of a custom device using agarose pads with micropillars.
• Application of external weights to induce defined physical confinement.
• Validation through assessment of nuclear deformation, nuclear area changes, and cell viability.
• Compatibility with live and fixed cell imaging.

Main Results:

• Successful induction of precisely defined physical confinement on cells.
• Confirmation of nuclear deformation and changes in nuclear area.
• Demonstration of maintained cell viability after confinement.
• Validation of the device for both short- and long-term studies.

Conclusions:

• The developed device offers a versatile and robust approach to studying 3D cell confinement.
• It facilitates the investigation of nuclear deformation's impact on cellular functions.
• The protocols support various applications, including live imaging and analysis of fixed cells.