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Human bone marrow mesenchymal stem cells (hMSCs) present a promising cell source with the potential to be used for curing various intractable diseases, and it is expected that the development of regenerative medicine employing cell-based therapy would be significantly accelerated when such methods are established. For that, powerful methods for selective growth and differentiation of hMSCs should be developed.We developed an efficient method for hMSC proliferation and differentiation into osteoblasts and adipocytes using gravity-controlled environments.The results indicate that the average doubling time of hMSCs cultured in a regular maintenance medium under microgravity conditions (0.001 G) was 1.5 times shorter than that of cells cultured under natural gravity conditions (1.0 G). Furthermore, 99.2% of cells grown in the microgravity environment showed the expression of hMSC markers, as indicated by flow cytometry analysis. Osteogenic and adipogenic differentiation of hMSCs expanded in the microgravity environment was enhanced under microgravity and hypergravity conditions, respectively, as evidenced by the downregulation of hMSC markers and upregulation of osteoblast and adipocyte markers, respectively. Most cells differentiated into osteoblasts in the microgravity environment after 14 days (~80%) and adipocytes in the hypergravity environment after 12 days (~90%).Our results indicate that hMSC proliferation and selective differentiation into specific cell lineages could be promoted under microgravity or hypergravity conditions, suggesting that cell culture in the gravity-controlled environment is a useful method to obtain cell preparations for potential clinical applications.

Patterning of a surface modified with the copolymer of ω-methoxy-(ethylene glycol)8 acrylamide and 3-methacryloxypropyltrimethoxysilane by UV irradiation and the creation of a heterologous cell patterning surface.

In order to develop new three-dimensional (3D) cell culture systems for articular cartilage regeneration, concentrated poly(styrene sulfonate sodium salt) brush-modified cellulose nanofibers were employed as building blocks for the self-assembly of human mesenchymal stem cells (hMSCs). Unique 3D cellular structures, such as giant spheres and sheets, were formed by controlling hMSC self-assembly.

We report a next-generation, biocide-free, and durable marine antifouling coating technology. To achieve this, we combined two different polymers previously developed by us. First, we synthesized well-defined 2-hydroxypropyl acrylamide (HPA) based bottlebrush polymers with concentrated polymer brush (CPB) structures, which exhibit excellent bioinertness, and second, we synthesized photoreactive copolymers of 2-hydroxypropyl acrylamide (HPA) and

Quantum dots (QDs) can be delivered efficiently inside macrophages using a freeze-concentration approach. In this study, we introduced a new, facile, high concentration-based freezing technology of low toxicity. We also developed QD-conjugated new hydrophobic polyampholytes using poly-L-lysine (PLL), a naturally derived polymer, which showed sustained biocompatibility, stability over one week, and enhanced intracellular delivery. When freeze-concentration was applied, the QD-encapsulated hydrophobic polyampholytes showed a higher tendency to adsorb onto the cell membrane than the non-frozen molecules. Interestingly, we observed that the efficacy of adsorption of QDs on RAW 264.7 macrophages was higher than that on fibroblasts. Furthermore, the intracellular delivery of QDs using hydrophobic polyampholytes was higher than those of PLL and QDs. In vitro studies revealed the efficient endosomal escape of QDs in the presence of hydrophobic polyampholytes and freeze-concentration. Collectively, these observations indicated that the promising combination of freeze-concentration and hydrophobic polyampholytes may act as an effective and versatile strategy for the intracellular delivery of QDs, which can be used for biological diagnosis and therapeutic applications.

This study was conducted to develop biodegradable cellulose scaffolds by oxidising porous cellulose sponges for tissue engineering applications. Cellulose powder was dissolved in ionic liquid using a salt leaching method, and porous cellulose scaffolds of various pore sizes were prepared. The scaffolds were oxidised with periodate to introduce aldehyde at a rate controlled by the periodate concentration. Oxidised scaffolds exhibited weight loss in cell culture medium, but not in phosphate buffer. Therefore, we confirmed that Schiff base formation between the aldehyde and amino groups through a Maillard reaction triggered cellulose molecular degradation. The degradation rate was controlled by the oxidation degree, whereas the aldehyde content controlled protein adsorption and cell proliferation. Additionally, in vivo implantation tests revealed that optimising the oxidation ratio not only improved biodegradability but also reduced inflammation. In conclusion, our results suggest that simple oxidised cellulose is useful as a low-toxicity biodegradable scaffold.

Wound healing is a complex process involving an intricate cascade of body responses. A composite dressing that would effectively target different stages of wound healing and regeneration is urgently needed. In the current study, we tested the efficacy of a previously prepared micelle-hydrogel composite loaded with two drugs, in full-thickness excision wound model in rat. We found that the composite elicited almost no inflammation and effectively enhanced healing at all stages of the healing process. An initial burst of the first drug, amphotericin B, eliminated any preliminary infection. This burst was followed by a gradual release of curcumin as the healing and anti-inflammatory agent. Better healing was observed in rats treated with the drug-loaded composites than in blank and control groups. Wounds showed up to 80% closure in the treated group, with high collagen deposition. Re-epithelialization and granulation were also better in the treated group than in the non-treated control and blank groups. Histopathological examination revealed that drug-loaded composites improved cutaneous wound healing and regeneration. In conclusion, the micelle-hydrogel composite is an effective dressing and might have major applications in wound healing. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1094-1106, 2019.

A novel surface modification method was developed using a polymer brush and a semi-interpenetrating polymer network (semi-IPN). First, a temperature-responsive copolymer brush of methoxydiethyleneglycol methacrylate and 2-hydroxyethyl methacrylate (P(MDM-co-HEMA)) was constructed on a substrate by surface-initiated atom transfer radical polymerization. The HEMA residues in the grafted chains of the brushes were cross-linked by hexamethylenediisocyanate. Then, functional polymers—zwitterionic, cationic, and anionic polymers used in this study as a model case—were synthesized in the presence of the cross-linked polymer brush-modified substrate, which resulted in the semi-IPN. The thin gel layer having functional polymers as a component of the semi-IPN imparted high elasticity (15–25 MPa) as compared to simple polymer brushes (1 MPa) and specific abilities derived from the semi-IPN polymer. In particular, the adsorption and adhesion of proteins and cells, respectively, to the surface of the thin gel layer incorporating the zwitterionic polymer were drastically suppressed. In contrast, proteins and cells significantly adsorbed and adhered to semi-IPN layers having cationic and anionic polymers. Furthermore, the thin gel layer had switching function because P(MDM-co-HEMA) having lower critical solution temperature was used as the brush component. When the surface density of the brushes was decreased (0.03 chains nm−2), the thin gel layer surface interpenetrated with a zwitterionic polymer clearly demonstrated the switching of cell adhesion according to the temperature: cells were detached from the surface when the temperature was changed from 37 to 15 °C. High elasticity, specific function derived from the interpenetrating polymer, durability, and thermo-responsiveness of the thin gel layer would be applicable in biomedical and environmental fields.

