関 実

Technologies for efficiently expanding Chinese hamster ovary (CHO) cells, the primary host cells for antibody production, are of growing industrial importance. Various processes for the use of microcarriers in CHO suspension cultures have been developed, but there have been very few studies on cell-adhesive microcarriers that are similar in size to cells. In this study, we proposed a new approach to suspension cultures of CHO cells using cell-sized condensed and crosslinked gelatin microparticles (GMPs) as carriers. Unlike commercially available carriers with sizes typically greater than 100 μm, each cell can adhere to the surface of multiple particles and form loose clusters with voids. We prepared GMPs of different average diameters (27 and 48 μm) and investigated their effects on cell adhesion and cluster formation. In particular, small GMPs promoted cell proliferation and increased IgG4 production by the antibody-producing CHO cell line. The data obtained in this study suggest that cell-sized particles, rather than larger ones, enhance cell proliferation and function, providing useful insights for improving suspension-culture-based cell expansion and cell-based biologics production for a wide range of applications.

Spheroid formation assisted by microengineered chambers is a versatile approach for morphology-controlled three-dimensional (3D) cell cultivation with physiological relevance to human tissues. However, the limitation in diffusion-based oxygen/nutrient transport has been a critical issue for the densely packed cells in spheroids, preventing maximization of cellular functions and thus limiting their biomedical applications. Here, we have developed a multiscale microfluidic system for the perfusion culture of spheroids, in which porous microchambers, connected with microfluidic channels, were engineered. A newly developed process of centrifugation-assisted replica molding and salt-leaching enabled the formation of single micrometer-sized pores on the chamber surface and in the substrate. The porous configuration generates a vertical flow to directly supply the medium to the spheroids, while avoiding the formation of stagnant flow regions. We created seamlessly integrated, all PDMS/silicone-based microfluidic devices with an array of microchambers. Spheroids of human liver cells (HepG2 cells) were formed and cultured under vertical-flow perfusion, and the proliferation ability and liver cell-specific functions were compared with those of cells cultured in non-porous chambers with a horizontal flow. The presented system realizes both size-controlled formation of spheroids and direct medium supply, making it suitable as a precision cell culture platform for drug development, disease modelling, and regenerative medicine.

Numerous attempts have been made to organize isolated primary hepatocytes into functional three-dimensional (3D) constructs, but technologies to introduce extracellular matrix (ECM) components into such assemblies have not been fully developed. Here we report a new approach to forming hepatocyte-based 3D tissues using fibrillized collagen microparticles (F-CMPs) as intercellular binders. We created thick tissues with a thickness of w200 mm simply by mixing FCMPs with isolated primary rat hepatocytes and culturing them in cell culture inserts. Owing to the incorporated FCMPs, the circular morphology of the formed tissues was stabilized, which was strong enough to be manually manipulated and retrieved from the chamber of the insert. We confirmed that the F-CMPs dramatically improved the cell viability and hepatocyte-specific functions such as albumin production and urea synthesis in the formed tissues. The presented approach provides a versatile strategy for hepatocyte-based tissue engineering, and will have a significant impact on biomedical applications and pharmaceutical research. (c) 2021, The Society for Biotechnology, Japan. All rights reserved.

Numerous attempts have been made to develop efficient systems to purify trace amounts of circulating tumor cells (CTCs) from blood samples. However, current technologies are limited by complexities in device fabrication, system design, and process operability. Here we describe a facile, scalable, and highly efficient approach to physically capturing CTCs using a rationally designed microfluidic isolator with an array of microslit channels. The wide but thin microslit channels with a depth of several micrometers selectively capture CTCs, which are larger and less deformable than other blood cells, while allowing other blood cells to just flow through. We investigated in detail the effects of the microchannel geometry and operating parameters on the capture efficiency and selectivity of several types of cultured tumor cells spiked in blood samples as the CTC model. Additionally, in situ post-capture staining of the captured cells was demonstrated to investigate the system's applicability to clinical cancer diagnosis. The presented approach is simple in operation but significantly effective in capturing specific cells and hence it may have great potential in implementating cell physics-based CTC isolation techniques for cancer liquid biopsy.

