Direct Laser Writing of Tubular Microtowers for 3D Culture of Human Pluripotent Stem Cell-Derived Neuronal Cells

Turunen, Sanna; Joki, Tiina; Hiltunen, Maiju; Ihalainen, Teemu O.; Narkilahti, Susanna; Kellomäki, Minna

doi:10.1021/acsami.7b05536

Cited by 37 publications

(28 citation statements)

References 62 publications

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“…Cells formed 3D neural networks and suspended bridges throughout the micropillar height and along and between the micropillar walls. A similar formation of suspended neural process bridges has also been reported with microtowers, microfibres and 2PP-DLW fabricated microstructures [54][55][56][57]. In order to visualize the cells seeded on the silicon micropillar arrays with an inverted microscope, the device has to be set upside down, facing the microscope objectives.…”

Section: Culturing Human Cortical Progenitors On Vertically-aligned Smentioning

confidence: 74%

Vertically-Aligned Functionalized Silicon Micropillars for 3D Culture of Human Pluripotent Stem Cell-Derived Cortical Progenitors

Cutarelli

Ghio

Zasso

et al. 2019

Cells

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Silicon is a promising material for tissue engineering since it allows to produce micropatterned scaffolding structures resembling biological tissues. Using specific fabrication methods, it is possible to build aligned 3D network-like structures. In the present study, we exploited vertically-aligned silicon micropillar arrays as culture systems for human iPSC-derived cortical progenitors. In particular, our aim was to mimic the radially-oriented cortical radial glia fibres that during embryonic development play key roles in controlling the expansion, radial migration and differentiation of cortical progenitors, which are, in turn, pivotal to the establishment of the correct multilayered cerebral cortex structure. Here we show that silicon vertical micropillar arrays efficiently promote expansion and stemness preservation of human cortical progenitors when compared to standard monolayer growth conditions. Furthermore, the vertically-oriented micropillars allow the radial migration distinctive of cortical progenitors in vivo. These results indicate that vertical silicon micropillar arrays can offer an optimal system for human cortical progenitors' growth and migration. Furthermore, similar structures present an attractive platform for cortical tissue engineering.

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Section: Culturing Human Cortical Progenitors On Vertically-aligned Smentioning

confidence: 74%

Vertically-Aligned Functionalized Silicon Micropillars for 3D Culture of Human Pluripotent Stem Cell-Derived Cortical Progenitors

Cutarelli

Ghio

Zasso

et al. 2019

Cells

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“…[ 28,29 ] On the untreated, planar areas, the native Ormocomp surface supported strong adhesion of both cells (Figure 1f) and metals (Figure 1g), which facilitated integration of cell‐compatible gold electrodes for impedance detection. The cell compatibility of native Ormocomp [ 22–26 ] as well as the Ormocomp metallization protocols [ 30 ] has been thoroughly established in previous literature. In this study, the impedance spectra were measured after cell seeding, once every hour for 96 h, using frequency range of 5–100 000 Hz (Figure 1h).…”

Section: Resultsmentioning

confidence: 99%

“…The developed microfluidic platform is made from an organically modified ceramic material (Ormocomp), which is optically transparent down to near‐UV range [ 21 ] and inherently biocompatible supporting cell adhesion without any additional coating. [ 22–26 ] Thus, the developed platform also enables parallel culturing of 2D cell monolayers, under identical growth conditions to those of 3D spheroids, in a single microfluidic channel. For the fabrication of the round (cross‐section profile) microwells, we exploit the controlled overexposure of Ormocomp, [ 27 ] which facilitates straightforward customization of the microwell shape and depth in a single lithographic step.…”

Section: Introductionmentioning

confidence: 99%

Simultaneous Culturing of Cell Monolayers and Spheroids on a Single Microfluidic Device for Bridging the Gap between 2D and 3D Cell Assays in Drug Research

Järvinen

Bonabi

Jokinen

et al. 2020

Adv Funct Materials

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(1 of 11)Two-dimensional (2D) cell cultures have been the primary screening tools to predict drug impacts in vitro for decades. However, owing to the lack of tissuespecific architecture of 2D cultures, secondary screening using three-dimensional (3D) cell culture models is often necessary. A microfluidic approach that facilitates side-by-side 2D and 3D cell culturing in a single microchannel and thus combines the benefits of both set-ups in drug screening; that is, the uniform spatiotemporal distributions of oxygen, nutrients, and metabolic wastes in 2D, and the tissuelike architecture, cell-cell, and cell-extracellular matrix interactions only achieved in 3D. The microfluidic platform is made from an organically modified ceramic material, which is inherently biocompatible and supports cell adhesion (2D culture) and metal adhesion (for integration of impedance electrodes to monitor cell proliferation). To induce 3D spheroid formation on another area, a single-step lithography process is used to fabricate concave microwells, which are made cellrepellant by nanofunctionalization (i.e., plasma porosification and hydrophobic coating). Thanks to the concave shape of the microwells, the spheroids produced on-chip can also be released, with the help of microfluidic flow, for further off-chip characterization after culturing. In this study, the methodology is evaluated for drug cytotoxicity assessment on human hepatocytes.

