Koji Kadowaki

Control of cell surface and functions by layer-by-layer nanofilms.

Various nanometer-sized multilayers were directly prepared onto the surface of mouse L929 fibroblast cells by a layer-by-layer (LbL) assembly technique to control the cell surface microenvironment and cell functions, such as viability, morphology, and proliferation. The species of LbL nanofilms strongly affected the cell morphology and growth. Polyelectrolyte (PE) multilayers induced a round-shaped morphology of the adhered cells, although each component of the multilayers had high cytocompatibility, whereas fibronectin (FN)-gelatin (G) and -dextran sulfate (DS) multilayers with FN-binding domain interactions (FN films) showed extended morphologies of the cells similar to that of control cells (without films). A clear difference in cell proliferation was observed for PE and FN films. The cells with FN films on their surfaces showed good proliferation profiles independent of the film thickness, but cell proliferation was not observed using the PE films although the cells survived during the culture period. Fluorescence microscopic and scanning electron microscopic observations clearly suggested a nanometer-sized meshwork morphology of the FN films on the cell surface after 24 h of incubation, whereas the PE films showed homogeneous film morphologies on the cell surface. These nanomeshwork morphologies seemed to be similar to the fibrous structure of the natural extracellular matrix. The results of this study demonstrated that the components, charge, and morphology of LbL nanofilms prepared directly on the cell surface strongly affected cell functions, and the effects of these LbL nanofilms on cell functions differed vastly as compared to PE films prepared on a substrate. The preparation of LbL nanofilms onto a cell surface might be a novel and interesting technique to control cell functions.

Immunomodulatory nanoparticles as adjuvants and allergen-delivery system to human dendritic cells: Implications for specific immunotherapy.

Novel adjuvants and antigen-delivery systems with immunomodulatory properties that shift the allergenic Th2 response towards a Th1 or regulatory T cell response are desired for allergen-specific immunotherapy. This study demonstrates that 200-nm sized biodegradable poly(gamma-glutamic acid) (gamma-PGA) nanoparticles (NPs) are activators of human monocyte-derived dendritic cells (MoDCs). Gamma-PGA NPs are efficiently internalized by immature MoDCs and strongly stimulate production of chemokines and inflammatory cytokines as well as up-regulation of co-stimulatory molecules and immunomodulatory mediators involved in efficient T cell priming. Furthermore, MoDCs from allergic subjects stimulated in vitro with a mixture of gamma-PGA NPs and extract of grass pollen allergen Phleum pratense (Phl p) augment allergen-specific IL-10 production and proliferation of autologous CD4(+) memory T cells. Thus, gamma-PGA NPs are promising as sophisticated adjuvants and allergen-delivery systems in allergen-specific immunotherapy.

Engineering fibrotic tissue in pancreatic cancer: A novel three-dimensional model to investigate nanoparticle delivery

Pancreatic cancer contains both fibrotic tissue and tumor cells with embedded vasculature. Therefore anti-cancer nanoparticles need to extravasate from tumor vasculature and permeate thick fibrotic tissue to target tumor cells. To date, permeation of drugs has been investigated in vitro using monolayer models. Since three-dimensional migration of nanoparticles cannot be analyzed in a monolayer model, we established a novel, three-dimensional, multilayered, in vitro model of tumor fibrotic tissue, using our hierarchical cell manipulation technique with K643f fibroblasts derived from a murine pancreatic tumor model. NIH3T3 normal fibroblasts were used in comparison. We analyzed the size-dependent effect of nanoparticles on permeation in this experimental model using fluorescent dextran molecules of different molecular weights. The system revealed permeation decreased as number of layers of cultured cells increased, or as molecule size increased. Furthermore, we showed changes in permeation depended on the source of the fibroblasts. Observations of this sort cannot be made in conventional monolayer culture systems. Thus our novel technique provides a promising in vitro means to investigate permeation of nanoparticles in fibrotic tissue, when both type and number of fibroblasts can be regulated.

Quantitative 3D analysis of nitric oxide diffusion in a 3D artery model using sensor particles.