Although many types of technologies for hydrogel-based cell cultivation have recently been developed, strategies to integrate cell-adhesive micrometer-sized supports with bulk-scale hydrogel platforms have not been fully established. Here, we present a highly unique approach to produce cell-adhesive, protein-based microfibers assisted by the sacrificial template of alginate; we applied these fibers as microengineered scaffolds for hydrogel-based cell encapsulation. Two types of microfluidic devices were designed and fabricated: a single-layered device for producing relatively thick (φ of 10-60 μm) alginate-protein composite fibers with a uniform cross-sectional morphology and a four-layered device for preparing thinner (φ of ∼4 μm) ones through the formation of patterned microfibers with eight distinct alginate-protein composite regions. Following chemical cross-linking of protein molecules and the subsequent removal of the sacrificial alginate from the double-network matrices, microfibers composed only of cross-linked proteins were obtained. We used gelatin, albumin, and hemoglobin as the protein material, and the gelatin-based cell-adhesive fibers were further encapsulated in hydrogels together with the mammalian cells. We clarified that the thinner fibers were especially effective in promoting cell proliferation, and the shape of the constructs was maintained even after removing the hydrogel matrices. The presented approach offers cells with biocompatible solid supports that enhance cell adhesion and proliferation, paving the way for the next generation of techniques for tissue engineering and multicellular organoid formation.

Although many types of technologies for hydrogel-based cell cultivation have recently been developed, strategies to integrate cell-adhesive micrometer-sized supports with bulk-scale hydrogel platforms have not been fully established. Here, we present a highly unique approach to produce cell-adhesive, protein-based microfibers assisted by the sacrificial template of alginate; we applied these fibers as microengineered scaffolds for hydrogel-based cell encapsulation. Two types of microfluidic devices were designed and fabricated: a single-layered device for producing relatively thick (Phi of 10-60 mu m) alginate-protein composite fibers with a uniform cross-sectional morphology and a four-layered device for preparing thinner (Phi of similar to 4 mu m) ones through the formation of patterned microfibers with eight distinct alginate-protein composite regions. Following chemical cross-linking of protein molecules and the subsequent removal of the sacrificial alginate from the double-network matrices, microfibers composed only of cross-linked proteins were obtained. We used gelatin, albumin, and hemoglobin as the protein material, and the gelatin-based cell-adhesive fibers were further encapsulated in hydrogels together with the mammalian cells. We clarified that the thinner fibers were especially effective in promoting cell proliferation, and the shape of the constructs was maintained even after removing the hydrogel matrices. The presented approach offers cells with biocompatible solid supports that enhance cell adhesion and proliferation, paving the way for the next generation of techniques for tissue engineering and multicellular organoid formation.

Although many types of technologies for hydrogel-based cell cultivation have recently been developed, strategies to integrate cell-adhesive micrometer-sized supports with bulk-scale hydrogel platforms have not been fully established. Here, we present a highly unique approach to produce cell-adhesive, protein-based microfibers assisted by the sacrificial template of alginate; we applied these fibers as microengineered scaffolds for hydrogel-based cell encapsulation. Two types of microfluidic devices were designed and fabricated: a single-layered device for producing relatively thick (Φ of 10-60 μm) alginate-protein composite fibers with a uniform cross-sectional morphology and a four-layered device for preparing thinner (Φ of ∼4 μm) ones through the formation of patterned microfibers with eight distinct alginate-protein composite regions. Following chemical cross-linking of protein molecules and the subsequent removal of the sacrificial alginate from the double-network matrices, microfibers composed only of cross-linked proteins were obtained. We used gelatin, albumin, and hemoglobin as the protein material, and the gelatin-based cell-adhesive fibers were further encapsulated in hydrogels together with the mammalian cells. We clarified that the thinner fibers were especially effective in promoting cell proliferation, and the shape of the constructs was maintained even after removing the hydrogel matrices. The presented approach offers cells with biocompatible solid supports that enhance cell adhesion and proliferation, paving the way for the next generation of techniques for tissue engineering and multicellular organoid formation.

