Shinya Yamahira

Photocontrollable Dynamic Micropatterning of Non‐adherent Mammalian Cells Using a Photocleavable Poly(ethylene glycol) Lipid

Cell micropatterning has become an important technology for a wide variety of applications, ranging from tissue engineering and cell microarrays to fundamental studies in cell biology. In addition to conventional patterning methods, such as photolithography, soft lithography, and inkjet printing, patterning methods with dynamic substrates, in which cell adhesive properties can be changed by an external stimulus, such as heat, voltage, and light, at any desired position and any point in time, are currently the focus of many studies. These spatiotemporal patterning methods can easily construct patterns of multiple cell lines and be useful for analyzing dynamic cellular activity. In particular, in contrast to heat and voltage, light can be readily applied to anywhere in transmissive spaces with high spatial and temporal resolution, and light-induced fine control of biomolecules or living cells has been widely reported, 8] even at a single-molecular level. Therefore, cell patterning with light-responsive substrates potentially offers a practical tool for biologists. However, almost all reported cell micropatterning methods have a major limitation in target cells. In conventional methods, the adhesiveness of cells is used to attach them onto bare or ligand-coated surfaces. Therefore, the existing methods cannot be applied to nonor weakly adherent cells, which include blood cells (especially immunocytes), some cancer cells, and stem cells. These cell lines are important as research targets in biological and medical fields, and for this reason expansion of an applicable range of current micropatterning methods to non-adherent cells is an important challenge. We report herein a light-induced in situ cell micropatterning method that can be applied to non-adherent cells. Recently, we reported a cell patterning method for nonadherent cells using a cell membrane binding reagent consisting of poly(ethylene glycol) (PEG) and an oleyl group. This compound can bind to any type of cell without cytotoxicity, because the oleyl moiety can be inserted into ubiquitous lipid bilayer membranes in a noncovalent manner. In the current study, a photocleavable PEG-lipid was newly designed and synthesized for light-induced cell patterning, and then cell immobilization on the substrate coated with the photocleavable PEG-lipid was confirmed to be regulated by the dose of light exposure. Moreover, the present method allows the preparation of arbitrary and fine patterns of non-adherent cells. Furthermore, the cell micropattern on the present light-responsive substrate can be altered by light irradiation at a desired point in time. First, we designed and synthesized a photocleavable PEGlipid. In our design, a photocleavable unit was incorporated between the PEG and oleyl moieties, and at the opposite end of the PEG segment an amino-reactive ester group was added for attachment onto the substrate through an amide coupling reaction (Figure 1 a). After coating, the oleyl moieties are expected to be exposed and to anchor living cells (Figure 1b). Moreover, this molecule can be cleaved by irradiation, and then the PEG moiety is exposed at the light-irradiated area (Figure 1b). It has been reported that a PEG-coated surface inhibits cell adhesion. Therefore, cell-adhesive and nonadhesive surfaces were expected to be prepared by light irradiation (Figure 1b). A photocleavable PEG-lipid was synthesized from a commercially available o-nitrobenzyl photocleavable linker and characterized by using standard methods (see the Supporting Information). The photolytic property of the PEG-lipid in solution was confirmed by means of H NMR spectroscopy after irradiation with UV light at 365 nm (see the Supporting Information). Furthermore, substrate coating by the photocleavable PEG-lipid and its photolytic degradation on substrates were confirmed by water-drop contact-angle measurements (see the Supporting Information). Cell immobilization on a dish coated with photocleavable PEG-lipid was investigated by fluorescent microscopic observation before and after irradiating the dish with UV light (365 nm). On the nonirradiated dishes, a non-adherent cell line, the human hematopoietic cell line BaF3, was densely [*] Dr. S. Yamaguchi, Prof. T. Nagamune Department of Chemistry and Biotechnology School of Engineering, The University of Tokyo 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656 (Japan) E-mail: yamaguchi@bio.t.u-tokyo.ac.jp nagamune@bioeng.t.u-tokyo.ac.jp