The provision of adhesive scaffolding and protection from inflammatory responses are important for enhancing graft survival. We previously developed a functional hydrogel that significantly enhances the survival of cells transplanted into the midbrain striatum. Although graft survival reached approximately 40% using this hydrogel, the survival of transplanted cells required further enhancements because it ultimately produced a decrease in the number of transplanted cells. Therefore, we developed a hydrogel system that can locally prevent the inflammatory response. This hydrogel was modified by the addition of the interleukin 10 chimeric protein (IL10CP), which is selectively released from the hydrogel when triggered by an inflammatory response. This design protects transplanted cells from inflammatory response, while other host cells remain unaffected. The IL10 domains are selectively released from the hydrogel, which act locally on the immune cells to prevent the inflammatory response without the administration of an immune suppressor. The selective release of IL10 domains from the hydrogel and their activity to prevent immune responses were evaluated using various approaches. Moreover, the ability of the IL10CP-modified hydrogel to protect cells was investigated using an in vitro co-culture with activated microglia. The IL10 incorporated into the hydrogel was selectively released by the activity of matrix-metalloproteinase 9 (MMP9), and the neural progenitor cells encapsulated in the IL10CP-immobilized hydrogel were protected from activated microglia by the release of IL10s from the hydrogel by the MMP9, produced by the activated microglia. These results show that the IL10CP-modified hydrogel will be useful as a biomaterial for improving the survival of transplanted cells.

A novel type of shape memory polyurethane (SMPU) with high mechanical properties and biodegradability was constructed using a lactone copolymer (poly(e-caprolactone-co-γ-butyrolactone), PCLBL), a diol- or triol-based chain extender (1,5-pentanediol, glycerol and 2-amino-2-hydroxymethyl-1,3-propanediol) and a diisocyanate cross-linker (1,6-hexamethylene diisocyanate). All types of SMPUs possessed high mechanical properties, and the shape recovery test indicated that the SMPU sheets prepared using a triol-chain extender with an amine group recovered completely the original shape at 80 °C. Moreover, the degradation products of the SMPUs were innoxious, which is an important property for use in the biomedical field. Furthermore, the SMPU sheets were interpenetrated with a zwitterionic polymer, poly(carboxymethyl betaine) (PCMB), using the interpenetrating polymer network (IPN) method to additionally introduce an anti-biofouling property. Water contact angle measurements of the surface of PCMB-introduced SMPU sheets showed a drastic reduction from 87° to approximately 30° due to the exposure of the PCMB chains from the SMPU sheets. These SMPU-IPN sheets suppressed significantly both protein adsorption and cell adhesion. Consequently, the PCLBL-PU-based SMPUs interpenetrated with PCMB are promising materials for biomedical devices because of their high mechanical, shape memory, biodegradable, and anti-biofouling properties. These materials are expected to be applied to biomaterials such as embolization materials for aneurysms and a novel type of membrane for postoperative adhesion prevention.

Circulating tumor cells (CTCs) are tumor cells present in the blood. CTCs have attracted much attention as a new tumor marker, because their analysis provides useful information for monitoring cancer progress. In this study, we developed cell-capture and release methods using three-dimensional (3D) microfiber fabrics without damaging the cells. Using functional peptides containing sequences from a polystyrene-binding site and a cleavable site for collagenase type IV, immobilized antibodies on the peptides were able to specifically capture MCF-7 cells in a few minutes and release the captured cells from 3D microfiber fabrics incorporating a vacuum system. The efficiency of cell capture was around 80% and that of the cell release was over 90%. The released cells proliferated normally in culture medium, suggesting that our system will be applicable for the culture and analysis of CTCs. Statement of Significance In this paper, we report cell-capture and release methods using enzyme-cleavable peptides immobilized on microfiber fabrics which has microporous polymeric three-dimensional structures. Detachment and collection of the selectively captured cancer cells are required for ex vivo culture and their further analysis, whereas the cell detachment methods developed so far might cause cell damage, even if cell viability is high enough. Therefore, specific attachment and gentle detachment from the device are required for the accurate analysis of cells. In this study, for capture and release of cancer cells we designed the peptide cleavable by collagenase type IV, which has no target molecule in cells. Our system will be useful for further CTC analysis and might lead to more accurate cancer diagnosis.

A mixed self-assembled monolayer (SAM) of an initiator (3-(2-bromo-2-isobutyryloxy)propyl triethoxysilane) for atom transfer radical polymerization (ATRP) and an agent (6-(triethoxysilyl)hexyl 2-(((methylthio)carbonothioyl)thio)-2-phenylacetate) for reversible addition-fragmentation chain transfer (RAFT) polymerization was constructed on the surface of a silicon wafer or glass plate by a silane coupling reaction. When a UV light at 254nm was irradiated at the mixed SAM through a photomask, the surface density of the bromine atom at the end of BPE in the irradiated region was drastically reduced by UV-driven scission of the BrC bond, as observed by X-ray photoelectron spectroscopy. Consequently, the surface-initiated (SI)-ATRP of 2-ethylhexyl methacrylate (EHMA) was used to easily construct the poly(EHMA) (PEHMA) brush domain. Subsequently, SI-RAFT polymerization of a zwitterionic vinyl monomer, carboxymethyl betaine (CMB), was performed. Using the sequential polymerization, the PCMB and PEHMA brush domains on the solid substrate could be very easily patterned. Patterning proteins and cells with the binary polymer brush is expected because the PCMB brush indicated strong suppression of protein adsorption and cell adhesion, and the PEHMA brush had non-polar properties. This technique is very simple and useful for regulating the shape and size of bio-fouling and anti-biofouling domains on solid surfaces.

