Construction of a spheroid array culture system on a suspended permeable hydrogel membrane scaffold for improving the expression of a liver-specific drug-metabolizing enzyme of HepG2 cells.

Herein, we constructed a spheroid array culture system on a flexible hydrogel membrane suspended in the culture medium. When we applied this culture system to HepG2 cells, the results suggested that an aerobic culture environment was implemented, and the gene expression of a liver-specific drug-metabolizing enzyme was improved in comparison with that of the conventional immobilized monolayer culture.

Gravity-driven microfluidic device placed on a slow-tilting table enables constant unidirectional perfusion culture of human induced pluripotent stem cells.

Gravity-driven microfluidics, which utilizes gravity force to drive liquid flow, offers portability and multi-condition setting flexibility because they do not require pumps or connection tubes to drive the flow. However, because the flow rate decreases with time in gravity-driven microfluidics, it is not suitable for stem cell experiments, which require long-term (at least a day) stability. In this study, gravity-driven microfluidics and a slow-tilting table were developed to culture cells under constant unidirectional perfusion. The microfluidic device was placed on a slow-tilting table, which tilts unidirectionally at a rate of approximately 7° per day to compensate for the reduction in the flow rate. Computational simulations showed that the pulsation of the flow arising from the stepwise movement of the table was less than 0.2%, and the flow was laminar. Hydrophilization of the tanks increased the flow rate, which is consistent with the theoretical values. We showed that vitronectin is better than laminin 511 fragments as a coating material for adhering human induced pluripotent stem cells on a microchamber made of polydimethylsiloxane, and succeeded in culturing the cells for 3 days. It is believed that the system offers easy-to-use cell culture tools, such as conventional multiwell culture vessels, and enables the control of the cell microenvironment.

Perfusion culture of endothelial cells under shear stress on microporous membrane in a pressure-driven microphysiological system.

This paper reports perfusion culture of human umbilical vein endothelial cells (HUVECs) on a microporous membrane in a pressure-driven microphysiological system (PD-MPS), which we developed previously as a multi-throughput perfusion culture platform. We designed fluidic culture unit with microporous membrane to culture HUVECs under fluidic shear stress and constructed a perfusion culture model in the PD-MPS platform. Four fluidic culture units were arranged in the microplate-sized device, which enables four-throughput assay for characterization of HUVECs under flow. Medium flow was generated above and below the membrane by sequential pneumatic pressure to apply physiological shear stress to HUVECs. HUVECs exhibited aligned morphology to the direction of the flow with shear stress of 11.5-17.7 dyn/cm2 under the flow condition, while they randomly aligned under static culture condition in a 6 well plate. We also observed 3.3- and 5.0-fold increase in the expression levels of the thrombomodulin and endothelial nitric oxide synthase mRNAs, respectively, under the flow condition in the PD-MPS compared to the static culture in 6 well plate. We also observed actin filament aligned to the direction of flow in HUVECs cultured under the flow condition.

Perfusion culture of multi-layered HepG2 hepatocellular carcinoma cells in a pressure-driven microphysiological system.

Here we report the perfusion culture of a multi-layered tissue composed of HepG2 cells (a human hepatoma line) in a pressure-driven microphysiological system (PD-MPS), which we developed previously as a multi-throughput perfusion culture platform. The perfusion culture of multi-layered tissue model was constructed by inserting a modified commercially available permeable membrane insert into the PD-MPS. HepG2 cells were layered on the membrane, and culture medium was perfused both through and below the membrane. The seeded density (number of cells/cm2) of the culture model is 70 times that of static culture in a conventional 35-mm culture dish. Pressure-driven circulation of the medium in our compact device (8.6 × 7.0 × 4.5 cm3), which comprised two perfusion-culture modules and a pneumatic connection port, enabled perfusion culture of two multi-layered tissues (initially 1 × 105 cells). To obtain insight into the basic functionality of the multi-layered tissues as hepatocytes, we compared albumin production and urea synthesis between perfusion cultures and static cultures. The HepG2 cells grew and secreted increasing amounts of albumin throughout 20 days of perfusion culture, whereas albumin secretion did not increase under static culture conditions. In addition, on day 20, the amount of albumin secreted by the HepG2 cells in the microfluidic device was 68% of that in the conventional culture dish, which was seeded with the same number of cells but had a 70 times larger culture area. These features of high-density culture of functioning cells in a compact device support the application of PD-MPS in single- and multi-organ MPS.