Visualization of the pH-dependent dynamic distribution of G2A in living cells

G2A (from G2 accumulation) receptor is a member of the proton‐sensing G‐protein coupled receptor (GPCR) family and induces signal transduction events that regulate the cell cycle, proliferation, oncogenesis, and immunity. The mechanism by which G2A‐mediated signal transduction is regulated by the extracellular pH remains unresolved. Here, we first visualize the pH‐dependent G2A distribution change in living cells by a sortase A‐mediated pulse labeling technology: the short‐peptide tag‐fused human G2A on human embryo kidney HEK293T cell surfaces was labeled with a small fluorescent dye in the presence of lysophosphatidylcholine, and the labeled G2A was chased at acidic and neutral pHs in real time by microscope time course observations. G2A internalization from cell surfaces into intracellular compartments was observed to be inhibited under acidic pH conditions, and this inhibition was relieved at neutral pH. Additionally, the internalized G2A was redistributed onto cell surfaces by jumping from a neutral to an acidic pH. From quantitative image analysis data, we conclude the amount of G2A on the cell surface was controlled by suppressing the G2A internalization rate by one‐tenth in response to the extracellular acidic pH, and this acidic pH‐induced G2A accumulation on cell surfaces may be explained by proton‐induced dissociation of G2A from endocytic machinery.—Lan, W., Yamaguchi, S., Yamamoto, T., Yamahira, S., Tan, M., Murakami, N., Zhang, J., Nakamura, M., Nagamune, T. Visualization of the pH‐dependent dynamic distribution of G2A in living cells. FASEB J. 28, 3965‐3974 (2014). www.fasebj.org

Collagen Surfaces Modified with Photo-Cleavable Polyethylene Glycol-Lipid Support Versatile Single-Cell Arrays of Both Non-adherent and Adherent Cells

Cell patterning on photo-responsive materials are a promising tool for preparing unique single-cell arrays. However, most conventional single-cell arrays on such smart materials can be applied only to adherent cells and limit cellular functions such as extension and migration within the patterned adhesive surfaces. In this study, a versatile single cell array that works with both non-adherent and adherent cells was constructed using a photo-cleavable polyethylene glycol (PEG)-lipid/collagen surface. On this single-cell array, cells behaved similar to their native functions without limitation from the patterned surface. Furthermore, quantitative imaging analyses of cellular motility and morphological changes were performed in a high-throughput manner.

Photosensitizer and polycationic peptide-labeled streptavidin as a nano-carrier for light-controlled protein transduction

Transductions of exogenous proteins into cells enable the precise study of the effect of the transduced proteins on cellular functions. Accordingly, the protein transduction technique, which can control the release of proteins into the cytosol with certainty and high-throughput, is highly desired in various research fields. In this study, streptavidin (SA) labeled with a photosensitizer and cell-permeable peptides (CPP) was proposed as a nano-carrier for light-controlled protein transduction. SA was modified with biotinylated oligo-arginine peptides (Rpep), which were functionalized with Alexa Fluor 546 (AF546), to achieve cell penetrating and endosomal escape functionalities. The SA-Rpep complex was efficiently internalized into living HeLa cells corresponding to the length and the modification number of Rpep. SA conjugated with more than three equimolar AF546-modified Rpep consisting of fifteen arginine residues was achieved to diffuse throughout the cytosol without cytotoxicity by irradiation of the excitation light for AF546. The optimized nano-carrier was confirmed to transduce a biotinylated model cargo protein, enhanced green fluorescent protein fused with thioredoxin (tEGFP) into the cytosol at the light-irradiated area. The results provided proof-of-principle that SA possessing multiple AF546-modified Rpep has the potential to be a versatile and facile carrier for light-controlled protein transduction into the cytosol of mammalian cells.