Physical methodologies such as electroporation and the gene-gun technology have been widely used for transfection; however, their applicability is limited because they lead to cell damage and low cell viability. Therefore, to address these limitations we developed a new freeze concentration-based gene transfection system that provides enhanced in vitro gene delivery compared to that provided by the commercially available systems. The system employs a facile freeze concentration step, whereby cells are simply frozen to very low temperatures in the presence of polymer-pDNA complexes. As part of system development, we also synthesized a low toxicity polyethyleneimine (PEI)-based polyampholyte prepared through succinylation with butylsuccinic anhydride. In aqueous solution, this modified polyampholyte self-assembles to form small (20 nm diameter), positively charged (net surface charge of 35 mV), nanoparticles through a combination of hydrophobic and electrostatic interactions. Agarose gel electrophoresis analysis indicated that the polyampholyte nanoparticle was able to form a complex with pDNA that provided stability against nuclease degradation. Using transfection of HEK-293T cells, we demonstrated that a combination of polyampholyte: pDNA, at an appropriate ratio, and the freeze concentration method resulted in significant enhancement of GFP and luciferase expression compared to commercially available carriers. Endosomal escape of pDNA was also found to be increased when using the modified polyampholyte compared to branched PEI. This study suggests that the efficient combination of freeze concentration and the modified polyampholyte described here has great potential for in vitro gene therapy.

Contact lens users very often become patients of allergic conjunctivitis, which is caused by protein and bacteria adsorption to the eye, because contact lenses easily adsorb proteins and bacteria. However, even if contact lens users develop eye diseases such as allergic conjunctivitis, most of them continue to use contact lenses to avoid interference to daily life or a decrease in their quality of life. If novel contact lenses able to prevent and additionally cure eye diseases can be manufactured, they could improve the quality of life of contact lens users worldwide. Thus, we aim to develop a novel material for contact lenses to prevent diseases by incorporating a zwitterionic polymer with the ability to suppress protein and bacteria adsorption. In addition, we also aim to effectively introduce and release a drug against allergic conjunctivitis from the contact lens material. Because the poorly water-soluble drug for allergic conjunctivitis (pranoprofen) forms a rigid crystal structure, we developed the novel “hot-melt press method” to construct a contact lens able to effectively release it. In the present study, polymer sheets containing carboxymethyl betaine (a kind of zwitterionic monomer), 2-hydroxyethyl methacrylate, and 1-vinyl-2-pyrrolidone were prepared using three different procedures. The sheets were hydrophilic and showed a strong resistance against protein and bacteria adsorption. The sheets prepared by the hot-melt press method were transparent and seemed to have potential as a material for contact lenses. In addition, the drug introduced into the sheets during preparation was observed to release at a practically appropriate dose. Therefore, it is expected that the sheets could possibly be used as a material for contact lenses which not only protect against the development of eye trouble due to protein and bacterial adsorption, but also heal allergic conjunctivitis.

A self-assembled monolayer (SAM) of a 2-bromoisobutyryl end group-carrying initiator for atom transfer radical polymerization (ATRP) was constructed on the surface of silicon wafer or glass substrates via a silane-coupling reaction. When the initiator SAM was irradiated with UV light at 254nm, the surface density of bromine atoms was reduced by the scission of CBr bonds as observed by XPS. With the surface-initiated ATRP of the zwitterionic vinyl monomer, carboxymethyl betaine (CMB), the surface density of PCMB brushes could be easily varied by changing the irradiation period of UV light prior to the polymerization. Furthermore, by using a UV-cut shutter sliding above the initiator SAM-modified substrate at a constant speed, the degree of bromine atom removal could be linearly varied along the direction of movement of the shutter. Consequently, the amount of both proteins adsorbed and cells adhered to the PCMB brush-covered substrate could easily be controlled by the gradation of the surface density of PCMB brushes, which suppressed protein adsorption and cell adhesion. Such a technique is very simple and useful for the regulation of the surface density of adsorbed proteins and adhered cells on an originally bio-inert surface.

Random copolymers (S-PCMBx) of the zwitterionic monomer carboxymethylbetaine (CMB) and a small percentage of 3-methacryloyloxypropyl trimethoxysilane with various composition ratios were synthesized in ethanol using 2,2'-azobisisobutyronitrile as the initiator. An S-PCMBx layer formed on the glass substrate after soaking in the copolymer solution and had a thickness of 2-3 nm. The S-PCMBx-modified surface was highly hydrophilic and suppressed both the non-specific adsorption of protein (bovine serum albumin) and NIH3T3-fibroblast adhesion. The degree of adsorption suppression increased with increasing copolymer CMB content with a maximum at 90 mol % CMB. In contrast, the modification of the glass substrate with a PCMB homopolymer terminally modified with a trimethoxysilyl group did not effectively suppress protein adsorption and cell adhesion due to the low graft density. The importance of balancing the number of fixation points and the length of the zwitterionic polymer loops to produce bio-inactive metal oxide surfaces is suggested. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2029-2036, 2016.

A polymer film composed of a mixture of chitosan (Ch) and carboxymethyl cellulose sodium salt (CMC) nanofibers was deposited on a glass surface. The thin film of the Ch–CMC mixture obtained was stable, and fibroblast adhesion to the film was lowest when the weight ratio of Ch to CMC was 4:6. The ζ-potential and contact angle of the mixture film indicated that a polyion complex of Ch and CMC was formed. The mechanical strength of the film composed of Ch–CMC nanofiber complexes was much higher than that of the film composed of Ch–water-soluble CMC complexes (non-nanofiber), likely because the entanglement of nanofibers was enhanced by electrostatic attractions. These results indicate that the charge-neutralized nanofiber film was highly effective in suppressing cell adhesion and therefore is a promising material for biomedical applications.

The effective control of biointerfacial interactions is of outstanding interest in a broad range of biomedical applications, ranging from cell culture tools to biosensors and implantable medical devices. For many of these applications, highly specific interactions between cells and material surfaces are desired. Sophisticated control over these interactions requires reducing or preventing non-specific interactions on the one hand and displaying highly specific signals that can be recognized by extracellular receptors on the other. We have recently developed ultra-low fouling coatings that can be applied in a single step using photoreactive copolymers of 2-hydroxypropyl acrylamide and N-benzophenone acrylamide. Here, we have expanded this approach by incorporating polymerizable peptide monomers into these copolymers. The monomers QQGWFGAGK(acrylamide) and acrylamide-GAGQQGWF were synthesized after identifying the QQGWF sequence as a binding motif for CD44 by phage display for the first time. Our results demonstrate that UV-crosslinked coatings fabricated using the QQGWFGAGK(acrylamide) monomer are effective at selectively binding hMSC in the presence of HepG2 and HEK293 cells due to the difference in CD44 expression. Our results also demonstrate that the peptide modified coatings retain their low biofouling character using a BCA protein binding assay as well as an E. coli bacterial attachment assay over a 24 h period. Our approach provides an alternative to traditional integrin-mediated selective cell binding on surfaces and opens the door to new diagnostic applications, exploiting the fact that the transmembrane protein CD44 is highly expressed in multiple diseases.

