Akihiro Nishiguchi

Rapid Construction of Three‐Dimensional Multilayered Tissues with Endothelial Tube Networks by the Cell‐Accumulation Technique

The in vitro construction of living tissue or organ models, which are highly organized with various types of cells and extracellular matrices (ECM) and allow for the evaluation of tissue functions, have attracted increasing attention in the fi elds of tissue engineering and drug assessment. [ 1–3 ] In particular, the fabrication of vascularized thick and complex tissues is a key challenge for transplantable constructs or angiogenesis models. [ 4 ] Although various functional scaffolds possessing specifi c cell-adhesive and degradability-controllable properties have achieved notable advances in tissue regeneration, [ 5–8 ] three-dimensional (3D) tissue models which precisely control the cell type, cell alignment, and cell-cell interactions in all three dimensions have not been developed yet. A conventional approach using biodegradable scaffolds has several limitations in developing 3D tissue constructs which satisfy the above requirements. Recently, several bottom-up approaches such as a cell sheet, [ 9–11 ] magnetic liposomes, [ 12 , 13 ] and cell-containing gel layers [ 14 ] have been reported for the construction of multilayered tissues. These methods are intriguing examples of a bottom-up approach, but have limitations due to the complicated manipulation of fragile cell sheets or the remains of magnetic particles in the cells. We have developed a simple bottom-up approach by preparing nanometer-sized ECM fi lms on cell surfaces. [ 15–17 ] Less than 10 nm-thick fi bronectin-gelatin (FN-G) fi lms prepared by layer-by-layer (LbL) assembly [ 18–20 ] promoted cell-cell interactions like a natural ECM, and thus we successfully fabricated over fi ve layered (5L) tissue models such as blood vessels, skeletal muscle, and connective tissue. Although this technique is simple and versatile enough to develop the multilayered constructs while controlling the cellular type and location, the fabrication of 2L tissues is limited due to the time required for stable cell adhesion. Therefore, a simple and rapid approach is strongly desired for the in vitro construction of thick multilayered tissue models. In this study, we developed a simple and rapid bottom-up approach, called the cell-accumulation technique, by a single

Development of vascularized iPSC derived 3D-cardiomyocyte tissues by filtration Layer-by-Layer technique and their application for pharmaceutical assays

UNLABELLED In vitro development of three-dimensional (3D) human cardiomyocyte (CM) tissues derived from human induced pluripotent stem cells (iPSCs) has long been desired in tissue regeneration and pharmaceutical assays. In particular, in vitro construction of 3D-iPSC-CM tissues with blood capillary networks have attracted much attention because blood capillaries are crucial for nutrient and oxygen supplies for CMs. Blood capillaries in 3D-iPSC-CM tissues will also be important for in vitro toxicity assay of prodrugs because of the signaling interaction between cardiomyocytes and endothelial cells. Here, we report construction of vascularized 3D-iPSC-CM tissues by a newly-discovered filtration-Layer-by-Layer (LbL) technique for cells, instead of our previous centrifugation-LbL technique. The filtration-LbL allowed us to fabricate nanometer-sized extracellular matrices (ECM), fibronectin and gelatin (FN-G), films onto iPSC-CM surfaces without any damage and with high yield, although centrifugation-LbL induced physical stress and a lower yield. The fabricated FN-G nanofilms interacted with integrin molecules on the cell membrane to construct 3D-tissues. We found that the introduction of normal human cardiac fibroblasts (NHCFs) into the iPSC-CM tissues modulated organization and synchronous beating depending on NHCF ratios. Moreover, co-culture with normal human cardiac microvascular endothelial cells (NHCMECs) successfully provided blood capillary-like networks in 3D-iPSC-CM tissues, depending on NHCF ratios. The vascularized 3D-iPSC-CM tissues indicated significantly different toxicity responses as compared to 2D-iPSC-CM cells by addition of doxorubicin as a model of a toxic drug. The constructed vascularized 3D-iPSC-CM tissues would be a promising tool for tissue regeneration and drug development. STATEMENT OF SIGNIFICANCE In vitro fabrication of vascularized three-dimensional (3D) human cardiomyocyte (CM) tissues derived from human induced pluripotent stem cells (iPSCs) has attracted much attention owing to their requirement of much amount of nutrition and oxygen, but not yet published. In this manuscript, we report construction of vascularized 3D-iPSC-CM tissues by a newly-discovered filtration-Layer-by-Layer (LbL) technique. The filtration-LbL fabricates nanometer-sized fibronectin and gelatin (FN-G) films onto iPSC-CM surfaces. The FN-G nanofilms induce cell-cell interactions via integrin molecules on cell surfaces, leading to construction of 3D-tissues. The constructed vascularized 3D-iPSC-CM tissues would be a promising tool for tissue regeneration and drug development. We believe that this manuscript has a strong impact and offers important suggestions to researchers concerned with biomaterials and tissue engineering.