A new process is presented to fabricate basement membrane-mimetic cell co-culture platforms using microfabricated PDMS stencil plates. An aqueous solution of extracellular matrices (ECM) was introduced into the through-holes of the microstencil plate and then dried, to form vitrified and condensed ECM films. Because the microstencil plate physically supported the films, we were able to produce significantly thin ECM films with a thickness of 0.5-2 μm, which is comparable to the basement membranes in vivo (< 1 μm). We co-cultured heterotypic cells on both sides of the film, demonstrating the usability of the presented platform in investigating cellular physiology.

Here we present a new approach to rapid formation of collagen gel tubes using microfluidic devices made of phosphate particle-embedding PDMS (PP-PDMS). An acidic solution of collagen, introduced into the PP-PDMS channel, was rapidly transformed into a hydrogel layer on the channel surface because it was neutralized by the phosphate ions supplied from the PP-PDMS substrate. After removing the non-gelled collagen solution at the center, collagen gel tubes were obtained. Based on this concept, we successfully fabricated vascular tissue-like multilayered structures incorporating vascular endothelial cells (ECs) and smooth muscle cells (SMCs). The presented technique is highly useful for preparing functional tubular tissues that reconstituted the in vivo tissue-like extracellular matrix environments.

Here we present an efficient microfluidic system for selectively capturing circulating tumor cells (CTCs) from blood samples, employing parallelized structures of cell trappers. The thin configuration of the trapper (depth of several micrometers) enables capture of CTCs based on physical characteristics; CTCs are larger and less-deformable than other blood cells. Parallelization of the cell trappers achieved high-throughput processing of the blood sample (~100 µL/min) and a high capture efficiency (~90%) of CTCs despite the simple configuration of the trapper. The presented system would be applicable to easy, low-cost, and highly sensitive cancer diagnosis for tumor patients.

Here we present a medium exchange system for cell culture applications using microfluidic devices partially made of a micropored substrate as fluid drainage. The microdevices were fabricated by bonding a microchannel-embedding PDMS plate with a partially-pored plate, which was created via particle leaching technique. The medium of a cell/particle suspension was removed from the main channel through the micropores, achieving efficient exchange of the medium. We examined the medium-exchange behaviors using model microparticles and dyed solutions, demonstrating that liquid switching was rapidly achieved. The presented system would be highly useful as a new tool for various cell culture experiments.

Here we propose a new scheme for particle sorting using cross-flow microfluidic devices incorporating a porous PDMS substrate. The porous region, prepared by dissolving sacrificial particles encapsulated in the PDMS prepolymer, allows small particles selectively to flow through the pores, resulting in continuous size-based particle sorting. We designed and fabricated a parallelized channel system to enhance the throughput of particle sorting. As a cell-sorting application, direct enrichment of leukocytes from a diluted blood sample was demonstrated. The presented system does not require complicated fabrication processes of precise microstructures, and hence it would provide new insights into microfluidic particle/cell sorting technologies.

Here we present a unique perfusion cell culture system capable of forming uniformly-sized multicellular aggregates utilizing microcavities embedded in a spongelike PDMS matrix. The combination of a particle leaching method and a replica molding technique allows us to integrate microfluidic channels and the microcavities, where the medium flow could be directly supplied through the cavities via the surrounding continuous micropores. We cultured mammalian cells and achieved high cell viability because of the efficient supply of the culture medium to the cells. The presented system offers a versatile approach to the efficient perfusion culture of mammalian cells for various biochemical applications.

Here we propose an efficient approach to processing mammalian cells using microfluidic devices integrated with spongious PDMS matrices having continuous micropores. The micropores on the sponge surface created by dissolving sacrificial microparticles were able to gently trap cells, followed by multistep cell treatment and staining by repeated introduction of reagents. Intercellular molecules were successfully stained with minimal reagent consumption and minimal cell loss. Further cell recovery was also possible by reverse infusion of a buffer. The presented system would help biological researchers in processing cells for biological studies and clinical diagnostics because of its simplicity in operation and versatility.