A blood vessel is crucial not only for circulatory diseases and treatments, but also for the biological evaluation of drug diffusion to target tissues, the penetration of cancer cells or pathogens, and the control of blood pressure. A blood vessel is generally composed of three distinct layers: the intima, an inner single layer of endothelial cells (ECs); the media, medium layers of smooth muscle cells (SMCs); and the adventitia, an outer layer of fibroblast cells. The ECs act as a sensing surface to transduce hydrodynamic forces and chemical stimuli into extracellular signal molecules such as endothelin-1, prostacyclin, and nitric oxide, which affect vascular events such as the contraction and relaxation of SMCs. Nitric oxide (NO), produced by the nitric oxide synthase (NOS) protein family, is a lipophilic, highly diffusible, and short-lived physiological messenger. It is well known that NO regulates a variety of important physiological responses including vasodilation, respiration, cell migration, and apoptosis. The NO produced from ECs diffuses into SMCs through their cell membranes, and activates guanylate cyclase to produce intracellular cyclic guanosine monophosphate (cGMP), which induces a signaling pathway mediated by kinase proteins leading to SMC relaxation. Accordingly, quantitative, kinetic, and spatial analyses of the extracellular delivery of NO molecules from the EC layer to the SMC layers upon drug stimulation are crucial for pharmaceutical and biomedical evaluations of hypertension and diabetes. So far, pharmaceutical assays of NO production have been performed by in vivo animal experiments, but low reproducibility and difference of NO production depending on animal types are unsolved issues. Malinski and co-workers reported the in situ analysis of NO diffusion using the extracted animal aorta, but special equipment and techniques were necessary for their analysis method. Thus, the development of a convenient and versatile method for the in vitro quantitative and spatial analyses of NO diffusion inside the artery wall instead of animal experiments is important for biological and pharmaceutical applications. In a previous study, we reported biocompatible and highly sensitive NO sensor particles prepared by layer-by-layer (LbL) assembly. The mesoporous, micrometer-sized silica particles encapsulating 4,5-diaminofluorescein (DAF-2), NO fluorescent indicator dye, were covered with biocompatible chitosan (CT)–dextran sulfate (DS) LbL films to provide cytocompatibility and to inhibit leakage of DAF-2. The NO sensor particles (SPs) showed high NO sensitivity at 5– 500 nm, which is sufficient to detect NO at concentrations of hundreds of nm (EC production level). If an artificial three-dimensional (3D) artery model allocating these SPs can be developed, the extracellular diffusion of NO from the EC layer to the SMC layers with chemical and physical stimuli is expected to be observed in vitro fluorescently by using confocal laser scanning microscopy (CLSM). Herein, we demonstrate for the first time spatial and quantitative analyses of NO diffusion from the EC layer to the SMC layers using a 3D artery model with SPs allocated into each cellular layer (Figure 1). Recently, we reported an in vitro hierarchical cell manipulation technique to develop 3D cellular multilayers by the fabrication of nanometer-sized extracellular matrix (ECM) films on the surface of each cell layer. Approximately 6 nm thick fibronectin (FN)–gelatin (G) LbL films prepared on the surface of the first layer of cells can provide a suitable cell-adhesive surface that is similar to the natural ECM for the second layer of cells. Furthermore, 3D blood vessel models consisting of human ECs and SMCs were successfully developed, and their morphology and histology were evaluated in detail. Herein, we developed five-layered (5L) artery models including SPs using human aortic smooth muscle cells (AoSMCs) and human aortic endothelial cells (HAECs). The 3D structural effect of HAECs and AoSMCs on NO production from the HAECs was clarified in relation to the direction of interaction between these cells. Furthermore, a graded concentration change of NO from the uppermost HAEC layer to the underlying AoSMC layers was elucidated by 3D analysis using confocal laser scanning microscopy. The SPs were prepared based on our previous report. The encapsulated DAF-2 in the mesoporous silica particles was stable even after 1 month of incubation in buffer (see Figure S1 in the Supporting Information) or 1 week of incubation in culture medium containing serum (data not shown). Since DAF-2 has a weak negative charge under neutral pH conditions, the electrostatic interaction with CT is [*] Dr. M. Matsusaki, S. Amemori, K. Kadowaki, Prof. M. Akashi Department of Applied Chemistry Graduate School of Engineering, Osaka University Yamada-oka, Suita, Osaka 565-0871 (Japan) E-mail: akashi@chem.eng.osaka-u.ac.jp Homepage: http://www.chem.eng.osaka-u.ac.jp/~akashi-lab/

Morphological and histological evaluations of 3D-layered blood vessel constructs prepared by hierarchical cell manipulation.

