Hidenori Otsuka

Nanofabrication of Nonfouling Surfaces for Micropatterning of Cell and Microtissue

Surface engineering techniques for cellular micropatterning are emerging as important tools to clarify the effects of the microenvironment on cellular behavior, as cells usually integrate and respond the microscale environment, such as chemical and mechanical properties of the surrounding fluid and extracellular matrix, soluble protein factors, small signal molecules, and contacts with neighboring cells. Furthermore, recent progress in cellular micropatterning has contributed to the development of cell-based biosensors for the functional characterization and detection of drugs, pathogens, toxicants, and odorants. In this regards, the ability to control shape and spreading of attached cells and cell-cell contacts through the form and dimension of the cell-adhesive patches with high precision is important. Commitment of stem cells to different specific lineages depends strongly on cell shape, implying that controlled microenvironments through engineered surfaces may not only be a valuable approach towards fundamental cell-biological studies, but also of great importance for the design of cell culture substrates for tissue engineering. To develop this kind of cellular microarray composed of a cell-resistant surface and cell attachment region, micropatterning a protein-repellent surface is important because cellular adhesion and proliferation are regulated by protein adsorption. The focus of this review is on the surface engineering aspects of biologically motivated micropatterning of two-dimensional surfaces with the aim to provide an introductory overview described in the literature. In particular, the importance of non-fouling surface chemistries is discussed.

Micropatterned co-culture of hepatocyte spheroids layered on non-parenchymal cells to understand heterotypic cellular interactions

Abstract Microfabrication and micropatterning techniques in tissue engineering offer great potential for creating and controlling cellular microenvironments including cell–matrix interactions, soluble stimuli and cell–cell interactions. Here, we present a novel approach to generate layered patterning of hepatocyte spheroids on micropatterned non-parenchymal feeder cells using microfabricated poly(ethylene glycol) (PEG) hydrogels. Micropatterned PEG-hydrogel-treated substrates with two-dimensional arrays of gelatin circular domains (φ = 100 μm) were prepared by photolithographic method. Only on the critical structure of PEG hydrogel with perfect protein rejection, hepatocytes were co-cultured with non-parenchymal cells to be led to enhanced hepatocyte functions. Then, we investigated the mechanism of the functional enhancement in co-culture with respect to the contributions of soluble factors and direct cell–cell interactions. In particular, to elucidate the influence of soluble factors on hepatocyte function, hepatocyte spheroids underlaid with fibroblasts (NIH/3T3 mouse fibroblasts) or endothelial cells (BAECs: bovine aortic endothelial cells) were compared with physically separated co-culture of hepatocyte monospheroids with NIH3T3 or BAEC using trans-well culture systems. Our results suggested that direct heterotypic cell-to-cell contact and soluble factors, both of these between hepatocytes and fibroblasts, significantly enhanced hepatocyte functions. In contrast, direct heterotypic cell-to-cell contact between hepatocytes and endothelial cells only contributed to enhance hepatocyte functions. This patterning technique can be a useful experimental tool for applications in basic science, drug screening and tissue engineering, as well as in the design of artificial liver devices.

Chondrocyte spheroids on microfabricated PEG hydrogel surface and their noninvasive functional monitoring

Abstract A two-dimensional microarray of 10 000 (100 × 100) chondrocyte spheroids was constructed with a 100 μm spacing on a micropatterned gold electrode that was coated with poly(ethylene glycol) (PEG) hydrogels. The PEGylated surface as a cytophobic region was regulated by controlling the gel structure through photolithography. In this way, a PEG hydrogel was modulated enough to inhibit outgrowth of chondrocytes from a cell adhering region in the horizontal direction, which is critical for inducing formation of three-dimensional chondrocyte aggregations (spheroids) within 24 h. We further report noninvasive monitoring of the cellular functional change at the cell membrane using a chondrocyte-based field effect transistor. This measurement is based on detection of extracellular potential change induced as a result of the interaction between extracellular matrix protein secreted from spheroid and substrate at the cell membrane. The interface potential change at the cell membrane/gate interface can be monitored during the differentiation of spheroids without any labeling materials. Our measurements of the time evolution of the interface potential provide important information for understanding the uptake kinetics for cellular differentiation.