Nanometer‐sized extracellular matrix coating on polymer‐based scaffold for tissue engineering applications

Surface modification can play a crucial role in enhancing cell adhesion to synthetic polymer-based scaffolds in tissue engineering applications. Here, we report a novel approach for layer-by-layer (LbL) fabrication of nanometer-size fibronectin and gelatin (FN-G) layers on electrospun fibrous poly(carbonate urethane)urea (PCUU) scaffolds. Alternate immersions into the solutions of fibronectin and gelatin provided thickness-controlled FN-G nano-layers (PCUU(FN-G) ) which maintained the scaffolds 3D structure and width of fibrous bundle of PCUU as evidenced by scanning electron miscroscopy. The PCUU(FN-G) scaffold improved cell adhesion and proliferation of bladder smooth muscles (BSMCs) when compared to uncoated PCUU. The high affinity of PCUU(FN-G) for cells was further demonstrated by migration of adherent BSMCs from culture plates to the scaffold. Moreover, the culture of UROtsa cells, human urothelium-derived cell line, on PCUU(FN-G) resulted in an 11-15 μm thick multilayered cell structure with cell-to-cell contacts although many UROtsa cells died without forming cell connections on PCUU. Together these results indicate that this approach will aid in advancing the technology for engineering bladder tissues in vitro. Because FN-G nano-layers formation is based on nonspecific physical adsorption of fibronectin onto polymer and its subsequent interactions with gelatin, this technique may be applicable to other polymer-based scaffold systems for various tissue engineering/regenerative medicine applications.

Secretions from placenta, after hypoxia/reoxygenation, can damage developing neurones of brain under experimental conditions.

Some psychiatric diseases in children and young adults are thought to originate from adverse exposures during foetal life, including hypoxia and hypoxia/reoxygenation. The mechanism is not understood. Several authors have emphasised that the placenta is likely to play an important role as the key interface between mother and foetus. Here we have explored whether a first trimester human placenta or model barrier of primary human cytotrophoblasts might secrete factors, in response to hypoxia or hypoxia/reoxygenation, that could damage neurones. We find that the secretions in conditioned media caused an increase of [Ca(2+)]i and mitochondrial free radicals and a decrease of dendritic lengths, branching complexity, spine density and synaptic activity in dissociated neurones from embryonic rat cerebral cortex. There was altered staining of glutamate and GABA receptors. We identify glutamate as an active factor within the conditioned media and demonstrate a specific release of glutamate from the placenta/cytotrophoblast barriers invitro after hypoxia or hypoxia/reoxygenation. Injection of conditioned media into developing brains of P4 rats reduced the numerical density of parvalbumin-containing neurones in cortex, hippocampus and reticular nucleus, reduced immunostaining of glutamate receptors and altered cellular turnover. These results show that the placenta is able to release factors, in response to altered oxygen, that can damage developing neurones under experimental conditions.

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.

Dynamic Nano‐Interfaces Enable Harvesting of Functional 3D‐Engineered Tissues

Functional 3D-engineered tissues are successfully harvested from a substrate using stimuli-responsive hydrogel films with dynamic nano-interface. The dynamic wettability control at the interfaces allows cellular detachment, leading to tissue harvesting without serious damage and remaining polymers. This method can be applied to various types of organs and used for tissue transplantation in regenerative medicine.

High-Throughput Blood- and Lymph-Capillaries with Open-Ended Pores Which Allow the Transport of Drugs and Cells.