Coculture with hiPS-derived intestinal cells enhanced human hepatocyte functions in a pneumatic-pressure-driven two-organ microphysiological system.

Examining intestine-liver interactions is important for achieving the desired physiological drug absorption and metabolism response in in vitro drug tests. Multi-organ microphysiological systems (MPSs) constitute promising tools for evaluating inter-organ interactions in vitro. For coculture on MPSs, normal cells are challenging to use because they require complex maintenance and careful handling. Herein, we demonstrated the potential of coculturing normal cells on MPSs in the evaluation of intestine-liver interactions. To this end, we cocultured human-induced pluripotent stem cell-derived intestinal cells and fresh human hepatocytes which were isolated from PXB mice with medium circulation in a pneumatic-pressure-driven MPS with pipette-friendly liquid-handling options. The cytochrome activity, albumin production, and liver-specific gene expressions in human hepatocytes freshly isolated from a PXB mouse were significantly upregulated via coculture with hiPS-intestinal cells. Our normal cell coculture shows the effects of the interactions between the intestine and liver that may occur in vivo. This study is the first to demonstrate the coculturing of hiPS-intestinal cells and fresh human hepatocytes on an MPS for examining pure inter-organ interactions. Normal-cell coculture using the multi-organ MPS could be pursued to explore unknown physiological mechanisms of inter-organ interactions in vitro and investigate the physiological response of new drugs.

Membrane properties of ether-type phosphatidylcholine bearing partially fluorinated C18-monoacetylenic chains and their applicability to membrane protein reconstitution matrices.

To examine the applicability of fluorinated membrane-forming phospholipids to reconstitution matrices for functional membrane proteins, the membrane properties of a synthetic ether-type phosphatidylcholine (PC) bearing partially fluorinated C18-monoacetylenic (9-octadecynyl) chains, DF8CCH8PC, were compared with those of its non-fluorinated counterpart, DH8CCH8PC. Light-harvesting complex 2 (LH2) and the light-harvesting 1‒reaction center core complex (LH1-RC) isolated from purple photosynthetic bacteria were employed as probe membrane proteins to evaluate the extent to which their reconstitution into DF8CCH8PC membranes could proceed. DF8CCH8PC formed more expanded and more stable fluid monolayers than DH8CCH8PC at the air-water interface at 25 °C; the former PC molecule occupied an area of ca. 0.70 nm2 at a collapse pressure, πc, of 52 mN/m, while the latter occupied an area of ca. 0.55 nm2 at a πc of 45 mN/m. In contrast, the molecular motion detected using fluorescent probes was much more restricted in DF8CCH8PC bilayers than in DH8CCH8PC ones. Although the reconstitution efficiencies of both LH2 and LH1-RC into DF8CCH8PC bilayers were lower than those into DH8CCH8PC bilayers, the membrane proteins incorporated into DF8CCH8PC bilayers showed increased thermostability. The increased thermostability of these proteins in fluorinated PC membranes might be due to the restricted molecular motion in the hydrophobic chains. The results of this study suggest that partially fluorinated PCs can be useful materials for the construction of lipid‒functional membrane protein assemblies including large membrane protein complexes, such as LH1-RC, for biotechnological applications.

Stepwise construction of dynamic microscale concentration gradients around hydrogel-encapsulated cells in a microfluidic perfusion culture device.

Inside living organisms, concentration gradients dynamically change over time as biological processes progress. Therefore, methods to construct dynamic microscale concentration gradients in a spatially controlled manner are needed to provide more realistic research environments. Here, we report a novel method for the construction of dynamic microscale concentration gradients in a stepwise manner around cells in micropatterned hydrogel. In our method, cells are encapsulated in a photodegradable hydrogel formed inside a microfluidic perfusion culture device, and perfusion microchannels are then fabricated in the hydrogel by micropatterned photodegradation. The cells in the micropatterned hydrogel can then be cultured by perfusing culture medium through the fabricated microchannels. By using this method, we demonstrate the simultaneous construction of two dynamic concentration gradients, which allowed us to expose the cells encapsulated in the hydrogel to a dynamic microenvironment.

Interstitial flow regulates in vitro three-dimensional self-organized brain micro-vessels.

Cell culture under medium flow has been shown to favor human brain microvascular endothelial cells function and maturation. Here a three-dimensional in vitro model of the human brain microvasculature, comprising brain microvascular endothelial cells but also astrocytes, pericytes and a collagen type I microfiber - fibrin based matrix, was cultured under continuous medium flow in a pressure driven microphysiological system (10 kPa, in 60-30 s cycles). The cells self-organized in micro-vessels perpendicular to the shear flow. Comparison with static culture showed that the resulting interstitial flow enhanced a more defined micro-vasculature network, with slightly more numerous lumens, and a higher expression of transporters, carriers and tight junction genes and proteins, essential to the blood-brain barrier functions.

