P.F.J. van Altena
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3 records found
1
Journal article
(2026)
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P.F.J. van Altena, Lucía Castillo Ransanz, R.H. Guis, Giulia Bergamaschi, Nanne J. Paauw, U. Staufer, Vivi M. Heine, A. Accardo
During neural development, cells generate and respond to mechanical forces within their microenvironment while they migrate and differentiate. These forces are central to the development of the human brain, as they are transduced into intracellular signals that regulate key neurobiological processes and may be involved in pathological processes. However, measurement of such forces in the nanonewton (nN) range within a three-dimensional (3D) microenvironment remains technically challenging and computationally intensive. Here, we present a solution to this challenge, based on elastomeric 3D microstructures fabricated via two-photon polymerization (2PP) and a tailored wet-etching process. The resulting free-standing beam architectures enable the quantification of nN forces exerted by pluripotent stem cell-derived neuroepithelial progenitor cells. We characterized the morphology of the cells and the forces they exerted using live-cell confocal imaging microscopy, scanning electron microscopy (SEM), supported by an in-house routine for intensity-based deflection measurements with sub-pixel resolution, and atomic force microscopy (AFM). Using this platform, we precisely quantified neuronal traction forces down to 1.2 nN from both healthy neural cells and those carrying a TSC2 gene mutation linked to tuberous sclerosis complex (TSC). The proposed elastomeric microenvironment paves the way for investigating cytoskeletal mechanics, neuromechanobiology in 3D, and for developing in vitro disease treatment models.
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During neural development, cells generate and respond to mechanical forces within their microenvironment while they migrate and differentiate. These forces are central to the development of the human brain, as they are transduced into intracellular signals that regulate key neurobiological processes and may be involved in pathological processes. However, measurement of such forces in the nanonewton (nN) range within a three-dimensional (3D) microenvironment remains technically challenging and computationally intensive. Here, we present a solution to this challenge, based on elastomeric 3D microstructures fabricated via two-photon polymerization (2PP) and a tailored wet-etching process. The resulting free-standing beam architectures enable the quantification of nN forces exerted by pluripotent stem cell-derived neuroepithelial progenitor cells. We characterized the morphology of the cells and the forces they exerted using live-cell confocal imaging microscopy, scanning electron microscopy (SEM), supported by an in-house routine for intensity-based deflection measurements with sub-pixel resolution, and atomic force microscopy (AFM). Using this platform, we precisely quantified neuronal traction forces down to 1.2 nN from both healthy neural cells and those carrying a TSC2 gene mutation linked to tuberous sclerosis complex (TSC). The proposed elastomeric microenvironment paves the way for investigating cytoskeletal mechanics, neuromechanobiology in 3D, and for developing in vitro disease treatment models.
Micro 3D Printing Elastomeric IP-PDMS Using Two-Photon Polymerisation
A Comparative Analysis of Mechanical and Feature Resolution Properties
The mechanical properties of two-photon-polymerised (2PP) polymers are highly dependent on the employed printing parameters. In particular, the mechanical features of elastomeric polymers, such as IP-PDMS, are important for cell culture studies as they can influence cell mechanobiological responses. Herein, we employed optical-interferometer-based nanoindentation to characterise two-photon-polymerised structures manufactured with varying laser powers, scan speeds, slicing distances, and hatching distances. The minimum reported effective Young’s modulus (YM) was 350 kPa, while the maximum one was 17.8 MPa. In addition, we showed that, on average, immersion in water lowered the YM by 5.4%, a very important point as in the context of cell biology applications, the material must be employed within an aqueous environment. We also developed a printing strategy and performed a scanning electron microscopy morphological characterisation to find the smallest achievable feature size and the maximum length of a double-clamped freestanding beam. The maximum reported length of a printed beam was 70 µm with a minimum width of 1.46 ± 0.11 µm and a thickness of 4.49 ± 0.05 µm. The minimum beam width of 1.03 ± 0.02 µm was achieved for a beam length of 50 µm with a height of 3.00 ± 0.06 µm. In conclusion, the reported investigation of micron-scale two-photon-polymerized 3D IP-PDMS structures featuring tuneable mechanical properties paves the way for the use of this material in several cell biology applications, ranging from fundamental mechanobiology to in vitro disease modelling to tissue engineering.