We herein propose a new hydrodynamic mechanism of particle separation using dual-depth, lattice-patterned asymmetric microchannel networks. This mechanism utilizes three-dimensional (3D) laminar flow profiles formed at intersections of lattice channels. Large particles, primarily flowing near the bottom surface, frequently enter the shallower channels (separation channels), whereas smaller particles flowing near the microchannel ceiling primarily flow along the deeper channels (main channels). Consequently, size-based continuous particle separation was achieved in the lateral direction in the lattice area. We confirmed that the depth of the main channel was a critical factor dominating the particle separation efficiencies, and the combination of 15-μm-deep separation channels and 40-μm-deep main channels demonstrated the good separation ability for 3-10-μm particles. We prepared several types of microchannels and successfully tuned the particle separation size. Furthermore, the input position of the particle suspension was controlled by adjusting the input flow rates and/or using a Y-shaped inlet connector that resulted in a significant improvement in the separation precision. The presented concept is a good example of a new type of microfluidic particle separation mechanism using 3D flows and may potentially be applicable to the sorting of various types of micrometer-sized objects, including living cells and synthetic microparticles.

We herein propose a new hydrodynamic mechanism of particle separation using dual-depth, lattice-patterned asymmetric microchannel networks. This mechanism utilizes three-dimensional (3D) laminar flow profiles formed at intersections of lattice channels. Large particles, primarily flowing near the bottom surface, frequently enter the shallower channels (separation channels), whereas smaller particles flowing near the microchannel ceiling primarily flow along the deeper channels (main channels). Consequently, size-based continuous particle separation was achieved in the lateral direction in the lattice area. We confirmed that the depth of the main channel was a critical factor dominating the particle separation efficiencies, and the combination of 15-μm-deep separation channels and 40-μm-deep main channels demonstrated the good separation ability for 3-10-μm particles. We prepared several types of microchannels and successfully tuned the particle separation size. Furthermore, the input position of the particle suspension was controlled by adjusting the input flow rates and/or using a Y-shaped inlet connector that resulted in a significant improvement in the separation precision. The presented concept is a good example of a new type of microfluidic particle separation mechanism using 3D flows and may potentially be applicable to the sorting of various types of micrometer-sized objects, including living cells and synthetic microparticles.

We herein propose a new hydrodynamic mechanism of particle separation using dual-depth, lattice-patterned asymmetric microchannel networks. This mechanism utilizes three-dimensional (3D) laminar flow profiles formed at intersections of lattice channels. Large particles, primarily flowing near the bottom surface, frequently enter the shallower channels (separation channels), whereas smaller particles flowing near the microchannel ceiling primarily flow along the deeper channels (main channels). Consequently, size-based continuous particle separation was achieved in the lateral direction in the lattice area. We confirmed that the depth of the main channel was a critical factor dominating the particle separation efficiencies, and the combination of 15-mu m-deep separation channels and 40-mu m-deep main channels demonstrated the good separation ability for 3-10-mu m particles. We prepared several types of microchannels and successfully tuned the particle separation size. Furthermore, the input position of the particle suspension was controlled by adjusting the input flow rates and/or using a Y-shaped inlet connector that resulted in a significant improvement in the separation precision. The presented concept is a good example of a new type of microfluidic particle separation mechanism using 3D flows and may potentially be applicable to the sorting of various types of micrometer-sized objects, including living cells and synthetic microparticles.

With the recent progress in three-dimensional (3D) cell culture techniques for regenerative medicine and drug development, hydrogel-based tissue engineering approaches that can precisely organize cells into functional formats have attracted increasing attention. However, challenges remain in creating continuous microconduits within hydrogels to effectively deliver oxygen and nutrients to the embedded cells. Here we propose a one-step, fully liquid state, and all-aqueous process to create porous hydrogels that can encapsulate living cells without the need for extensive processing protocols, including the incorporation and removal of sacrificial materials. An unusual bicontinuous state of aqueous two-phase dispersion was utilized, and one of the two phases, encapsulating living cells, was rapidly photo-cross-linked to form hydrogel sponges. We optimized the volumetric mixing ratio of gelatin methacrylate (GelMA)-rich and polyethylene glycol (PEG)-rich solutions and investigated the effects of the formed continuous microconduits on the cell functions by creating liver-tissue mimetic 3D constructs. The presented technology provides a facile and versatile strategy for fabricating microstructured hydrogels for cell culture and would bring new insights for the development of porous materials by fully aqueous bicontinuous dispersions.