Cell-Based Odorant Sensor Array for Odor Discrimination Based on Insect Odorant Receptors

The olfactory system of living organisms can accurately discriminate numerous odors by recognizing the pattern of activation of several odorant receptors (ORs). Thus, development of an odorant sensor array based on multiple ORs presents the possibility of mimicking biological odor discrimination mechanisms. Recently, we developed novel odorant sensor elements with high sensitivity and selectivity based on insect OR-expressing Sf21 cells that respond to target odorants by displaying increased fluorescence intensity. Here we introduce the development of an odorant sensor array composed of several Sf21 cell lines expressing different ORs. In this study, an array pattern of four cell lines expressing Or13a, Or56a, BmOR1, and BmOR3 was successfully created using a patterned polydimethylsiloxane film template and cell-immobilizing reagents, termed biocompatible anchor for membrane (BAM). We demonstrated that BAM could create a clear pattern of Sf21 sensor cells without impacting their odorant-sensing performance. Our sensor array showed odorant-specific response patterns toward both odorant mixtures and single odorant stimuli, allowing us to visualize the presence of 1-octen-3-ol, geosmin, bombykol, and bombykal as an increased fluorescence intensity in the region of Or13a, Or56a, BmOR1, and BmOR3 cell lines, respectively. Therefore, we successfully developed a new methodology for creating a cell-based odorant sensor array that enables us to discriminate multiple target odorants. Our method might be expanded into the development of an odorant sensor capable of detecting a large range of environmental odorants that might become a promising tool used in various applications including the study of insect semiochemicals and food contamination.

Transfer printing of transfected cell microarrays from poly(ethylene glycol)‐oleyl surfaces onto biological hydrogels

We have developed a novel technique for constructing microarrays of transfected mammalian cells on or in extracellular matrix (ECM) hydrogels by transfer printing from patterned poly(ethylene glycol) (PEG)-oleyl surfaces. A mixed solution of small interfering RNA (siRNA) and a transfection reagent was spotted on PEG-oleyl-coated glass slides using an ink-jet printer, and the cells were then transiently immobilized on the patterned transfection mixtures. After overlaying an ECM hydrogel sheet onto the immobilized cells, the cells sandwiched between the glass slide and the hydrogel sheet were incubated at 37°C for simultaneous transfection of siRNA into cells and adhesion of cells to the hydrogel sheet. Transfer of the adhered, transfected cells was completed by peeling off the hydrogel sheet. The knockdown of a model gene in the transferred cell microarray by the transfected siRNA was successfully confirmed. Transfected cell microarrays were also embedded within three-dimensional ECM hydrogels. In the three-dimensional hydrogel, the inhibition effect of siRNA on cancer cell invasion was evaluated by quantifying the size of cell clusters on the microarrays. These results indicate that transfection of cell microarrays on or in a biological matrix is a promising technique for high-throughput screening of disease-related genes by direct observation of cellular phenomena in a physiologically relevant environment.

Selective intracellular vaporisation of antibody-conjugated phase-change nano-droplets in vitro

While chemotherapy is a major mode of cancer therapeutics, its efficacy is limited by systemic toxicities and drug resistance. Recent advances in nanomedicine provide the opportunity to reduce systemic toxicities. However, drug resistance remains a major challenge in cancer treatment research. Here we developed a nanomedicine composed of a phase-change nano-droplet (PCND) and an anti-cancer antibody (9E5), proposing the concept of ultrasound cancer therapy with intracellular vaporisation. PCND is a liquid perfluorocarbon nanoparticle with a liquid–gas phase that is transformable upon exposure to ultrasound. 9E5 is a monoclonal antibody targeting epiregulin (EREG). We found that 9E5-conjugated PCNDs are selectively internalised into targeted cancer cells and kill the cells dynamically by ultrasound-induced intracellular vaporisation. In vitro experiments show that 9E5-conjugated PCND targets 97.8% of high-EREG-expressing cancer cells and kills 57% of those targeted upon exposure to ultrasound. Furthermore, direct observation of the intracellular vaporisation process revealed the significant morphological alterations of cells and the release of intracellular contents.