Various techniques and systems have been reported for the efficient differentiation of neural stem/progenitor cells into dopaminergic neurons. Although a comparatively high percentage of dopaminergic neurons can be obtained using these techniques, the differentiated cells display varied cellular phenotypes such as astrocytes and oligodendrocytes. Generation of highly pure dopaminergic neurons is important for cell-based therapy and in vitro evaluation of dopaminergic neuron function. In this study, we developed a culture surface anchored with several neurotrophic factors and a neuronal cell-adhesive protein for efficient differentiation of neural stem/progenitor cells into dopaminergic neurons. Oligohistidine-fused brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor, synthesized using genetic engineering, were co-immobilized on the surface via metal chelation. To facilitate cell adhesion, a cell-adhesive chimeric protein derived from laminin (LN-G) was also immobilized on the surface. Approximately 40% of the cells cultured for 14 days with these protein-immobilized substrates expressed tyrosine hydroxylase, a marker of dopaminergic neurons, with a three-fold increase in differentiation efficiency than that reported previously. In addition, the number of tyrosine hydroxylase-positive cells increased to approximately 80% of the culture after 30 days. These cells secreted dopamine and expressed dopaminergic neuron-specific genes. Interestingly, cell types (glial cells and oligodendrocytes) other than neuronal cells (immature and mature dopaminergic neurons) were not detected on the protein-anchored surface. Our results demonstrate that highly pure dopaminergic neurons can be exclusively obtained using the novel substrate without extra purification steps such as cell sorting. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 860-871, 2019.

Biomaterials modified with proteins such as growth and trophic factors are known to precisely regulate various cell and tissue functions. However, the mechanisms for regulation with proteins anchored to a substrate have not been extensively studied. Although we previously evaluated specific signal transduction from epidermal growth factor (EGF) anchored to a substrate to neural stem/progenitor cells (NSPCs), the internalization of immobilized-EGF and the continuity of signaling transduction were not discussed in detail. This information is important to determine the value of growth factor-anchored biomaterials in the regulation of cells. Here, we tried to clarify the mechanisms underlying immobilized-growth factor in NSPC regulation using approaches from materials science and cell biology. In this evaluation, we used EGF chimeric protein (EGF-His) and NSPCs, and found that EGF anchored to a substrate facilitated continuous signal transduction in NSPCs attached to the substrate. In addition, the anchored-EGFs were finally internalized into cells only when the proteins formed a complex with their receptors on cell membranes detached from the substrate. Finally, we concluded that continuous signal transduction by anchoring to the substrate and final internalization into cells with the detachment of anchored-proteins from a substrate are important events for efficient regulation of cell function.

A silane coupling agent having a zwitterionic end group was covalently bound to a semi-cylindrical fused silica prism for sum-frequency generation (SFG) analyses and to a flat glass for estimating biological affinity. It was found that total intensity of the -derived from water in contact with a positively or negatively charged SAM-modified surface and a bare silica prism. These results indicated that water molecules in the vicinity of the zwitterionic SAM-modified surface are not strongly oriented in comparison with those of lopsidedly charged SAMs and bare silica. Moreover, the zwitterionic SAM surface suppressed non-specific adsorption of bovine serum albumin in contrast to the significant adsorption to lopsidedly charged SAMs and the bare cover glass. On the other hand, fibroblasts gradually adhered to the SAM surfaces and extended regardless of the electrical charge of the SAM, though the number of cells that adhered to the zwitterionic SAM was the smallest. The results strongly suggested that the charge neutralization of a solid material surface is very important for anti-biofouling properties.

Neural progenitor cells derived from induced pluripotent stem cells have been considered as a potential source for cell-transplantation therapy of central nervous disorders. However, efficient methods to expand neural progenitor cells are further required for their clinical applications. In this study, a protein array was fabricated with nine extracellular matrices and used to screen substrates suitable for the expansion of neural progenitor cells derived from mouse induced pluripotent stem cells. The results showed that neural progenitor cells efficiently proliferated on substrates with immobilized laminin-1, laminin-5, or Matrigel. Based on this result, further attempts were made to develop clinically compliant substrates with immobilized polypeptides that mimic laminin-1, one of the most effective extracellular matrices as identified in the array-based screening. We used here recombinant DNA technology to prepare polypeptide containing the globular domain 3 of laminin-1 and immobilized it onto glass-based substrates. Our results showed that neural progenitor cells selectively proliferated on substrate with the immobilized polypeptide while maintaining their differentiated state. Biotechnol. Bioeng. 2015;112: 2388–2396. © 2015 Wiley Periodicals, Inc.

Hydrophilic epoxy resin-based monoliths were employed as cell culture substrates. The monoliths were made of a porous material with a bicontinuous structure that consisted of a porous channel and a resin skeleton. Monolith disks were prepared with a skinless surface through polymerization-induced spinodal decomposition-type phase separation. The pore sizes, which were well controlled by the polymerization temperature, ranged from 70 to 380 nm. The quantity of protein adsorbed per unit area and the early-stage adhesion of HepG2 cells on the monolith substrates were independent of pore size, meaning they were not affected by surface topology. Long-term cell adhesion, as indicated by adherent cell number and shape, as well as liver-specific gene expression were significantly affected by pore size. In terms of cell shape, number, and gene expression, pores of approximately 200 nm were most suitable for HepG2 cell growth. These results highlight the importance of monolith morphology for use as a cell culture substrate. The well-controlled morphology demonstrated in this work indicates monoliths are capable of supporting growth for various types of cells in a range of applications.