Thin membrane-based cell culture platforms have recently gained much attention for facilitating separated but adjacent cocultures of heterotypic cells in in vivo tissue-mimetic microenvironments. Here we propose a new approach to preparing significantly thin but highly stable membranes composed of extracellular matrix (ECM) components, mainly type I collagen, that are supported by PDMS microstencil plates. A collagen solution was introduced into the through holes of a microstencil plate and dried to form condensed membranes with a thickness of less than 1 μm. A coculture of HepG2 and Swiss-3T3 cells was performed on both membrane surfaces, and gene expression assays revealed the superiority of the presented thin membranes. Release of the membranes from the plate was possible, as was the integration of the membranes into microfluidic systems to perform perfusion cultures of cells. We were also able to prepare Matrigel membranes by the same procedure. The presented thin membrane-based cell culture platforms would provide physiologically suitable conditions for cells and thus would be widely applicable to a variety of in vitro cell culture systems.

With the recent progress in three-dimensional (3D) cell culture techniques for regenerative medicine and drug development, hydrogel-based tissue engineering approaches that can precisely organize cells into functional formats have attracted increasing attention. However, challenges remain in creating continuous microconduits within hydrogels to effectively deliver oxygen and nutrients to the embedded cells. Here we propose a one-step, fully liquid state, and all-aqueous process to create porous hydrogels that can encapsulate living cells without the need for extensive processing protocols, including the incorporation and removal of sacrificial materials. An unusual bicontinuous state of aqueous two-phase dispersion was utilized, and one of the two phases, encapsulating living cells, was rapidly photo-cross-linked to form hydrogel sponges. We optimized the volumetric mixing ratio of gelatin methacrylate (GelMA)-rich and polyethylene glycol (PEG)-rich solutions and investigated the effects of the formed continuous microconduits on the cell functions by creating liver-tissue mimetic 3D constructs. The presented technology provides a facile and versatile strategy for fabricating microstructured hydrogels for cell culture and would bring new insights for the development of porous materials by fully aqueous bicontinuous dispersions.

Thin membrane-based cell culture platforms have recently gained much attention for facilitating separated but adjacent cocultures of heterotypic cells in in vivo tissue-mimetic microenvironments. Here we propose a new approach to preparing significantly thin but highly stable membranes composed of extracellular matrix (ECM) components, mainly type I collagen, that are supported by PDMS microstencil plates. A collagen solution was introduced into the through holes of a microstencil plate and dried to form condensed membranes with a thickness of less than 1 mu m. A coculture of HepG2 and Swiss-3T3 cells was performed on both membrane surfaces, and gene expression assays revealed the superiority of the presented thin membranes. Release of the membranes from the plate was possible, as was the integration of the membranes into microfluidic systems to perform perfusion cultures of cells. We were also able to prepare Matrigel membranes by the same procedure. The presented thin membrane-based cell culture platforms would provide physiologically suitable conditions for cells and thus would be widely applicable to a variety of in vitro cell culture systems.

Here we proposed a new high-throughput, continuous microfluidic device for particle/cell separation based on pinched flow fractionation. In the newly-proposed system, a wide and shallow microchannel is fabricated and particles are separated in the depth-direction of the microchannel. We successfully increased the throughput of the particle separation with high accuracy simply by enlarging the microchannel only in the width direction without the fabrication difficulty. The presented particle/cell separation design (Vertical Slit-Fractionation) would be useful for versatile high-throughput systems for particle/cell separation.

Here we present lateral flow immunoassay platforms consisted of polymeric sheets with micro/nanoimprinted multiscale architectures. Microcone arrays with nanometer-scale surface roughness were prepared by thermal nano-imprinting. The dramatically enhanced surface area of the microcones contributed to effective immobilization of primary antibodies. Liquid samples were transported through the sheet with the help of a surfactant added to the sample/reagent solutions. We successfully demonstrated immunoassays of C-reactive protein spiked in serum with the detection limit of ~0.1 μg mL . The presented immunoassay platforms are advantageous because of the high reproducibility and sensitivity, and would be suitable for point-of-care testing. -1

Here we present a unique process to fabricate type-I collagen hydrogels integrated in microfluidic devices, with the help of gelation agent-incorporating composite PDMS substrates. We fabricated microfluidic devices made of phosphate particle-embedding polydimethylsiloxane (PP-PDMS). Simply by introducing an acidic collagen solution into the channels, the solution near the PP-PDMS surface was selectively gelled, because of the neutralization effects of the phosphate ions. We successfully fabricated cell-encapsulating collagen microgels, and performed perfusion cultivation for the cells, showing the high usability of the presented approach. The presented collagen based microgels would be useful as cell culture platforms for organs-on-a-chip applications.