Fabrication of Hollow Structures in Photodegradable Hydrogels Using a Multi-Photon Excitation Process for Blood Vessel Tissue Engineering.

Engineered blood vessels generally recapitulate vascular function in vitro and can be utilized in drug discovery as a novel microphysiological system. Recently, various methods to fabricate vascular models in hydrogels have been reported to study the blood vessel functions in vitro; however, in general, it is difficult to fabricate hollow structures with a designed size and structure with a tens of micrometers scale for blood vessel tissue engineering. This study reports a method to fabricate the hollow structures in photodegradable hydrogels prepared in a microfluidic device. An infrared femtosecond pulsed laser, employed to induce photodegradation via multi-photon excitation, was scanned in the hydrogel in a program-controlled manner for fabricating the designed hollow structures. The photodegradable hydrogel was prepared by a crosslinking reaction between an azide-modified gelatin solution and a dibenzocyclooctyl-terminated photocleavable tetra-arm polyethylene glycol crosslinker solution. After assessing the composition of the photodegradable hydrogel in terms of swelling and cell adhesion, the hydrogel prepared in the microfluidic device was processed by laser scanning to fabricate linear and branched hollow structures present in it. We introduced a microsphere suspension into the fabricated structure in photodegradable hydrogels, and confirmed the fabrication of perfusable hollow structures of designed patterns via the multi-photon excitation process.

Biology-inspired microphysiological systems to advance patient benefit and animal welfare in drug development

The first microfluidic microphysiological systems (MPS) entered the academic scene more than 15 years ago and were considered an enabling technology to human (patho)biology in vitro and, therefore, provide alternative approaches to laboratory animals in pharmaceutical drug development and academic research. Nowadays, the field generates more than a thousand scientific publications per year. Despite the MPS hype in academia and by platform providers, which says this technology is about to reshape the entire in vitro culture landscape in basic and applied research, MPS approaches have neither been widely adopted by the pharmaceutical industry yet nor reached regulated drug authorization processes at all. Here, 46 leading experts from all stakeholders - academia, MPS supplier industry, pharmaceutical and consumer products industries, and leading regulatory agencies - worldwide have analyzed existing challenges and hurdles along the MPS-based assay life cycle in a second workshop of this kind in June 2019. They identified that the level of qualification of MPS-based assays for a given context of use and a communication gap between stakeholders are the major challenges for industrial adoption by end-users. Finally, a regulatory acceptance dilemma exists against that background. This t4 report elaborates on these findings in detail and summarizes solutions how to overcome the roadblocks. It provides recommendations and a roadmap towards regulatory accepted MPS-based models and assays for patients' benefit and further laboratory animal reduction in drug development. Finally, experts highlighted the potential of MPS-based human disease models to feedback into laboratory animal replacement in basic life science research.

Kinetic analysis of sequential metabolism of triazolam and its extrapolation to humans using an entero-hepatic two-organ microphysiological system.

The microphysiological system (MPS) is a promising tool for predicting drug disposition in humans, although limited information is available on the quantitative assessment of sequential drug metabolism in MPS and its extrapolation to humans. In the present study, we first constructed a mechanism-based pharmacokinetic model for triazolam (TRZ) and its metabolites in the entero-hepatic two-organ MPS, composed of intestinal Caco-2 and hepatic HepaRG cells, and attempted to extrapolate the kinetic information obtained with the MPS to the plasma concentration profiles in humans. In the two-organ MPS and HepaRG single culture systems, TRZ was found to be metabolized into α- and 4-hydroxytriazolam and their respective glucuronides. All these metabolites were almost completely reduced in the presence of a CYP3A inhibitor, itraconazole, confirming sequential phase I and II metabolism. Both pharmacokinetic model-dependent and -independent analyses were performed, providing consistent results regarding the metabolic activity of TRZ: clearance of glucuronidation metabolites in the two-organ MPS was higher than that in the single culture system. The plasma concentration profile of TRZ and its two hydroxy metabolites in humans was quantitatively simulated based on the pharmacokinetic model, by incorporating several scaling factors representing quantitative gaps between the MPS and humans. Thus, the present study provided the first quantitative extrapolation of sequential drug metabolism in humans by combining MPS and pharmacokinetic modeling.