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“…Using 2PP‐DLW, nearly any mechanically stable structure can be realized as real 3D culturing substrate consisting of a biocompatible polymer within only a few hours. 2PP‐DLW has already been utilized for neuronal growth studies . Note, while most of these applications try to mimic the natural brain environment to a certain structural degree, the adhesions spots of the cell somata and/or the direction of neurite outgrowth on 3D substrates have been statistical so far.…”

Section: Introductionmentioning

confidence: 99%

Microscaffolds by Direct Laser Writing for Neurite Guidance Leading to Tailor‐Made Neuronal Networks

et al. 2019

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cone. [7,8] Topological cues such as stiffness, [4] hydrophobicity, [9] and modulation of the surrounding surface, for example, through variations in roughness [10,11] or through guiding barriers [12][13][14] and channels, [15][16][17][18][19][20] have also been found to be effective methods to direct the neurite outgrowth on artificial substrates.A significant drawback of classical 2D neuronal in vitro cultures is their inability to sufficiently mimic the distinct 3D connections in a nervous system. [21] Most of the standard lithography techniques are only available in 2D or 2.5D. [22] However, recent advances in micro-and nanoscale fabrication, especially direct laser writing by two-photon polymerization (2PP-DLW), now enable the construction of complex resist-based 3D substrates with feature sizes down to ≈150 nm. [23] Using 2PP-DLW, nearly any mechanically stable structure can be realized as real 3D culturing substrate consisting of a biocompatible polymer within only a few hours. 2PP-DLW has already been utilized for neuronal growth studies. [24][25][26][27][28][29] Note, while most of these applications try to mimic the natural brain environment to a certain structural degree, the adhesions spots of the cell somata and/or the direction of neurite outgrowth on 3D substrates have been statistical so far. As a first step toward 2PP-DLW application for neurite guidance, Turunen et al. used DLW in 2014 to print walls on planar glass substrates to form cavities and channels, and applied laminin with a microinjection system inside the neurocages. [27] The design lacked efficient cell confinement properties, but potential function of printed channels as neurite guides was shown.Other approaches, which enable in vitro neuronal network growth in a 3D environment in different ways, such as synthetic hydrogel platforms, [30][31][32] network growth on micro beads, [21,33] polymer scaffolds fabricated by gaseous salt leaching, [34,35] or microfibrous scaffolds [36] feature only statistically oriented network growth.Here, we present an approach for guided neuronal network growth that features single neurites specifically grown through rectangular tubes in a 3D scaffold. The design we show herein combines the tunability and the precision of a 3D design utilizing 2PP-DLW manufacturing with topologically and chemically predefined adhesion areas as well as guidance paths for While modern day integrated electronic circuits are essentially designed in a 2D fashion, the brain can be regarded as a 3D circuit. The thus enhanced connectivity enables much more complex signal processing as compared to conventional 2D circuits. Recent technological advances in the development of nano/microscale 3D structuring have led to the development of artificial neuron culturing platforms, which surpass the possibilities of classical 2D cultures. In this work, in vitro culturing of neuronal networks is demonstrated by determining predefined pathways through topological and chemical neurite guiding. Tailor-made culturing substrates of microtowers ...

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Direct Laser Writing of Tubular Microtowers for 3D Culture of Human Pluripotent Stem Cell-Derived Neuronal Cells

Cited by 37 publications

References 62 publications

Vertically-Aligned Functionalized Silicon Micropillars for 3D Culture of Human Pluripotent Stem Cell-Derived Cortical Progenitors

Vertically-Aligned Functionalized Silicon Micropillars for 3D Culture of Human Pluripotent Stem Cell-Derived Cortical Progenitors

Simultaneous Culturing of Cell Monolayers and Spheroids on a Single Microfluidic Device for Bridging the Gap between 2D and 3D Cell Assays in Drug Research

Microscaffolds by Direct Laser Writing for Neurite Guidance Leading to Tailor‐Made Neuronal Networks

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