Here we present a particle separation system utilizing microengineered PDMS plates with micrometer-sized continuous pores as the sieving matrix. The continuous micropores were formed using sacrificial microparticles embedded in and dissolved from the PDMS substrate. Filtration devices were fabricated by sandwiching the porous PDMS plate with upper/lower plates having planar microchannels. We confirmed that the separation of submicrometer-sized particles was possible even when we employed sacrificial NaCl particles with several ten micrometers, showing the high sieving ability of the microporous PDMS. This system would be significantly useful as a new tool for separating/sorting various types of synthetic and biological microparticles.

Although various types of hydrogel-based 3D cell culture systems have been developed, creation of micrometer-sized capillaries as the conduits in bulk-scale hydrogels still remains a challenge. Here we present a highly efficient strategy to form cell-encapsulating hydrogels equipped with densely-packed continuous micropores. Sacrificial alginate-based microfibers were prepared using microfluidic devices, which were fragmented and incorporated in the hydrogel precursor solutions. Simply by dissolving the fibers after hydrogel formation, microcapillaries with diameters of 10-30 μm were formed. The presented approach would be useful for cell culture applications including cellular physiological studies, drug development, and tissue/organ regeneration.

Here we present a new process to prepare cell co-culture platforms incorporating thin extracellular matrix (ECM) membranes. We utilized PDMS microstencil plates to generate thin ECM membranes, which were physically stabilized in the micro through holes of the stencil plate. An aqueous solution of ECM (type I collagen or Matrigel) was introduced into the micro-holes of the stencil plate, and then it was dried to form a condensed ECM membrane. The thickness of the ECM membrane was controlled from 0.1 to 0.5 μm simply by changing the initial concentration of the ECM solution and/or the depth of the micro-holes. We performed heterotypic cell co-culture on both surfaces of the ECM membranes, demonstrating the high usability for mammalian cell culture. The presented thin ECM membranes would be applicable to the reconstruction of cell-friendly environment mimicking the in vivo tissue structures and to the cell-based drug assay systems.

Although various types of hydrogel-based 3D cell culture platforms have been developed, it is difficult to prepare porous hydrogels encapsulating intact cells and having capillary networks with a one-step procedure. Here we propose a facile strategy to prepare microvasculature-embedding porous hydrogels for 3D cell culture, utilizing an aqueous two-phase dispersion system. We used an aqueous solution of photo-crosslinkable gelatin (GelMA) and polyethylene glycol (PEG) to form an aqueous two-phase dispersion system. A bi-continuous state was generated by re-mixing these two phases at a proper mixing ratio, and a sponge-like, porous hydrogel could be formed by selectively crosslinking the GelMA-rich phase via UV irradiation. We successfully encapsulated mammalian cells in the hydrogel matrix with a high cell viability, and confirmed that the porous hydrogel was superior to homogeneous hydrogels for cell culture. The presented hydrogel would be applicable to 3D-cell culture for various types of biomedical applications.

In this study, we propose a new cell culture system using microengineered hierarchical hydrogel sheets, which enables guidance and evaluation of cancer cell invasion in 3D environments. Three-layered hydrogel sheets, which encapsulated cancer cells in the patterned middle layer, were placed on a collagen hydrogel. Because the direction of nutrition is only from the collagen gel to the sheets, cell invasion was guided to the basal collagen gel as in the case of epithelial tissues. In the experiment, we fabricated hierarchically-engineered alginate-based hydrogel sheets using multi-laminated microfluidic devices, and clearly demonstrated that embedded cancer cells invasion was guided to the collagen layer. By counting the number of cells reaching the collagen gel, the evaluation of cell invasion would become possible. The proposed system would be useful because it realizes facile and precise cell migration assays in 3D platforms.