A lot of research has been carried out in the last decade to find a cure for neurodegenerative diseases especially Parkinson's disease but to little avail. In this study we have demonstrated the use of poly(lactic-co-glycolic acid) (PLGA)/collagen biodegradable microparticles formed using water-in-oil-in-water (W/O/W) double emulsion method, as a neurotrophic factor delivery vehicle. The microparticles were encapsulated with glial cell-derived neurotrophic factor (GDNF) fused with collagen binding peptide (CBP) immobilized to the inner collagen phase. The novelty lies in the strict regulation of release of GDNF-CBP from the microparticles as compared to a burst release from standard microparticles. The microparticles were demonstrated to be non-cytotoxic till 300 μg/2 × 10⁵ cells and revealed a maximum release of 250 ng GDNF-CBP/mg microparticles in 0.3% collagenase. Differentiation of neural progenitor cells (NPCs) into mature neurons was demonstrated by co-culturing microparticles with cells in a medium containing collagenase which enabled the release of encapsulated GDNF-CBP, signaling the differentiation of NPCs into microtubule-associated protein 2 (MAP2)-expressing neurons. The successful ability of these microparticles to deliver neurotrophic factors and allow differentiation of NPCs into mature neurons provides some scope in its use for the treatment of Parkinson's disease and other neurodegenerative diseases.

The surface of a titanium (Ti) alloy was modified with a self-assembled monolayer of poly(ethylene glycol) methacrylate phosphate (Phosmer PE). A zwitterionic monomer (carboxymethyl betaine, CMB) could be copolymerized with the surface-bound Phosmer PE due to a flexible linker between the Ti alloy surface and a methacryloyl group of Phosmer PE. The poly(CMB) (PCMB)-modified Ti alloy plate exhibited strong suppression of protein adsorption and cell adhesion, and induced approximately twice the amount of calcium (Ca2+) deposition as compared to the unmodified Ti alloy plate. The zwitterionic polymer-modified surfaces not only showed enhanced mineralization clusters creation and growth, but they were also highly non-responsive to biologically derived materials such as proteins and cells. Therefore, it is possible to easily form highly pure and rigid hydroxyapatite layers on Ti alloy surfaces without the incorporation of organic molecules such as proteins. The present surface modification technique and strategy can be applied to implantable orthopedic materials as a means of encouraging integration with host tissues, such as the thigh bone.

To develop biomaterials that serve to improve the survival of neural cells transplanted into central nervous tissues, type I collagen-based hydrogels were prepared as a cell carrier. The hydrogels were modified with a laminin-derived peptide that is known to have an affinity for alpha3beta1 integrin, to transduce antiapoptotic signaling in embedded cells. For the modification of collagen, the peptide was fused to the N- or C-terminus, or both termini of a collagen-binding polypeptide domain by means of recombinant DNA technology. The chimeric proteins were characterized by polyacrylamide gel electrophoresis and circular dichroism spectroscopy, while binding of chimeric proteins to collagen-coated substrates was verified by surface plasmon resonance analysis under physiological conditions. Cell culture assays revealed that the adhesion of neurosphere-forming cells to collagen-coated polystyrene surfaces was significantly promoted by the incorporation of the chimeric proteins in a peptide-density dependent manner. The live/dead assays for cells cultured for 24 or 48 h in the hydrogels revealed that peptide incorporation improved the survival of cells embedded in collagen hydrogels. These results suggest that collagen hydrogel containing the laminin-derived peptide provides microenvironments suitable for the survival of neural cells.

Cell transplantation is a potential therapy for central nervous disorders such as Parkinson's disease. However, the therapeutic effect is limited by the low viability of transplanted cells. To solve this problem, we synthesized hyaluronic acid (HAc)-based hydrogel to be used as a carrier for neural cells. Another feature of the hydrogel synthesized here is to incorporate brain-derived neurotrophic factor (BDNF) for enhancing cell survival in the hydrogel. The cross-linking of HAc and the incorporation of BDNF were both achieved by employing the ability of a hexahistidine peptide to form stable chelate with metal ions. HAc was reacted with N-(5-amino-1-carboxypentyl) iminodiacetic acid and chelated with Zn(II) ions. An alpha-helical peptide of 70 amino acid residues carrying a hexahistidine peptide (His) at the both termini and BDNF carrying a His at the C-terminus were synthesized by recombinant DNA technology. These polypeptides were coordinated with the Zn(II) ions chelated to HAc chains for both the cross-linking of HAc and the tethering of BDNF. Viscosity measurements revealed that intermolecular cross-links were introduced in the Zn(II)-chelated HAc chains upon mixing with the His-containing helical peptide. It was shown by release tests that the coordinated BDNF was firmly bound to the Zn(II)-chelated HAc for more than 12 days. The results of cell culture experiments combined with live/dead assays demonstrated that a significantly higher fraction of neural cells survived 3 days post-seeding in the HAc-based hydrogel incorporating BDNF than in the control hydrogel lacking BDNF. These results suggest that the HAc-based hydrogel developed here has potential to improve survival of transplanted neural cells.

To develop culture substrates for use in selective expansion of neural stem cells (NSCs), epidermal growth factor (EGF)-containing chimeric proteins were designed and synthesized by means of recombinant DNA technology. The chimeric proteins consisted of three components including an EGF domain, an alpha-helical oligopeptide, and a hexahistidine sequence. Two different alpha-helical oligopeptides were separately incorporated into chimeric proteins. Structural analyses by native gel electrophoresis and circular dichroism spectroscopy revealed that the heterodimer of these proteins was spontaneously formed through coiled-coil association of the alpha-helical oligopeptides. The monomeric and dimeric forms of these chimeric proteins were immobilized to the glass-based substrate via coordinate bonding between the hexahistidine and Ni(II) ions fixed on a substrate. The results of cell culture assays with NSCs showed that cells proliferated most rapidly and selectively on a substrate with the surface-anchored EGF dimer. The rate of cell proliferation on the surface with dimeric EGF was 1.3-2.0 times higher on the surfaces with monomeric EGF. In addition, the content of stem cells, determined 96 h after cell seeding, was highest on the surface with dimeric EGF (98%) among the surfaces studied (90-97% on surfaces with monomeric EGF). The observed growth rate and the stem cell content on the surface with EGF dimer were far beyond those in the standard neurosphere culture. The effect of surface-anchored dimeric EGF may be attributed to the enhanced dimerization of EGF-EGF receptor complexes leading to efficient signaling for mitogenic activity. We conclude that surface-anchoring of the EGF dimer provides an excellent substrate that allows the highly efficient expansion of NSCs.