Self-assembly of poly(ethylene glycol)-block-polypyridine copolymer into micelles and at silica surface: effect of molecular architecture on silica dispersion

We have newly synthesized amphiphilic block copolymers composed of hydrophilic poly(ethylene glycol) (PEG) and hydrophobic pyridine segments (PEG-b-Py). Chain transfer agent-terminated PEG was subsequently chain-extended with 3-(4-pyridyl)-propyl acrylate to obtain PEG-b-Py by reversible additional-fragmentation chain transfer polymerization. Particularly, the effect of varying molecular weight (Mn) of PEG (Mnu2009=u20092,000 and 5,000) and Py in the block copolymers was investigated in terms of critical micelle concentration, pyrene solubilization, micelle size distribution, and association number per micelle. Based on the amphiphilic balance, PEG-b-Pys formed core-shell type polymer micelle. The association number of PEG2k-b-Py was higher than that of PEG5k-b-Py, suggesting the degree of phase separation strongly depended on PEG Mn. Furthermore, the adsorption of PEG-b-Py copolymer onto silica nanoparticles as dispersant was studied to estimate the effect of PEG Mn in the copolymers and their solubility in the medium on the adsorption. Adsorbed density of PEG2k-b-Py copolymer onto silica nanoparticle was higher than that of PEG5k-b-Py, which was significantly correlated with the degree of phase separation. Furthermore, the adsorbed amount of copolymer increased with the increase in ionic strength due to the reduced solubility of PEG in the buffer solution. The resultant dispersion stability was highly correlated with the graft density of copolymer onto silica surface. However, the stability of PEG2k-b-Py coated particles was lower than that with PEG5k-b-Py, this is attributed to the relatively thin layer of PEG at the silica surface, which cannot provide the system with sufficient steric stabilization as the salt concentration increases. These fundamental investigations for the surface modification of the nanoparticle provide the insight into the highly stable colloidal dispersion, particularly in the physiological condition with high ionic strength.

Highly robust protein production by co-culture of CHO spheroids layered on feeder cells in serum-free medium

Recombinant Chinese hamster ovary (rCHO) cells have been the most commonly used mammalian host for large-scale commercial production of therapeutic proteins. Although recent advances in 3D culture of rCHO cells is preferred to 2D monolayer culture for highly productive and robust expression of therapeutic proteins, there exists still limitation for efficient protein production. Therefore, a new cell culture system is essentially required for an efficient protein production. Here, we report on a new 3D cell culture system as a spheroid cell culture on the micropattern array for efficient production of protein by CHO cells. Particularly, cocultivation of CHO spheroids with bovine aortic endothelial cells (BAEC) as a feeder layer cells was essential to stably increase a protein production. We investigated the co-culture mechanism of functional enhancement with respect to the cell–cell interactions. Functional comparison between 2D and 3D co-cultures suggested the preferred configuration as spheroid for higher protein production. Specifically, to estimate the effect of respective cell constitution in co-cultured spheroids on the protein production per CHO cell, the number of viable cells in cell proliferation was determined with culture periods. These studies demonstrated the significant role of micropatterned BAEC as a feeder layer for the retained formation of CHO spheroids, resulting in predominantly enhanced production of proteins, although the functional enhancement of CHO cells was obtained by co-culture with BAECs in both 2D and 3D configurations. Thus, heterotypic cell communications that play indispensable roles in increasing CHO functions should be properly obtained in 3D cell configurations. Significantly, these spheroids in the serum-free medium drastically enhanced protein expression level up to sevenfold compared with CHO monospheroids, suggesting that a suitable culture conditions for heterotypic cell–cell interactions would allow improved protein secretion to occur unimpeded.

Nanofabrication technologies to control cell and tissue function for biomedical applications

Surface engineering techniques for cellular micropatterning are emerging as important tools to clarify the effects of the microenvironment on cellular behavior. Cells usually integrate with and respond to the microscale environment, and they are affected by the chemical and mechanical properties of the surrounding fluid and extracellular matrix, soluble protein factors, small signal molecules, and contacts with neighboring cells. Furthermore, recent progress in cellular micropatterning has contributed to the development of cell-based biosensors for the functional characterization and detection of drugs, pathogens, toxicants, and odorants. In this regard, the ability to control with high precision the shape and spreading of attached cells and cell-cell contacts through the form and dimension of cell-adhesive patches is important. Commitment of stem cells to different specific lineages depends strongly on cell shape, implying that controlling microenvironments through engineered surfaces may not only be a valuable approach toward fundamental cell-biological studies, but could also be of great importance to the design of cell culture substrates for tissue engineering. To develop this kind of cellular microarray composed of a cell-resistant surface and cell attachment region, micropatterning a protein-repellent surface is important because cellular adhesion and proliferation are regulated by protein adsorption. The focus of this review is on the surface engineering aspects of biologically motivated micropatterning of two-dimensional surfaces, with the goal of providing an introductory overview of what is described in the literature. In particular, the importance of nonfouling surface chemistries is discussed.

