Research ArticleMATERIALS SCIENCE

Mechanical stimulation of single cells by reversible host-guest interactions in 3D microscaffolds

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Science Advances  23 Sep 2020:
Vol. 6, no. 39, eabc2648
DOI: 10.1126/sciadv.abc2648
  • Fig. 1 Characterization of host-guest structures.

    (A) Chemical structure of components and products of the 3D laser lithography. (B) Array of hydrogel blocks fabricated by 3D laser lithography with schematic depiction of the host-guest dynamics. (C) Young’s modulus of a hydrogel block in 0 and 20 mM 1-adamantanecarboxylic acid (1-AdCA) in phosphate-buffered saline (PBS). (D) Block area as a function of time. After 100 s, the solution was exchanged to 20 mM 1-AdCA in PBS.

  • Fig. 2 Stimuli-responsive composite microscaffolds.

    (A) Schematic image of the microscaffolds, fabricated by 3D laser lithography with three different materials. Addition of 1-AdCA leads to a swelling of the hydrogel and displacement of the beams. (B) Optical micrographs of the two states with and without 1-AdCA. The red arrows indicate the displacement of the individual beams scaled by a factor of 3. (C) Average beam displacement relative to the center for an exemplary scaffold as a function of time. Addition of 1-AdCA leads to a strong positive displacement of the beams away from the center. Rinsing with pure PBS returns the scaffold into the original state. (D) Average displacement as a function of 1-AdCA concentration for multiple scaffolds. The table lists the resulting fit values and the lower and upper confidence limits LCL and UCL. (E) Average beam displacements for multiple cycles of solution exchange demonstrate complete reversibility of the system. (F) Scanning electron microscopy (SEM) micrographs of a typical sample with 360 scaffolds (without the host-guest hydrogel) with increasing magnifications from left to right.

  • Fig. 3 Cellular forces in the microscaffolds.

    (A) Schematic images of a cell that first invades the scaffold (1), builds up initial force in the process (2), and is later stretched by the scaffold (3). (B) Linear relationship of changes in displacement and changes in cellular traction forces, obtained by numerical calculations in COMSOL Multiphysics. Negative displacements are defined to point toward the center of the scaffold. (C) Exemplary images of displacements from numerical calculations before (left) and after (right) the swelling of the hydrogel. (D) Reaction of an exemplary cell in a scaffold as a function of time after addition of trypsin at minute 17. The black data points depict the displacement change, and the blue data points depict the change in traction force. (E) Optical micrographs of a cell before, during, and after addition of trypsin. The blue arrows indicate the displacement of the individual beams scaled by a factor of 10. The images below show a magnification of the left beam, with the green dashed line denoting the beam position of the initial frame and the blue dashed line denoting the position of the current frame. The numbers next to the arrow show the cellular reaction as a change in displacement and traction force relative to the first image. (F) Quantification of initial forces in the scaffolds for U2OS and NIH 3T3 cells. Each data point corresponds to one cell in a scaffold.

  • Fig. 4 Cellular reaction to external stimulation.

    (A) Optical micrographs from different time points during a stretching experiment. The arrows indicate the individual beam displacements due to the external stimulus (red) (scaled by factor of 3) and the cellular response (blue) (scaled by factor of 10). (B) Corresponding average beam displacement as a function of time for a scaffold with an attached cell during a stretch-release cycle. The individual numbers in the plot correspond to the optical micrographs in (A). (C) Zoom in of the reaction after the stretch/after the release respectively with the displacements in black and traction force changes in blue. (D) Quantification of the cellular response 30 min after the stretch and after the release. Each data point represents one cell during the depicted cycle in (B).

  • Fig. 5 Inhibition effects and actin filament reorganization.

    (A) Optical micrographs of ROCK inhibited and NM2A-KO cells in the unstretched and stretched state. Red arrows indicate the displacement of the individual beams scaled by a factor of 3. (B) Comparison of initial traction forces of WT cells, ROCK inhibitor–treated cells, and the NM2A-KO cell line. (C) Exemplary track of a NM2A-KO cell in a stretch-release cycle. The numbers correspond to the respective time points in Fig. 4B for comparison. (D) Quantification of the reaction after the stretch and after the release of the two cases with the WT situation. (E) Comparison between fixed and stained cells in the unstretched and the stretched state. For both cell types shown, actin and the cell nucleus are stained (the walls also show autofluorescence in the blue channel). For the U2OS WT cells, NM2A was stained additionally. The red arrows indicate the displacement of the individual beams, scaled by a factor of 3. DAPI, 4′,6-diamidino-2-phenylindole.

  • Fig. 6 Asymmetric stretch.

    (A) Left: Schematic images of a scaffold with three thick walls to enable an asymmetric stretch to the left side. Right: Comparison of immunocytochemical stainings of actin and the cell nucleus between an unstretched cell and a cell that has been stretched only to the left side. (B) Exemplary individual beam displacements as a function of time. (C) Quantification of force changes after stretching on the left beam.

Supplementary Materials

  • Supplementary Materials

    Mechanical stimulation of single cells by reversible host-guest interactions in 3D microscaffolds

    Marc Hippler, Kai Weißenbruch, Kai Richler, Enrico D. Lemma, Masaki Nakahata, Benjamin Richter, Christopher Barner-Kowollik, Yoshinori Takashima, Akira Harada, Eva Blasco, Martin Wegener, Motomu Tanaka, Martin Bastmeyer

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