Synthetic immunology has been a topic of research in both the immunology and tissue engineering fields. Since the first successful generation of an artificial lymph node (LN) in vivo by implantation of a collagen scaffold with stromal cells has been reported, tissue engineering of artificial LNs using several biological and engineering techniques has started. Recently, the advance engineering approaches using several manufacturing machines, including three-dimensional (3D) printers, 3D fabrication and assembling machines, have been addressed. Such approaches are in general called “Biofabrication”. As the use of machines has several potential capabilities, such as super-manufacturing beyond the capability of humans, direct tissue manufacturing by additive manufacturing and computer aided tissue engineering by applying high efficiency of the computer and digital technologies, the strategy of biofabrication can provide a potential of breakthrough to construct highly sophisticated 3D scaffolds and to generate several complex 3D tissues and organs. Based on this concept, we have ever developed several machines for this purpose such as an inkjet 3D bioprinter to construct complex and multi-composite 3D structures. In the present work, we applied this strategy to rationally design and construct artificial LNs. This research is still in its infancy, and it requires several improvements in terms of new technology. Here, we introduced our struggles and current works towards production of practical artificial LNs.

To develop a culture substrate that allows efficient expansion of neural stem cells (NSCs), epidermal growth factor (EGF) was immobilized onto the Ni(II)-chelated surface of a glass-based substrate through coordination of Ni(II) to the histidine tag that was fused to the C-terminal of EGF using recombinant technology. For the preparation of the nickel-chelated surface, a thin gold layer was deposited to the glass surface, and then the self-assembled monolayer of alkanethiol terminated with trivalent carboxylic acids was formed on gold and chelated with Ni(II) ions. In the preparation of a monolayer, triethylene glycol-terminated alkanethiol was mixed with carboxylic acid-terminated alkanethiol at various compositions in order to reduce the non-specific adsorption of EGF. The surface analysis of the monolayers was performed by X-ray photoelectron spectroscopy, infrared reflection–absorption spectroscopy, and contact angle measurements. Surface plasmon resonance analyses and protein assays were performed for characterizing EGF-immobilized surfaces. The proliferation and differentiation of rat fetal NSCs were examined on the EGF-chelated substrates to assess quantitatively the effects of alkanethiol composition on the efficiency of stem cell amplification. It was shown that the amplification efficiency was dependent on the alkanethiol composition. This result could be attributed to the difference in the surface density of chelated EGF. Under the optimal condition, 98% of proliferated cells expressed NSC marker. In addition, these cells could be subcultured for further expansion, while retained their multipotency. We concluded that the substrate developed here provides the efficient method for the highly selective expansion of NSCs.

Random copolymers (S-PCMBx) of the zwitterionic monomer carboxymethylbetaine (CMB) and a small percentage of 3-methacryloyloxypropyl trimethoxysilane with various composition ratios were synthesized in ethanol using 2,2'-azobisisobutyronitrile as the initiator. An S-PCMBx layer formed on the glass substrate after soaking in the copolymer solution and had a thickness of 2-3 nm. The S-PCMBx-modified surface was highly hydrophilic and suppressed both the non-specific adsorption of protein (bovine serum albumin) and NIH3T3-fibroblast adhesion. The degree of adsorption suppression increased with increasing copolymer CMB content with a maximum at 90 mol % CMB. In contrast, the modification of the glass substrate with a PCMB homopolymer terminally modified with a trimethoxysilyl group did not effectively suppress protein adsorption and cell adhesion due to the low graft density. The importance of balancing the number of fixation points and the length of the zwitterionic polymer loops to produce bio-inactive metal oxide surfaces is suggested. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2029-2036, 2016.

The molecular recognition of various kinds of bisphenols (BPs) and a bisphenol A-polymer conjugate (BPA-polymer) by a self-assembled monolayer (SAM) of thiolated beta-cyclodextrin (CD) on a gold electrode was examined using cyclic voltammetry (CV). Based on the inhibitory effect of BPs on the inclusion of hydroquinone (HQ) as a probe by the surface-confined CD, the association constants (K(assoc)) of BPs with the immobilized beta-CD were estimated. The K(assoc) values for BPs with the SAM of 3-dithiobis(undecanoylamido)-3-deoxy-beta-cyclodextrin (DTUA-beta-CD) were smaller than those in the free beta-CD system reported previously. A similar tendency was obtained when 6-(lipoylamido)-6-deoxy-beta-cyclodextrin (LP-beta-CD) was used in place of DTUA-beta-CD. The K(assoc) values for all the BPs except for bisphenol B with the SAM of LP-beta-CD were always larger than those with the SAM of DTUA-beta-CD, due to a difference in the orientation of the beta-CD moiety in the SAMs. Furthermore, adsorption and desorption processes of the BPA-polymer from the surface-confined beta-CD was followed using local surface plasmon resonance spectroscopy.

Single-walled carbon nanotubes (SWNTs)mechanically dispersed in HEPES buffer were incubated with a dispersion of A-B-A block telomers, composed of 2-methacryloyloxyethyl-D)glucopyranoside telomers (A block) and a styrene telomer (B block), which had been prepared by the macroinitiator technique. After the incubation, the polymer-wrapped SWNT could be stably dispersed in the buffer without any precipitation for several months. The glucose residue above the SWNT was definitely recognized by lectin (Concanavalin A) as proven by the increase in turbidity. Using a similar procedure, a block telomer composed of N-isopropylacrylamide telomers (A block) and a styrene telomer (B block) could easily be deposited onto the SWNT. A temperature-responsive aggregation and the subsequent precipitation of the modified SWNT could clearly be observed. The usage of the block-telomer examined here would be promising for the preparation of stable dispersions of SWNTs with various functions.

Neural progenitor cells derived from induced pluripotent stem cells have been considered as a potential source for cell-transplantation therapy of central nervous disorders. However, efficient methods to expand neural progenitor cells are further required for their clinical applications. In this study, a protein array was fabricated with nine extracellular matrices and used to screen substrates suitable for the expansion of neural progenitor cells derived from mouse induced pluripotent stem cells. The results showed that neural progenitor cells efficiently proliferated on substrates with immobilized laminin-1, laminin-5, or Matrigel. Based on this result, further attempts were made to develop clinically compliant substrates with immobilized polypeptides that mimic laminin-1, one of the most effective extracellular matrices as identified in the array-based screening. We used here recombinant DNA technology to prepare polypeptide containing the globular domain 3 of laminin-1 and immobilized it onto glass-based substrates. Our results showed that neural progenitor cells selectively proliferated on substrate with the immobilized polypeptide while maintaining their differentiated state.