High-throughput screening of drug diffusion and cell transports from the blood-/lymph-capillary (BC/LC) networks to the peripheral cells in 3D engineered tissues using a microplate would make a powerful tool for in vitro pharmacokinetic assessments. Here, perfusable BC/LC networks embedded in 3D-tissues inside a 24-microplate using a cell-coating technology are reported which allows location control of cell layers. Arrangement of an endothelial cell layer at the top, middle, and bottom of dermal fibroblast tissues provides an interconnected BC/LC networks possessing open pores on both surfaces. When fluorescently labeled dextran, microparticles, and red blood cells are applied to the top surfaces, diffusion and penetration through the networks are observed depending on the size of the substances. Moreover, BC networks mimick a series of in vivo processes of cancer metastasis, extravasation, growth, and growth suppression with drug treatment. The perfusable networks existing in 3D-tissues show great potential for in vitro pharmacokinetic studies.

In‐Gel Direct Laser Writing for 3D‐Designed Hydrogel Composites That Undergo Complex Self‐Shaping

Abstract Self‐shaping and actuating materials inspired by biological system have enormous potential for biosensor, microrobotics, and optics. However, the control of 3D‐complex microactuation is still challenging due to the difficulty in design of nonuniform internal stress of micro/nanostructures. Here, we develop in‐gel direct laser writing (in‐gel DLW) procedure offering a high resolution inscription whereby the two materials, resin and hydrogel, are interpenetrated on a scale smaller than the wavelength of the light. The 3D position and mechanical properties of the inscribed structures could be tailored to a resolution better than 100 nm over a wide density range. These provide an unparalleled means of inscribing a freely suspended microstructures of a second material like a skeleton into the hydrogel body and also to direct isotropic volume changes to bending and distortion motions. In the combination with a thermosensitive hydrogel rather small temperature variations could actuate large amplitude motions. This generates complex modes of motion through the rational engineering of the stresses present in the multicomponent material. More sophisticated folding design would realize a multiple, programmable actuation of soft materials. This method inspired by biological system may offer the possibility for functional soft materials capable of biomimetic actuation and photonic crystal application.

In vitro placenta barrier model using primary human trophoblasts, underlying connective tissue and vascular endothelium

Fetal development may be compromised by adverse events at the placental interface between mother and fetus. However, it is still unclear how the communication between mother and fetus occurs through the placenta. In vitro - models of the human placental barrier, which could help our understanding and which recreate three-dimensional (3D) structures with biological functionalities and vasculatures, have not been reported yet. Here we present a 3D-vascularized human primary placental barrier model which can be constructed in 1 day. We illustrate the similarity of our model to first trimester human placenta, both in its structure and in its ability to respond to altered oxygen and to secrete factors that cause damage cells across the barrier including embryonic cortical neurons. We use this model to highlight the possibility that both the trophoblast and the endothelium within the placenta might play a role in the fetomaternal dialogue.

Three-dimensional cultured tissue constructs that imitate human living tissue organization for analysis of tumor cell invasion

Abstract Preventing cancer metastasis requires a thorough understanding of cancer cell invasion. These phenomena occur in human 3‐D living tissues. To this end, we developed a human cell‐based three‐dimensional (3‐D) cultured tissue constructs that imitate in vivo human tissue organization. We investigated whether our 3‐D cell culture system can be used to analyze the invasion of human oral squamous cell carcinoma (OSCC) cells. The 3‐D tissue structure consisted of five layers of normal human dermal fibroblasts along with human dermal lymphatic endothelial cell tubes and was generated by the cell accumulation technique and layer‐by‐layer assembly using fibronectin and gelatin. OSCC cells with different lymph metastatic capacity were inoculated on the 3‐D tissues and their invasion through the 3‐D tissue structure was observed. Conventional methods of analyzing cell migration and invasion, that is, 2‐D culture‐based transwell and Matrigel assays were also used for comparison. The results using the 3‐D cultured tissue constructs were comparable to those obtained using conventional assays; moreover, use of the 3‐D system enabled visualization of differential invasion capacities of cancer cells. These results indicate that our 3‐D cultured tissue constructs can be a useful tool for analysis of cancer cell invasion in a setting that reflects the in vivo tissue organization.