Effect of the fluorination degree of partially fluorinated octyl-phosphocholine surfactants on their interfacial properties and interactions with purple membrane as a membrane protein model.

Interfacial properties and membrane protein solubilization activity of a series of partially fluorinated octyl-phosphocholine (PC) surfactants were investigated from the viewpoint of the fluorination degree of the hydrophobic chain. The critical micelle concentration (CMC), surface tension lowering activity, molecular occupied area at the CMC and free energy changes of micellization as well as adsorption to the air-water interface for each PC surfactant were estimated from surface tension measurements at 25 °C. The PCs with higher degree of fluorination exhibited low CMC and high surface activity, while the single trifluoromethyl group at the end of the chain appeared to enhance the hydrophilicity of the surfactant molecule. Under conditions where conventional short-chain surfactants, n-octyl-ß-D-glucoside, Triton X-100 and dioctanoylphosphatidylcholine significantly solubilize purple membranes (PM), none of the fluorinated-PCs solubilized PM. This suggests that fluorinated-PCs are low-invasive enough to maintain the structure of lipids/protein assemblies like PM.

[Microphysiological system as a promising technology for drug assay].

MPS (microphysiological system) is in-vitro cell-culture environment, which is precisely maintained in a micro space manufactured using MEMS (micro electro mechanical systems) technology, to derive in-vivo like functions from human cells. From the viewpoint of pharmacokinetics, it can be considered as a wet PBPK/PD simulator consisting of the micro organs (cell culture units) connected each other with a circulating medium, which mimic the organs responsible for the drug's ADME (absorption-distribution-metabolism-elimination). In this review, we identify two types of the cell culture units consisting of the MPS for pharmacokinetics, and overview the characteristics of each type. Then, we discuss about the technical requirements needed for the cell culture unit from the point of view of both cell-culture environmental design and cell function, and introduce the world-wide current situation of the commercialization of the MPS.

Raman Optical Activity on a Solid Sample: Identification of Atropisomers of Perfluoroalkyl Chains Having a Helical Conformation and No Chiral Center.

Perfluoroalkyl (Rf) chains have a specific helical conformation due to the steric repulsion between the adjacent CF2 units. Although Rf chains have no chiral center, two chiral structures, i.e., the right-handed (R) and left-handed (L) helices, are available as the most stable conformations, which are atropisomers to each other. According to the stratified dipole array (SDA) theory, the helical structure about the chain axis plays a key role in the spontaneous molecular aggregation of Rf chains in a two-dimensional manner, and the Rf chains having the same chirality tend to be aggregated spontaneously to generate molecular domains. This implies that an Rf compound in a solid state should be a mixture of the R and L domains, and each domain should exhibit distinguishable optical activity. To identify molecular domains with different atropisomers, in this study, Raman optical activity (ROA) measurements were performed on a Raman imaging spectrometer. Through the ROA measurements of recrystallized solid samples of an Rf compound, each particle exhibits an apparent optical activity, and the two atropisomers were readily distinguished. As a result, an Rf compound with the same helicity is found to be spontaneously aggregated as expected by the SDA theory.

Photolithographic Fabrication of Semi 3-D Microstructures Composed of Flexible Hydrogel Sheet for in Vivo-like Cell Culture System.

Insufficient reliability of current drug screening by cell-based assay has been one of the factors in the poor success rate in drug development. To improve the situation, we proposed a cell culture system using semi 3-D hydrogel microstructures as cell culture scaffolds. Because of its flexibility and permeability, the microstructure was expected to enhance the physiological function of cells. We developed a simple method of fabricating the unique hydrogel microstructures composed of hydroxypropyl cellulose through photolithography. Functionalization with poly(styrene-co-maleic anhydride) gained their cell adhesion, and it was demonstrated that several kinds of cells were incubated on the cell culture scaffolds.

Glass-based organ-on-a-chip device for restricting small molecular absorption.

The use of organ-on-a-chip (OOC) devices is a promising alternative to existing cell-based assays and animal testing in drug discovery. A rapid prototyping method with polydimethylsiloxane (PDMS) is widely used for developing OOC devices. However, because PDMS tends to absorb small hydrophobic molecules, the loss of test compounds in cell-based assays and increases in background fluorescence during observation often lead to biased results in cell-based assays. To address this issue, we have fabricated a glass-based OOC device and characterized the medium flow and molecular absorption properties in comparison with PDMS-based devices. Consequently, we revealed that the glass device generated a stable medium flow, restricted the absorption of small hydrophobic molecules, and showed enhanced cell adhesiveness. This glass device is expected to be applicable to precise cell-based assays to evaluate small hydrophobic molecules, for which PDMS devices cannot be applied because of their absorption of small hydrophobic molecules.