Surfaces of both a cover glass and the flat plane of a semi-cylindrical quartz prism were modified with a mixture of positively and negatively charged silane coupling reagents (3-aminopropyltriethoxysilane (APTES) and 3-(trihydroxysilyl)propylmethylphosphonate (THPMP), respectively). The glass surface modified with a self-assembled monolayer (SAM) prepared at a mixing ratio of APTES:THPMP = 4:6 was electrically almost neutral and was resistant to non-specific adsorption of proteins, whereas fibroblasts gradually adhered to an amphoteric (mixed) SAM surface probably due to its stiffness, though the number of adhered cells was relatively small. Sum frequency generation (SFG) spectra indicated that total intensity of the O H stretching region (3000–3600 cm −1 ) for the amphoteric SAM-modified quartz immersed in liquid water was smaller than those for the positively and negatively charged SAM-modified quartz prisms and a bare quartz prism in contact with liquid water. These results suggested that water molecules at the interface of water and an amphoteric SAM-modified quartz prism are not strongly oriented in comparison with those at the interface of a lopsidedly charged SAM-modified quartz prism and bare quartz. The importance of charge neutralization for the anti-biofouling properties of solid materials was strongly suggested.

Poor viability of cells transplanted into the brain has been the critical problem associated with stem cell-based therapy for Parkinson's disease. To overcome this problem, a collagen hydrogel incorporating an integrin-binding protein complex was prepared and used as a carrier for neural stem cells. The protein complex consisted of two polypeptides containing the G3 domain of a laminin α1 chain and the C-terminal oligopeptide of a laminin γ1 chain. These polypeptides were fused with α-helical segments which spontaneously formed a coiled-coil heterodimer and with the collagen-binding peptide that facilitated the binding of the heterodimer to collagen networks. In this study, neural stem cells stably expressing the enhanced green fluorescent protein (EGFP) were suspended in the hydrogel and transplanted into the striatum of healthy rats. The viability of transplanted cells was evaluated by histological analysis and quantitative reverse-transcriptase polymerase chain reaction for EGFP mRNA present in the tissue explants. Our results showed that the collagen hydrogel incorporating the integrin-binding protein complex serves to improve the viability of neural stem cells (NSCs) in the early stage after transplantation into the striatum.

Human neural progenitor cells (hNPCs) are a potential source for cell transplantation therapy in central nervous disorders. Neurosphere culture, the standard method for obtaining hNPCs, suffers from several limitations including the heterogeneity of cells in a neurosphere and the limitation of growth rate due to the presence of differentiated cells in the neurospheres. To overcome these limitations, we developed culture substrates that enable the selective expansion of hNPCs in adherent culture. Epidermal growth factor and basic fibroblast growth factor were fused with hexahistidine (EGF-His and bFGF-His, respectively) and were immobilized alone or in combination onto Ni ion-bound glass through coordination. When hNPCs derived from human fetal brain were cultured on these substrates, adhesion and proliferation of hNPCs took place most efficiently on the substrate with both EGF-His and bFGF-His compared to substrates with either factor alone and to a control substrate without growth factors. The rate of cell proliferation was two-fold higher in the adherent culture on the substrate immobilized with both EGF-His and bFGF-His than in the standard neurosphere culture. A cell population obtained after 5 days of culture on the substrate contained nestin-expressing progenitors (>90%). We conclude that the culture substrate with co-immobilized EGF and bFGF is effective for the selective expansion of hNPCs.

A thin layer (around 2 nm in thickness) composed of a random copolymer of zwitterionic carboxymethyl betaine (CMB) and p-trimethoxysilylstyrene (STMS) (9:1) is constructed on a glass substrate or a silicon wafer. The copolymer layer is highly resistant against nonspecific adsorption of bovine serum albumin (BSA). However, on UV irradiation at 193 nm, the layer becomes hydrophobic and BSA is significantly adsorbed on the substrate. Upon UV irradiation through a photomask, a patterning of the fluorophore-labeled protein with a resolution of about 1 µm can be clearly observed. On the other hand, the copolymer layer of CMB and 3-methacryloxypropyltrimethoxysilane (MPTMS) (9:1) without an aromatic group exhibit less distinct pattern. Further, the poly(CMB-r-STMS) layer decomposes more quickly compared with the poly(CMB-r-MPTMS) layer at low irradiation dose. The zwitterionic polymer layer with an aromatic anchor group will be used at the modification of substrates applicable to sensing devices.

Cell transplantation is a potential methodology for the treatment of Parkinson's disease. However, the therapeutic effect is limited by poor viability of transplanted cells. To overcome this problem, we hypothesized that a dual step approach, whereby providing an adhesive substrate for transplanted cells and, at the same time, by preventing the infiltration of activated microglia into the site of transplantation promotes the cell survival. To establish above conditions, attempts were made to prepare 3-D matrices using collagen hydrogels that incorporated integrin-binding polypeptides derived from laminin-1. Tandem combinations of laminin globular domains as well as a single globular domain 3 were prepared using recombinant DNA technology as a fusion with hexahistidine and bound to metal chelated surfaces to screen for the adhesion and proliferation of neural stem cells (NSCs). In addition, a small peptide derived from laminin γ1 chain was prepared and heterodimerized with the globular domain-containing chimeric proteins to evaluate for the enhancement of integrin-mediated cell adhesion. As a result, a heterodimer consisting of the globular domain 3 of the laminin α1 chain and the peptide from the laminin γ1 chain was selected as the best candidate among the polypeptides studied here for the incorporation into a collagen hydrogel. It was shown that the survival of NSCs was indeed promoted in the collagen hydrogel incorporating the heterodimer compared to the pure collagen hydrogel.

A random copolymer of zwitterionic monomer, carboxymethylbetaine (CMB), and 3-methacryloyloxypropyl trimethoxysilane was prepared in ethanol using 2,2'-azobisisobutyronitrile as initiator. The incubation of ethanol solution of the copolymer with a glass plate gave a layer of the copolymer with a thickness of about 2-3 nm. The copolymer-modified glass substrate became highly hydrophilic upon immersion in water, and showed a resistance against non-specific adsorption of proteins, and the degree of resistance increased with the content of CMB residues in the copolymer and leveled off. The adhesion of various cells to the glass substrate was also strongly suppressed by the surface modification with the copolymer layer. Further introduction of PolyCMB graft chains on the surface of the layer enhanced the suppression of cell adhesion due to the steric hindrance for the cells to approach the layer. The usefulness of the combination of zwitterionic polymer layer and graft chains to afford anti-biofouling properties to a solid surface of metal oxides was shown.

