Micropatterned Thermoresponsive Cell Culture Substrates for Dynamically Controlling Neurite Outgrowth and Neuronal Connectivity in Vitro

Behm, Laura; Gerike, Susanna; Grauel, M. Katharina; Uhlig, Katja; Pfisterer, Felix; Baumann, Werner; Bier, Frank F.; Duschl, Claus; Kirschbaum, Michael

doi:10.1021/acsabm.9b00246

Cited by 6 publications

(3 citation statements)

References 38 publications

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“…In contrast, the latter microprinting technology is simple stamping of adhesion factors on the flat substrate to control the spatial distribution of cells [ 5 , 6 ]. Adding to the simplicity, stepwise activation of stamped thermoresponsive molecules for direct control of neurite elongation in pre-designed patterns during cultivation was recently reported [ 7 ]. However, in both technologies, the designs are pre-determined by the photomasks or stamps and thus renders difficult the preparation of changes in complex structures and additional micropatterning after the cells are seeded onto the microengineered environment.…”

Section: Introductionmentioning

confidence: 99%

Photothermal Agarose Microfabrication Technology for Collective Cell Migration Analysis

et al. 2021

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Agarose photothermal microfabrication technology is one of the micropatterning techniques that has the advantage of simple and flexible real-time fabrication even during the cultivation of cells. To examine the ability and limitation of the agarose microstructures, we investigated the collective epithelial cell migration behavior in two-dimensional agarose confined structures. Agarose microchannels from 10 to 211 micrometer width were fabricated with a spot heating of a focused 1480 nm wavelength infrared laser to the thin agarose layer coated on the cultivation dish after the cells occupied the reservoir. The collective cell migration velocity maintained constant regardless of their extension distance, whereas the width dependency of those velocities was maximized around 30 micrometer width and decreased both in the narrower and wider microchannels. The single-cell tracking revealed that the decrease of velocity in the narrower width was caused by the apparent increase of aspect ratio of cell shape (up to 8.9). In contrast, the decrease in the wider channels was mainly caused by the increase of the random walk-like behavior of component cells. The results confirmed the advantages of this method: (1) flexible fabrication without any pre-designing, (2) modification even during cultivation, and (3) the cells were confined in the agarose geometry.

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Section: Introductionmentioning

confidence: 99%

Photothermal Agarose Microfabrication Technology for Collective Cell Migration Analysis

et al. 2021

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“…Recently, direct control of neurite elongation in predesigned patterns during cultivation, by using stamped patterns of thermoresponsive molecules and changing the temperature, and thus, the adhesivity of the pattern, was reported 13 . While these methods allow selective control over neuronal growth within the predesigned patterns, flexible control of cell-to-cell connections to form the desired networks was still not possible because of the limitations of microprinting technology.…”

Section: Introductionmentioning

confidence: 99%

Stepwise neuronal network pattern formation in agarose gel during cultivation using non-destructive microneedle photothermal microfabrication

Tanaka

Watanabe

Shimoda

et al. 2021

Sci Rep

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Conventional neuronal network pattern formation techniques cannot control the arrangement of axons and dendrites because network structures must be fixed before neurite differentiation. To overcome this limitation, we developed a non-destructive stepwise microfabrication technique that can be used to alter microchannels within agarose to guide neurites during elongation. Micropatterns were formed in thin agarose layer coating of a cultivation dish using the tip of a 0.7 $$\upmu \mathrm{m}$$ μ m -diameter platinum-coated glass microneedle heated by a focused 1064-nm wavelength infrared laser, which has no absorbance of water. As the size of the heat source was 0.7 $$\upmu \mathrm{m}$$ μ m , which is smaller than the laser wavelength, the temperature fell to 45 $$^\circ \hbox {C}$$ ∘ C within a distance of 7.0 $$\upmu \mathrm{m}$$ μ m from the edge of the etched agarose microchannel. We exploited the fast temperature decay property to guide cell-to-cell connection during neuronal network cultivation. The first neurite of a hippocampal cell from a microchamber was guided to a microchannel leading to the target neuron with stepwise etching of the micrometer resolution microchannel in the agarose layer, and the elongated neurites were not damaged by the heat of etching. The results indicate the potential of this new technique for fully direction-controlled on-chip neuronal network studies.

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“…For SH‐SY5Y and iPS cells, the protocol was adjusted to the requirements of the cell types: 2×10 5 SH‐SY5Y cells were seeded on Matrigel™‐coated microgel‐modified cell culture dishes. After 24 h, the medium was supplemented with 10 µM retinoic acid (R2625, Sigma‐Aldrich) for cell differentiation (Behm et al, 2019; Påhlman et al, 1984), and cells were cultivated for another 2–3 days at 37°C and 5% CO 2 . iPSC was detached using dispase (Stemcell Technologies), collected in 15 ml tubes, centrifuged at 300 g , and resuspended in FTDA medium.…”

Section: Methodsmentioning

confidence: 99%

Adhesion, proliferation, and detachment of various cell types on thermoresponsive microgel coatings

Flechner

Schaller

Stahl

et al. 2022

Biotech & Bioengineering

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Cutting-edge biomedical applications require increasingly complex and fastidious cell systems, for example, various classes of primary or stem cells. Their cultivation, however, still differs little from 30 years ago. This especially applies to the use of indiscriminative proteases for nonspecific cell detachment. A far more gentle alternative changes the adhesive properties of the cell culture substrates through coatings based on thermoresponsive polymers. Such polymers mediate cell adhesion at 37°C, but become repulsive upon a cell-compatible temperature drop to, for example, 32°C. While the high functionality of this method has already been well proven, it must also be easy and reproducible to apply. Here, we emphasize the potential of standard cell culture materials coated by spraying with thermoresponsive microgels for routine cultivation and beyond.On these surfaces, we successfully cultivated and detached various cell types, including induced pluripotent stem cells and cells in serum-free culture. In addition, we evaluated the compatibility of the microgel-sprayed surfaces with adhesion-promoting proteins, which are essential for, for example, stem cells or neuronal cells. Finally, we demonstrate that the microgel surfaces do not impair proliferation and show their long-term stability. We conclude that for cell detachment, thermoresponsive cell culture substrates can fully substitute proteases, like trypsin, by employing a comparably straightforward protocol that is compatible with many industrial processing lines.

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Micropatterned Thermoresponsive Cell Culture Substrates for Dynamically Controlling Neurite Outgrowth and Neuronal Connectivity in Vitro

Cited by 6 publications

References 38 publications

Photothermal Agarose Microfabrication Technology for Collective Cell Migration Analysis

Photothermal Agarose Microfabrication Technology for Collective Cell Migration Analysis

Stepwise neuronal network pattern formation in agarose gel during cultivation using non-destructive microneedle photothermal microfabrication

Adhesion, proliferation, and detachment of various cell types on thermoresponsive microgel coatings

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