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The mechanical properties of two-photon-polymerised (2PP) polymers are highly dependent on the employed printing parameters. In particular, the mechanical features of elastomeric polymers, such as IP-PDMS, are important for cell culture studies as they can influence cell mechanobiological responses. Herein, we employed optical-interferometer-based nanoindentation to characterise two-photon-polymerised structures manufactured with varying laser powers, scan speeds, slicing distances, and hatching distances. The minimum reported effective Young’s modulus (YM) was 350 kPa, while the maximum one was 17.8 MPa. In addition, we showed that, on average, immersion in water lowered the YM by 5.4%, a very important point as in the context of cell biology applications, the material must be employed within an aqueous environment. We also developed a printing strategy and performed a scanning electron microscopy morphological characterisation to find the smallest achievable feature size and the maximum length of a double-clamped freestanding beam. The maximum reported length of a printed beam was 70 µm with a minimum width of 1.46 ± 0.11 µm and a thickness of 4.49 ± 0.05 µm. The minimum beam width of 1.03 ± 0.02 µm was achieved for a beam length of 50 µm with a height of 3.00 ± 0.06 µm. In conclusion, the reported investigation of micron-scale two-photon-polymerized 3D IP-PDMS structures featuring tuneable mechanical properties paves the way for the use of this material in several cell biology applications, ranging from fundamental mechanobiology to in vitro disease modelling to tissue engineering.
The biomechanical properties of the brain microenvironment, which is composed of different neural cell types, the extracellular matrix, and blood vessels, are critical for normal brain development and neural functioning. Stiffness, viscoelasticity and spatial organization of brain tissue modulate proliferation, migration, differentiation, and cell function. However, the mechanical aspects of the neural microenvironment are largely ignored in current cell culture systems. Considering the high promises of human induced pluripotent stem cell- (iPSC-) based models for disease modelling and new treatment development, and in light of the physiological relevance of neuromechanobiological features, applications of in vitro engineered neuronal microenvironments should be explored thoroughly to develop more representative in vitro brain models. In this context, recently developed biomaterials in combination with micro- and nanofabrication techniques 1) allow investigating how mechanical properties affect neural cell development and functioning; 2) enable optimal cell microenvironment engineering strategies to advance neural cell models; and 3) provide a quantitative tool to assess changes in the neuromechanobiological properties of the brain microenvironment induced by pathology. In this review, we discuss the biological and engineering aspects involved in studying neuromechanobiology within scaffold-free and scaffold-based 2D and 3D iPSC-based brain models and approaches employing primary lineages (neural/glial), cell lines and other stem cells. Finally, we discuss future experimental directions of engineered microenvironments in neuroscience.
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The biomechanical properties of the brain microenvironment, which is composed of different neural cell types, the extracellular matrix, and blood vessels, are critical for normal brain development and neural functioning. Stiffness, viscoelasticity and spatial organization of brain tissue modulate proliferation, migration, differentiation, and cell function. However, the mechanical aspects of the neural microenvironment are largely ignored in current cell culture systems. Considering the high promises of human induced pluripotent stem cell- (iPSC-) based models for disease modelling and new treatment development, and in light of the physiological relevance of neuromechanobiological features, applications of in vitro engineered neuronal microenvironments should be explored thoroughly to develop more representative in vitro brain models. In this context, recently developed biomaterials in combination with micro- and nanofabrication techniques 1) allow investigating how mechanical properties affect neural cell development and functioning; 2) enable optimal cell microenvironment engineering strategies to advance neural cell models; and 3) provide a quantitative tool to assess changes in the neuromechanobiological properties of the brain microenvironment induced by pathology. In this review, we discuss the biological and engineering aspects involved in studying neuromechanobiology within scaffold-free and scaffold-based 2D and 3D iPSC-based brain models and approaches employing primary lineages (neural/glial), cell lines and other stem cells. Finally, we discuss future experimental directions of engineered microenvironments in neuroscience.