Photolytic Protein Aggregates: Versatile Materials for Controlled Release of Active Proteins

Photolytic protein aggregates are developed as a facile and versatile platform for light-induced release of active proteins. The proteins modified with biotin through a photo-cleavable linker rapidly form aggregates with streptavidin and biotinylated functional molecules simply by mixing. Light irradiation releases active proteins from the aggregates in high yields, and light-induced uptake of drug-modified transferrin into living cells is successfully demonstrated.

Dynamic photochemical lipid micropatterning for manipulation of nonadherent mammalian cells.

Cell micropatterning methods with stimuli-responsive dynamic surfaces are getting a lot of attention in a wide variety of research fields, ranging from cell engineering to fundamental studies in cell biology. The surface of a slide coated with photo-cleavable poly(ethylene glycol) (PEG)-lipid can be used to spatiotemporally control cell immobilization and release by light irradiation. On the basis of this surface, it is easy to design simple methods for making a fine micropattern of any kind of cell. Furthermore, target cells can be selectively and rapidly released from this surface by light irradiation. In this review, we first describe how to obtain the photo-cleavable PEG-lipid from commercially available compounds through a facile four-step synthesis. Next, as a cell-patterning method, the protocols of coating substrates with the PEG-lipid, irradiating a pattern of light onto the coated substrate, and loading cells onto the irradiated surface are described. These protocols require no expensive equipment and potentially apply to any substrates that can adsorb serum albumin or chemically expose amine moieties on their surfaces. Finally, as an advanced method, cell release from the PEG-lipid surface in microfluidic devices is introduced. We also discuss the advantages and the possible applications of the present dynamic cell-patterning method.

Stepwise phosphorylation of leukotriene B4 receptor 1 defines cellular responses to leukotriene B4

Sequential phosphorylation of the leukotriene B4 receptor determines specific cellular responses to low and high concentrations of LTB4. Phosphorylation order matters The inflammatory lipid LTB4 stimulates immune cells to migrate toward sites of inflammation and then degranulate. Like other G protein–coupled receptors, the LTB4 receptor BLT1 becomes phosphorylated after ligand-induced activation. Nakanishi et al. identified five residues in the cytoplasmic C-terminal domain of BLT1 that were variably phosphorylated in the absence of LTB4 and two that were phosphorylated upon stimulation with LTB4. Phosphorylation at the LTB4-dependent sites stimulated additional phosphorylation events at the basal phosphorylation sites. The sequence of these phosphorylation events depended on the concentration of LTB4 and was required for chemotaxis and degranulation in response to high concentrations of LTB4. These findings suggest a mechanism whereby increasing concentrations of LTB4 induce distinct responses as cells migrate up an LTB4 gradient. Leukotriene B4 (LTB4) receptor type 1 (BLT1) is abundant in phagocytic and immune cells and plays crucial roles in various inflammatory diseases. BLT1 is phosphorylated at several serine and threonine residues upon stimulation with the inflammatory lipid LTB4. Using Phos-tag gel electrophoresis to separate differentially phosphorylated forms of BLT1, we identified two distinct types of phosphorylation, basal and ligand-induced, in the carboxyl terminus of human BLT1. In the absence of LTB4, the basal phosphorylation sites were modified to various degrees, giving rise to many different phosphorylated forms of BLT1. Different concentrations of LTB4 induced distinct phosphorylation events, and these ligand-induced modifications facilitated additional phosphorylation events at the basal phosphorylation sites. Because neutrophils migrate toward inflammatory sites along a gradient of LTB4, the degree of BLT1 phosphorylation likely increases in parallel with the increase in LTB4 concentration as the cells migrate. At high concentrations of LTB4, deficiencies in these two types of phosphorylation events impaired chemotaxis and β-hexosaminidase release, a proxy for degranulation, in Chinese hamster ovary (CHO-K1) and rat basophilic leukemia (RBL-2H3) cells, respectively. These results suggest that an LTB4 gradient around inflammatory sites enhances BLT1 phosphorylation in a stepwise manner to facilitate the precise migration of phagocytic and immune cells and the initiation of local responses, including degranulation.