Three-dimensional (3D)-layered blood vessel constructs consisting of human umbilical artery smooth muscle cells (SMCs) and human umbilical vascular endothelial cells (ECs) were fabricated by hierarchical cell manipulation, and their basic morphology, histology and blood compatibility were evaluated in relation to the EC layers. For the hierarchical cell manipulation, fibronectin-gelatin (FN-G) nanofilms were prepared on the surface of SMC layers to provide a cell adhesive nano-scaffold for the second layer of cells. The layer number of blood vessel constructs was easily controllable from 2 to 7 layers, and the histological evaluation, scanning electron microscope (SEM) and transmission electron microscope (TEM) observations indicated a hierarchical blood vessel analogous morphology. The immunefluorescence staining revealed homogeneous and dense tight-junction of the uppermost EC layer. Furthermore, the nano-meshwork morphology of the FN-G films like a native extracellular matrix was observed inside the blood vessel constructs by SEM. Moreover, a close association between actin microfilaments and the nano-meshworks was observed on the SMC surface by TEM. The blood compatibility of the blood vessel constructs, 4-layered SMC/1-layered EC (4L-SMC/1L-EC), was clearly confirmed by inhibition of platelet adhesion, whereas the blood vessel constructs without EC layers (4L-SMC) showed high adhesion and activation of the platelet. The 3D-blood vessel constructs prepared by hierarchical cell manipulation technique will be valuable as a blood vessel model in the tissue engineering or pharmaceutical fields.

Three-dimensional constructs induce high cellular activity: Structural stability and the specific production of proteins and cytokines

The specific properties responsible for the stability and function induced by three-dimensional (3D) cellular constructs were evaluated and compared to a monolayer structure. 3D-cellular multilayers composed of human fibroblast cells (FCs) and human umbilical vascular endothelial cells (ECs) were fabricated by a hierarchical cell manipulation technique. Interestingly, the ECs adhered homogeneously onto four-layered (4L) FCs, and tight-junction formation was widely observed at the centimeter scale, while heterogeneous EC domain structures were observed on the monolayered (1L) FCs. The production of heat shock protein70 (Hsp70) and interleukin-6 (IL-6) from the cellular structures were investigated to elucidate any 3D-structural effect on cellular function. The Hsp70 expression of the ECs decreased after adhesion onto the 4L-FC structure as compared with the EC monolayer. Surprisingly, the Hsp70 production response to heat shock increased drastically by approximately 10-fold as compared with a non-heat shock by 3D structure formation, whereas the monolayer structures showed no change. Moreover, the production of the inflammatory cytokine IL-6 decreased significantly depending on the layer number of FCs. To the best of our knowledge, this is the first report on a basic, 3D-structural effect on cellular stability and function. These findings could be important for not only tissue engineering, but also for basic cell biology.

Scaffold-Free Tissue-Engineered Construct–Hydroxyapatite Composites Generated by an Alternate Soaking Process : Potential for Repair of Bone Defects

Mesenchymal stem cell (MSC)-based tissue-engineered construct (TEC)-hydroxyapatite (HAp) composites were developed by an alternate soaking process. The TEC derived from cultured synovial MSCs was alternately immersed in varying concentrations of CaCl(2)/Tris-HCl and Na(2)HPO(4)/Tris-HCl buffers, and HAp formation was analyzed by Fourier transform infrared spectroscopy (FT-IR), wide-angle X-ray diffraction, and scanning electron microscopy (SEM). These analyses clearly demonstrated HAp formation in the TEC. Specifically, SEM assessments showed that spherical HAp crystals of approximately 1 mum were directly formed on the surfaces of the cells and extracellular matrix (ECM) fibers. Cytotoxicity from exposure to calcium or phosphate buffers of >100 mM concentrations as assessed by LIVE/DEAD staining and total DNA assays was detected, but such cytotoxicity was not detected following exposure to concentrations of <50 mM. The HAp nanocrystals (ca. approximately 500 nm) were formed after 20 cycles in 10 mM calcium or phosphate buffers, and cell survival in the composites was confirmed. Moreover, preliminary implantation of TEC-HAp composites derived from rabbit synovial MSCs to rabbit osteochondral defects exhibited accelerated osteoinduction. These composites may be the first example of a hybrid material that consists of ECM, HAp nanocrystals, and living MSCs, and the TEC-HAp composite could be a unique and useful material for bone tissue engineering.

