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3D-Printed Structures Versus Drilled Cavities: A Comparison of Microconfinement Methods for Rheological

Isis V M Lima1, Chukwuma Chris Muoghalu1, Shruti G Kulkarni1

  • 1Institute for Biophysics, University of Bremen, Bremen, Germany.

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Summary

Researchers developed microconfinement strategies for atomic force microscopy (AFM) analysis of 3D cell cultures. These methods enable reproducible mechanical measurements of multicellular aggregates, crucial for understanding tumour microenvironments and developing cancer therapies.

Keywords:
3D‐printed truncated conical microstructuresPolyHEMAatomic force microscopycylindrical cavitieshuman pancreatic cancer cellsmicroconfinementmulticellular aggregatesviscoelasticity

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Area of Science:

  • Biomedical Engineering
  • Cell Biology
  • Materials Science

Background:

  • Three-dimensional (3D) cell cultures offer superior physiological relevance compared to 2D models for studying complex cellular behaviors and the tumor microenvironment.
  • Atomic force microscopy (AFM) is a powerful tool for mechanical characterization, but its application to 3D cell cultures is hindered by the instability of multicellular aggregates.

Purpose of the Study:

  • To develop and evaluate novel microconfinement strategies for stabilizing multicellular aggregates for reproducible AFM measurements.
  • To compare the efficacy of two distinct microconfinement methods in preserving cellular integrity and enabling mechanical property analysis.

Main Methods:

  • Development of two microconfinement strategies: 1) encapsulation in porous 3D-printed truncated conical microstructures, and 2) microfabrication of cylindrical cavities in Petri dishes.
  • Utilisation of two-photon polymerisation for 3D-printed structures and PolyHEMA coating for Petri dish cavities.
  • Application of AFM to measure stiffness and viscoelasticity of human pancreatic cancer cell (PANC-1) aggregates confined in both systems.

Main Results:

  • Both microconfinement strategies successfully stabilized multicellular aggregates without compromising cellular integrity, allowing for reproducible mechanical measurements.
  • Confined aggregates exhibited reduced stiffness and viscoelasticity compared to substrate-attached single cells, suggesting altered cytoskeletal tension in 3D environments.
  • Cylindrical cavities offered more robust aggregate retention, while 3D-printed structures showed potential for enhanced medium perfusion.

Conclusions:

  • Microconfinement strategies are essential for enabling reliable mechanical characterization of multicellular aggregates using AFM.
  • These methods significantly advance the study of tumour mechanobiology and the evaluation of anti-cancer therapies by extending AFM applicability to complex 3D models.
  • The choice of microconfinement method can be optimized based on the specific requirements for aggregate retention and medium perfusion.