Stimuli-Responsive Polymer Materials for Creation of Biointerfaces

In this review, we introduce the biorecognition-driven stimuli-responsive surface and hydrogels. The first attention focuses on recent advances in the development of functionalizable antifouling coatings and their applications in label-free optical biosensors. Approaches to the development of antifouling coatings, ranging from self-assembled monolayers and PEG derivatives to low-fouling polymer brushes and polymerized gels, are reviewed. Preparation of antifouling coatings and the functionalization of antifouling coatings with bioreceptors are introduced, and the application example of biofunctional coating with fouling properties is discussed. Special attention is given to biofunctional coatings for label-free bioanalysis of blood plasma and serum for medical diagnostics. The following focus is fed light on the biorecognition-based stimuli-responsive hydrogels. We will discuss on peptides and proteins recognition system of stimuli-responsive hybrid hydrogel composed of synthetic polymers and biopolymers.

Micropatterning of cell aggregate in three dimension for in vivo mimicking cell culture

Abstract Surface engineering techniques for cellular micropatterning are emerging as important tools to clarify the effects of the microenvironment on cellular behaviour, as cells usually integrate and respond to the microscale environment, such as chemical and mechanical properties of the surrounding fluid and extracellular matrix, soluble protein factors, small signal molecules, and contacts with neighbouring cells. Furthermore, recent progress in cellular micropatterning has contributed to the development of cell-based biosensors for the functional characterization and detection of drugs, pathogens, toxicants, and odorants. In this regard, the ability to control shape and spread of attached cells and cell–cell contacts through the form and dimension of cell-adhesive patches with high precision is important. Commitment of stem cells to different specific lineages depends strongly on cell shape, implying that controlled microenvironments through engineered surfaces may not only be a valuable approach towards fundamental cell-biological studies, but also be of great importance for the design of cell culture substrates for tissue engineering. In particular, surface engineering techniques for cellular micropatterning as spheroids are focused on in this review. To develop this kind of cellular microarray composed of a cell-resistant surface and cell attachment region, micropatterning a protein-repellent surface is important because cellular adhesion and proliferation are regulated by protein adsorption. The focus of this review is on the surface engineering aspects of biologically motivated micropatterning of two-dimensional surfaces with the aim to provide an introductory overview described in the literature. In particular, the importance of nonfouling surface chemistries is discussed.

PEGylation for biocompatible surface

Abstract Fouling corresponding to nonspecific protein adsorption is a key problem for many medical and biotechnological applications. The problem is most critical when complex biological media such as blood or blood plasma contact the surfaces of artificial materials. The proteins that adsorb on the biomaterial surface determine subsequent responses, including blood coagulation, platelet activation, complement activation and inflammation, and the final performance of the material. The development of technologies by which antifouling surfaces of biomedical materials can be prepared is a central challenge for contemporary research. Surface coatings based on poly(ethylene glycol) (PEG), a nontoxic and nonimmunogenic polymer, have been used for the modification of various biomedical surfaces (PEGylation). The high reactivity of PEG terminal makes the introduction of functional group easy to tether to the material’s surface. In this chapter, we highlight the construction of the PEG modified surface and its application to biological and biomedical fields. A PEG modified biocompatible surface is prepared by the various and effective surface coating method for substances and shows the characteristic features based on the properties of PEG.

Tripodal thiol-derivatives as a functional interface monolayer for immobilization of biomolecules

We designed interface molecules for immobilization of biomolecules on solid substrate and applied it to genetic field effect transistors. We have been investigating a new approach to realize a potentiometric detection for DNA chips. The concept of a genetic field effect transistor has been proposed for improving precision, standardization and miniaturization of a DNA chip system. We have designed and synthesized tripodal thiol-derivatives for stable immobilization of oligonucleotide probes on a gold surface. The genetic FET platform combined with the new interface molecule is suitable for a simple, accurate and inexpensive system for SNP typing in clinical diagnostics.