Neural stem cells (NSCs) have received much attention in cell-transplantation therapy for central nervous disorders such as Parkinson’s disease. However, poor engraftment of transplanted cells limits the efficacy of the treatments. To overcome this problem, collagen-based hydrogels were designed in this study to provide microenvironments for embedded cells to survive and proliferate. Our approach was to incorporate epidermal growth factor (EGF), known as a mitogen for NSCs, into a collagen hydrogel. For the stable binding of EGF with collagen under mild conditions, EGF was fused with a collagen-binding polypeptide domain by recombinant DNA technology. A cell population containing NSCs was derived from the fetal rat brain and cultured in the composite hydrogels for 7 d followed by analysis for cell proliferation. It was shown that the number of living cells was significantly higher in hydrogels incorporating collagen-binding EGF. This effect is largely owing to the collagen-binding domain that serves to sustain presentation of EGF toward cells within the hydrogel. It is further revealed by gene expression analysis that cells proliferated in the EGF-incorporating collagen hydrogel contained subpopulations expressing the marker of stem cells, neurons, astrocytes, or oligodendrocytes.

Neural stem cells (NSCs) have been frequently used to investigate in vitro the molecular and cellular mechanisms underlying the development of the central nervous system (CNS). In addition, NSCs are regarded as one of the potential sources for the cell replacement therapy of CNS disorders. Most of these studies have utilized NSCs prepared by neurosphere culture. However, this method normally yields a heterogeneous population containing differentiated neural cells as well as NSCs. In addition, the rate of cell expansion is not high enough for obtaining a large quantity of NSCs in a short period. Here we report the design of culture substrates that allow highly selective and rapid expansion of NSCs. We synthesize epidermal growth factor fused with a hexahistidine sequence (EGF-His) and a polystyrene-binding peptide (EGF-PSt), and these engineered growth factors were surface-anchored to a nickel-chelated glass plate and a polystyrene dish, respectively. The EGF-His-chelated glass substrate was further used to assemble a culture module. Neurosphere-forming cells prepared from the fetal rat striatum were used to examine the selective expansion of NSCs using the EGF-His-chelated module and the EGF-PSt-bound polystyrene dish. Our results show that the culture module enables to selectively expand NSCs in a closed system more efficiently than the standard neurosphere culture. The EGF-PSt-bound polystyrene dish also permits efficient expansion of NSCs, providing a straightforward means to acquire a large quantity of pure NSCs in standard laboratories.

The poor survival of neural stem/progenitor cells following transplantation into the brain is the major problem limiting the effect of cell-based therapy for Parkinson's disease. To overcome this problem, we are involved in designing keratin-based hydrogels that serve as physical barriers to prevent the infiltration of inflammatory cells. Another feature of the hydrogels is to contain a polypeptide that promotes integrin-mediated cell adhesion. To construct such hydrogels, a chimeric protein consisting of an alpha-helical polypeptide and a globular domain derived from laminin was synthesized by means of recombinant DNA technology and coassembled with extracted keratins that form hydrogels through intermolecular coiled-coil association of alpha-helical segments. It was found that neurosphere-forming cells specifically adhered to the keratin-based composite hydrogel and actively proliferated at a high survival rate. These results suggested that the composite hydrogel provides microenvironments suitable for the survival and proliferation of neural progenitor cells.

Controlling the dynamics of growth factor signaling is a challenge in regenerative medicine for various tissues including the central nervous system. Here, we report on the development of the biomolecular system that facilitates sequential regulation of growth factor signals acting on neural stem/progenitor cells. Recombinant technology was employed to synthesize the multifunctional chimeric protein that contained multiple domains, including epidermal growth factor (EGF), ciliary neurotrophic factor (CNTF), globular capping domain, thrombin-cleavable sequence, and substrate-binding domain with affinity for Ni(II) ions. The chimeric protein is expected to expose CNTF upon elimination of the capping domain by digestion with endogenous thrombin in vivo. When the multifunctional chimeric protein was immobilized onto a substrate through the coordination of the substrate-binding domain with surface-immobilized Ni(II) ions, the substrate served to proliferate neural stem cells, maintaining the population of undifferentiated cells at 85%. This effect is primarily due to the activity of EGF, while CNTF activity is temporally veiled with the capping domain. Upon digesting the thrombin-cleavable sequence to remove the capping domain, the activity of CNTF emerged to induce differentiation of astrocytes in situ from the proliferated neural stem cells. The fraction of differentiated astrocytes reached 68% of total cells. These results demonstrate the feasibility of the system for controlling the dynamics of growth factor signals.

The molecular recognition of various kinds of bisphenols (BPs) by a self-assembled monolayer (SAM) of thiolated calix[6]arene on a gold electrode was examined using cyclic voltammetry (CV). Based on the inhibitory effect of BPs on the inclusion of hydroquinone (HQ) as a probe bythe surface-confined calix[6]arene, the association constants (Kassoc) of BPs with the immobilized calix[6]arene were estimated. The Kassoc values for BPs with the SAM of thiolated hexasodium calix[6]arenehexasulfonic acid (thioSCX6) were much smaller than those in the free calix[6]arene derivative systems reported previously. The order of the Kassoc values for BPs with thioSCX6 was bisphenol A (BPA)bisphenol S (BPS)bisphenol B (BPB)bisphenol F (BPF). The Kassoc values for BPA and BPS with thioSCX6 were larger than that for BPB, despite the larger hydrophobicity of BPB than that of BPA and BPS. This is probably because the inclusion phenomena in this system are not simply driven by the hydrophobic interaction, but are significantly affected by several steric and structural factors with immobilization of the host. Those are (1) a decrease in flexibility of the SCX6 cavity by the formation of SAM, (2) a decrease in inclusion ability of the SCX6 by the presence of Au surface just beneath it, and (3) a repulsion of hydrophobic guests bythe presence of sulfonate groups at the top of the SAM. Moreover, the association phenomena (adsorption and desorption processes) of a bisphenol with the SCX6 SAM were examined directly by using localized surface plasmon resonance spectroscopy.