Survival and structural evaluations of three-dimensional tissues fabricated by the hierarchical cell manipulation technique

Mouse L929 fibroblasts and normal human dermal fibroblasts (NHDFs) were constructed into three-dimensional (3-D) multilayered tissues by directly coating them with nano-films consisting of fibronectin (FN) and gelatin (G) onto the surface of the cell layer using layer-by-layer assembly. The one-, two- and five-layered (1L, 2L and 5L) tissues were cultured for 1 month in order to evaluate their long-term survival and structural changes. L929 cells in the 3-D tissues showed excessive proliferation throughout the culture period, regardless of the starting layer number. The cross-sectional images stained with hematoxylin and eosin revealed heterogeneous and random increases in the thickness of their layered structures, probably due to the immortalized property of L929 fibroblasts. On the other hand, NHDFs, which are primary cells, showed high stability in their amount of DNA in the multilayered structures, and their thicknesses were completely maintained even after 1 month of incubation. To evaluate the living cell density in each layer of 5L tissues during the culture period, 5L NHDFs were fluorescently labeled with LIVE/DEAD reagent and analyzed by confocal laser scanning microscopy. Although the upper layers did not show any dead cells, the bottom layers showed pieces of dead cell nuclei depending on culture time. However, the living cell densities in all layers were almost the same, even after 1 month of culture, suggesting that the 5L structures were completely filled with living cells. These findings from the multilayered tissue constructs will provide important information not only for the construction of 3-D engineered tissues in tissue engineering but also on 3-D cell culture in biological science generally.

Protein nanoarrays on a highly-oriented lamellar surface

Well-aligned nanopatterns of various serum, antithrombogenic and cell adhesive proteins, such as gamma-globulin, fibrinogen, thrombomodulin, fibronectin and type I collagen, were fabricated on a highly-oriented block copolymer lamellar surface, and these bioactive protein nanoarrays will be useful in biological research.

3D-fibroblast tissues constructed by a cell-coat technology enhance tight-junction formation of human colon epithelial cells

Caco-2, human colon carcinoma cell line, has been widely used as a model system for intestinal epithelial permeability because Caco-2 cells express tight-junctions, microvilli, and a number of enzymes and transporters characteristic of enterocytes. However, the functional differentiation and polarization of Caco-2 cells to express sufficient tight-junctions (a barrier) usually takes over 21 days in culture. This may be due to the cell culture environment, for example inflammation induced by plastic petri dishes. Three-dimensional (3D) sufficient cell microenvironments similar to in vivo natural conditions (proteins and cells), will promote rapid differentiation and higher functional expression of tight junctions. Herein we report for the first time an enhancement in tight-junction formation by 3D-cultures of Caco-2 cells on monolayered (1L) and eight layered (8L) normal human dermal fibroblasts (NHDF). Trans epithelial electric resistance (TEER) of Caco-2 cells was enhanced in the 3D-cultures, especially 8L-NHDF tissues, depending on culture times and only 10 days was enough to reach the same TEER value of Caco-2 monolayers after a 21 day incubation. Relative mRNA expression of tight-junction proteins of Caco-2 cells on 3D-cultures showed higher values than those in monolayer structures. Transporter gene expression patterns of Caco-2 cells on 3D-constructs were almost the same as those of Caco-2 monolayers, suggesting that there was no effect of 3D-cultures on transporter protein expression. The expression correlation between carboxylesterase 1 and 2 in 3D-cultures represented similar trends with human small intestines. The results of this study clearly represent a valuable application of 3D-Caco-2 tissues for pharmaceutical applications.