Stability of the two-dimensional lattice of bacteriorhodopsin reconstituted in partially fluorinated phosphatidylcholine bilayers.

This study aims to investigate bacteriorhodopsin (bR) molecules reconstituted in lipid bilayers composed of di(nonafluorotetradecanoyl)-phosphatidylcholine (F4-DMPC), a partially fluorinated analogue of dimyristoyl-phosphatidylcholine (DMPC) to clarify the effects of partially fluorinated hydrophobic chains of lipids on protein's stability. Calorimetry measurements showed that the chain-melting transition of F4-DMPC/bR systems occurs at 3.5 °C, whereas visible circular dichroism (CD) and X-ray diffraction measurements showed that a two-dimensional (2D) hexagonal lattice formed by bR trimers in F4-DMPC bilayers remains intact even above 30 °C, similar to bR in a native purple membrane. Complete dissociation of the trimers into the monomers detected by visible CD almost coincides with the complete melting of 2D lattice observed by X-ray diffraction, in which both take place at around 65 °C (10 °C lower than that for bR in a native purple membrane). However, it is extremely high in comparison with the bR reconstituted in DMPC bilayers in which the dissociation of bR trimer in DMPC bilayers occurs near the chain-melting transition temperature of DMPC bilayers at approximately 18 °C. In order to explore the rationale behind the difference in stability, a further investigation of the detailed structural features of pure F4-DMPC bilayers was performed by analyzing the lamellar diffraction data using simple electron density models. The results suggested that the perfluoroalkyl groups do not exhibit any conformation change even if the chain-melting transition occurs, which is likely to contribute to the stability of the 2D hexagonal lattice formed by the bR trimers.

Automated adherent cell elimination by a high-speed laser mediated by a light-responsive polymer.

Conventional cell handling and sorting methods require manual labor, which decreases both cell quality and quantity. To purify adherent cultured cells, cell purification technologies that are high throughput without dissociation and can be utilized in an on-demand manner are expected. Here, we developed a Laser-induced, Light-responsive-polymer-Activated, Cell Killing (LiLACK) system that enables high-speed and on-demand adherent cell sectioning and purification. This system employs a visible laser beam, which does not kill cells directly, but induces local heat production through the trans-cis-trans photo-isomerization of azobenzene moieties. Using this system in each passage for sectioning, human induced pluripotent stem cells (hiPSCs) maintained their pluripotency and self-renewal during long-term culture. Furthermore, combined with deep machine-learning analysis on fluorescent and phase contrast images, a label-free and automatic cell processing system has been developed by eliminating unwanted spontaneously differentiated cells in undifferentiated hiPSC culture conditions.

Imaging cell picker: A morphology-based automated cell separation system on a photodegradable hydrogel culture platform.

Cellular morphology on and in a scaffold composed of extracellular matrix generally represents the cellular phenotype. Therefore, morphology-based cell separation should be interesting method that is applicable to cell separation without staining surface markers in contrast to conventional cell separation methods (e.g., fluorescence activated cell sorting and magnetic activated cell sorting). In our previous study, we have proposed a cloning technology using a photodegradable gelatin hydrogel to separate the individual cells on and in hydrogels. To further expand the applicability of this photodegradable hydrogel culture platform, we here report an image-based cell separation system imaging cell picker for the morphology-based cell separation on a photodegradable hydrogel. We have developed the platform which enables the automated workflow of image acquisition, image processing and morphology analysis, and collection of a target cells. We have shown the performance of the morphology-based cell separation through the optimization of the critical parameters that determine the system's performance, such as (i) culture conditions, (ii) imaging conditions, and (iii) the image analysis scheme, to actually clone the cells of interest. Furthermore, we demonstrated the morphology-based cloning performance of cancer cells in the mixture of cells by automated hydrogel degradation by light irradiation and pipetting.

Fabrication of pocket-like hydrogel microstructures through photolithography.

Photolithographic fabrication of unique microstructures composed of flexible hydrogel sheets is proposed and demonstrated by using photo-acid-generating poly(methyl methacrylate). Crosslinking of a hydroxyl-rich polymer and lifting off of the crosslinked polymer layer from the substrate are controlled respectively in an area-selective manner upon micropatterned light irradiation, and various pocket-like microstructures are fabricated resultantly.