Patterned with cells (area sum of microplates) from the whole area

Patterned with cells (area sum of microplates) from the whole area (area sum of microplates and areas of the bottom of the dish and of MPC coating) is ca.2 . The cell seeding concentration was 26104?6105 cells/ml, which corresponds to 4.26103?41.56103 cells/cm2. Non-adhered cells on the substrate were washed out after a 4 h culture period (Figures S1a-vi, S2vi).Detachment of the microplatesThere are two folding approaches. In the first approach, we used 0.05?.1 gelatin as the sacrificial layer, and the edges of individual microplates were pushed with a glass tip manipulated by a micromanipulator (NI2, Eppendorf), manually triggering detachment of the plates from the substrate (Figures 1D, S1 a-vii, movies S3, S4, S5). Thus, a large amount of the microstructures can be produced in order. In the second approach, we incorporated the flexible joint (Figures 1F, S2viii, movies S2, S6) with a 3? gelatin sacrificial layer. In this case, microplates were detached and self-folded by CTF spontaneously as the gelatin dissolved at 37uC, which is the temperature at the cell incubator. Consequently, many 3D microstructures were produced simultaneously. The optimum concentrations of the gelatin in both approaches were experimentally determined (Table S1).Figure 6. Batch process of folding cells-cultured microplates with flexible joint. (A) ITI-007 chemical information Sequential images of batch process of cubes. (B) Batch processing of tetrahedron before and after self-folding. (C) Ratio of the folded tetrahedron vs. culturing time. (D) Fluorescent microscopic image of the cells after culturing the cells for 4 days with the live/dead fluorescent staining. Live and dead cells are shown in green and red colors, respectively. NIH/3T3 cells were used. Scale bars, 50 mm. doi:10.1371/journal.pone.0051085.ga parylene film onto the MPC polymer and gelatin layers (Figure S2 ). In order to produce a flexible joint between the microplates, we patterned a photosensitive polymer SU-8 (Micro Chem, USA) and then deposited the parylene (Figure S2 i ii). Next, we etched the parylene film to produce the microplates using O2 [DTrp6]-LH-RH plasma with an Al mask that was patterned by standard lithography (Figure S2 v). After that, the MPC polymer was coated again on the glass substrate since the polymer was also etched and adversely affected by O2 plasma (Figure S2 ). Finally, the Al mask was removed using NMD, and the microplates with the flexible joint were produced (Figure S2 i). Figure 1G shows an image of the produced microplates with the flexible joint taken by scanning electron microscopy (SEM) (VHX-D510, Keyence, Japan). When the plates are folded, the joint works as a valley fold.Cell morphology and cell stainingIn order to visualize cell morphology, the cells were first fixed with 4 paraformaldehyde (PFA, Muto Pure Chemicals, Japan) for 15 min and rinsed three times with phosphate buffered saline (PBS, Sigma, USA). The cells were permeabilized with 0.1 TritonX-100 (Sigma, USA) for 2 min and rinsed three times with PBS. 1379592 In order to avoid non-specific binding, the substrate was immersed into 1 bovine serum albumin (BSA, Sigma, USA) solution for 30 minutes and rinsed once with PBS. The cells were then incubated with Alexa Fluor Phalloidin 488 conjugate (Molecular Probes; 1:200 dilution) and Hoechst 33342 (Molecular Probes; 1:400 dilution) to stain their actin filaments and nuclease with green and blue, respectively. After that the cells were rinsed three times with PBS.Cell OrigamiWe used the fluoresc.Patterned with cells (area sum of microplates) from the whole area (area sum of microplates and areas of the bottom of the dish and of MPC coating) is ca.2 . The cell seeding concentration was 26104?6105 cells/ml, which corresponds to 4.26103?41.56103 cells/cm2. Non-adhered cells on the substrate were washed out after a 4 h culture period (Figures S1a-vi, S2vi).Detachment of the microplatesThere are two folding approaches. In the first approach, we used 0.05?.1 gelatin as the sacrificial layer, and the edges of individual microplates were pushed with a glass tip manipulated by a micromanipulator (NI2, Eppendorf), manually triggering detachment of the plates from the substrate (Figures 1D, S1 a-vii, movies S3, S4, S5). Thus, a large amount of the microstructures can be produced in order. In the second approach, we incorporated the flexible joint (Figures 1F, S2viii, movies S2, S6) with a 3? gelatin sacrificial layer. In this case, microplates were detached and self-folded by CTF spontaneously as the gelatin dissolved at 37uC, which is the temperature at the cell incubator. Consequently, many 3D microstructures were produced simultaneously. The optimum concentrations of the gelatin in both approaches were experimentally determined (Table S1).Figure 6. Batch process of folding cells-cultured microplates with flexible joint. (A) Sequential images of batch process of cubes. (B) Batch processing of tetrahedron before and after self-folding. (C) Ratio of the folded tetrahedron vs. culturing time. (D) Fluorescent microscopic image of the cells after culturing the cells for 4 days with the live/dead fluorescent staining. Live and dead cells are shown in green and red colors, respectively. NIH/3T3 cells were used. Scale bars, 50 mm. doi:10.1371/journal.pone.0051085.ga parylene film onto the MPC polymer and gelatin layers (Figure S2 ). In order to produce a flexible joint between the microplates, we patterned a photosensitive polymer SU-8 (Micro Chem, USA) and then deposited the parylene (Figure S2 i ii). Next, we etched the parylene film to produce the microplates using O2 plasma with an Al mask that was patterned by standard lithography (Figure S2 v). After that, the MPC polymer was coated again on the glass substrate since the polymer was also etched and adversely affected by O2 plasma (Figure S2 ). Finally, the Al mask was removed using NMD, and the microplates with the flexible joint were produced (Figure S2 i). Figure 1G shows an image of the produced microplates with the flexible joint taken by scanning electron microscopy (SEM) (VHX-D510, Keyence, Japan). When the plates are folded, the joint works as a valley fold.Cell morphology and cell stainingIn order to visualize cell morphology, the cells were first fixed with 4 paraformaldehyde (PFA, Muto Pure Chemicals, Japan) for 15 min and rinsed three times with phosphate buffered saline (PBS, Sigma, USA). The cells were permeabilized with 0.1 TritonX-100 (Sigma, USA) for 2 min and rinsed three times with PBS. 1379592 In order to avoid non-specific binding, the substrate was immersed into 1 bovine serum albumin (BSA, Sigma, USA) solution for 30 minutes and rinsed once with PBS. The cells were then incubated with Alexa Fluor Phalloidin 488 conjugate (Molecular Probes; 1:200 dilution) and Hoechst 33342 (Molecular Probes; 1:400 dilution) to stain their actin filaments and nuclease with green and blue, respectively. After that the cells were rinsed three times with PBS.Cell OrigamiWe used the fluoresc.

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