Sho Yokoyama

Viscoelastic and optical properties of four different PDMS polymers

Polydimethylsiloxane (PDMS) is the most commonly used silicone elastomer with a wide range of applications including microfluidics and microcontact printing. Various types of PDMS are currently available, and their bulk material properties have been extensively investigated. However, because the properties are rarely compared in a single study, it is often unclear whether the large disparity of the reported data is attributable to the difference in methodology or to their intrinsic characteristics. Here we report on viscoelastic properties and optical properties of four different PDMS polymers, i.e. Sylgard-184, CY52-276, SIM-360, and KE-1606. Our results show that all the PDMSs are highly elastic rather than viscoelastic at the standard base/curing agent ratios, and their quantified elastic modulus, refractive index, and optical cleanness are similar but distinct in magnitude.

Microcontact peeling as a new method for cell micropatterning.

Micropatterning is becoming a powerful tool for studying morphogenetic and differentiation processes of cells. Here we describe a new micropatterning technique, which we refer to as microcontact peeling. Polydimethylsiloxane (PDMS) substrates were treated with oxygen plasma, and the resulting hydrophilic layer of the surface was locally peeled off through direct contact with a peeling stamp made of aluminum, copper, or silicon. A hydrophobic layer of PDMS could be selectively exposed only at the places of the physical contact as revealed by water contact angle measurements and angle-resolved X-ray photoelectron spectroscopy, which thus enabled successful micropatterning of cells with micro-featured peeling stamps. This new micropatterning technique needs no procedure for directly adsorbing proteins to bare PDMS in contrast to conventional techniques using a microcontact printing stamp. Given the several unique characteristics, the present technique based on the peel-off of inorganic materials may become a useful option for performing cell micropatterning.

New wrinkling substrate assay reveals traction force fields of leader and follower cells undergoing collective migration

Physical forces play crucial roles in coordinating collective migration of epithelial cells, but details of such force-related phenomena remain unclear partly due to the lack of robust methodologies to probe the underlying force fields. Here we develop a method for fabricating silicone substrates that detect cellular traction forces with a high sensitivity. Specifically, a silicone elastomer is exposed to oxygen plasma under heating. Removal of the heat shrinks the substrate so as to reduce its critical buckling strain in a spatially uniform manner. Thus, even small cellular traction forces can be visualized as micro-wrinkles that are reversibly emerged on the substrate in a direction orthogonal to the applied forces. Using this technique, we show that so-called leader cells in MDCK-II cell clusters exert significant magnitudes of traction forces distinct from those of follower cells. We reveal that the direction of traction forces is highly correlated with the long axis of the local, individual cells within clusters. These results suggest that the force fields in collective migration of MDCK-II cells are predominantly determined locally at individual cell scale rather than globally at the whole cell cluster scale.

Development of motorized plasma lithography for cell patterning

The micropatterning of cells, which restricts the adhesive regions on the substrate and thus controls cell geometry, is used to study mechanobiology-related cell functions. Plasma lithography is a means of providing such patterns and uses a spatially-selective plasma treatment. Conventional plasma lithography employs a positionally-fixed mask with which the geometry of the patterns is determined and thus is not suited for producing on-demand geometries of patterns. To overcome this, we have manufactured a new device with a motorized mask mounted in a vacuum chamber of a plasma generator, which we designate motorized plasma lithography. Our pilot tests indicate that various pattern geometries can be obtained with the control of a shielding mask during plasma treatment. Our approach can thus omit the laborious process of preparing photolithographically microfabricated masks required for the conventional plasma lithography.

Low-Power Laser Processing-Based Approach to Plasma Lithography for Cell Micropatterning

Plasma lithography is a technique to space-selectively create hydrophilic surfaces on silicone materials with oxygen plasma treatment. Cells can thus be micropatterned within the modified surfaces, allowing for artificially controlling the geometry of individual cell colonies. Conventional plasma lithography employs a photolithographically microfabricated mask with which the pattern geometry is determined. However, fast on-demand design change to the micropattern may be limited due to the time and cost associated with the sophisticated photolithographic fabrication. Here we attempted to microfabricate a mask for plasma lithography in a novel, quick, low-cost manner. An infrared absorption film was processed using a low-power Nd:YAG laser on an optical microscope to produce a mask of arbitrary pattern geometries. Our experiments indicate that plasma-shielding masks with various geometries are promptly obtained at a spatial resolution of several tens of microns with a laser power of below 200 mW. We demonstrate that cells are indeed micropatterned on functionalized silicone substrates so as to conform to the geometry of the laser-processed mask, thus suggesting the potential of this technique as a low-cost, fast on-demand means for cell micropatterning.

Microcontact Peeling: A Cell Micropatterning Technique for Circumventing Direct Adsorption of Proteins to Hydrophobic PDMS

Microcontact printing (μCPr) is one of the most popular techniques used for cell micropatterning. In conventional μCPr, a polydimethylsiloxane (PDMS) stamp with microfeatures is used to adsorb extracellular matrix (ECM) proteins onto the featured surface and transfer them onto particular areas of a cell culture substrate. However, some types of functional proteins other than ECM have been reported to denature upon direct adsorption to hydrophobic PDMS. Here we describe a detailed protocol of an alternative technique––microcontact peeling (μCPe)––that allows for cell micropatterning while circumventing the step of adsorbing proteins to bare PDMS. This technique employs microfeatured materials with a relatively high surface energy such as copper, instead of using a microfeatured PDMS stamp, to peel off a cell‐adhesive layer present on the surface of substrates. Consequently, cell‐nonadhesive substrates are exposed at the specific surface that undergoes the physical contact with the microfeatured material. Thus, although μCPe and μCPr are apparently similar, the former does not comprise a process of transferring biomolecules through hydrophobic PDMS.