Although many types of hydrogel-based cell culture platforms have been proposed, techniques for preparing microvasculature-embedded hydrogels haven’t been fully developed. Here we propose a facile and versatile strategy to fabricate hydrogel sponges, which can embed intact cells into the matrix, utilizing an aqueous two-phase system. GelMA- and PEG-rich phases were dispersed to form a bicontinuous state, then the GelMA-rich phase was selectively gelled to form the micro-sponge structures. We cultured mammalian cells in the sponges and evaluated the cell functions. Additionally, we performed perfusion culture, successfully demonstrating the usability of the presented platform in constructing tissue models or organs-on-a-chip systems.

Here we present a microfluidic system to produce linear cell assemblies, which were simultaneously wrapped with thin collagen membranes, using multilayered microfluidic devices. A cell suspension with a polymeric thickener (for core) and an aqueous solution of collagen (for shell) were concentrically patterned using the micronozzle-array structure. The shell of the collagen solution was subsequently gelled into thin membranes by phosphate-based pH modulation, resulting in the formation of linear assemblies of cells. We successfully generated hepatic-cord like microorganoids composed of liver cells. The presented strategy is highly useful as a unique technique to prepare linear tissue models for drug evaluation and cellular biological studies.

Although many researchers have been tackling on the sorting/capture of circulating tumor cells (CTCs) in blood samples, highly efficient, user-friendly, and cost-effective methodologies are still under development. Here we demonstrated that an extremely simple, thin and planar slit channel system effectively works as a deformabilitybased capture platform for CTCs. We found that the depth of the slit channels (2-5 μm) is a key parameter dominating the trapping efficiency. Furthermore, release of the captured CTCs was possible simply by applying a high flow rate. The presented microfluidic system would be useful as a new platform to detect/characterize various types of rare cell populations in a blood sample.

Although various types of hydrogel-based 3D cell culture platforms have been developed, it is difficult to prepare porous hydrogels encapsulating intact cells and having capillary networks with a one-step procedure. Here we propose a facile strategy to prepare microvasculature-embedding porous hydrogels for 3D cell culture, utilizing an aqueous two-phase dispersion system. We used an aqueous solution of photo-crosslinkable gelatin (GelMA) and polyethylene glycol (PEG) to form an aqueous two-phase dispersion system. A bi-continuous state was generated by re-mixing these two phases at a proper mixing ratio, and a sponge-like, porous hydrogel could be formed by selectively crosslinking the GelMA-rich phase via UV irradiation. We successfully encapsulated mammalian cells in the hydrogel matrix with a high cell viability, and confirmed that the porous hydrogel was superior to homogeneous hydrogels for cell culture. The presented hydrogel would be applicable to 3D-cell culture for various types of biomedical applications.

Here we present a new process to prepare cell co-culture platforms incorporating thin extracellular matrix (ECM) membranes. We utilized PDMS microstencil plates to generate thin ECM membranes, which were physically stabilized in the micro through holes of the stencil plate. An aqueous solution of ECM (type I collagen or Matrigel) was introduced into the micro-holes of the stencil plate, and then it was dried to form a condensed ECM membrane. The thickness of the ECM membrane was controlled from similar to 0.1 to similar to 0.5 mu m simply by changing the initial concentration of the ECM solution and/or the depth of the micro-holes. We performed heterotypic cell co-culture on both surfaces of the ECM membranes, demonstrating the high usability for mammalian cell culture. The presented thin ECM membranes would be applicable to the reconstruction of cell-friendly environment mimicking the in vivo tissue structures and to the cell-based drug assay systems.

In this study, we propose a new cell culture system using microengineered hierarchical hydrogel sheets, which enables guidance and evaluation of cancer cell invasion in 3D environments. Three-layered hydrogel sheets, which encapsulated cancer cells in the patterned middle layer, were placed on a collagen hydrogel. Because the direction of nutrition is only from the collagen gel to the sheets, cell invasion was guided to the basal collagen gel as in the case of epithelial tissues. In the experiment, we fabricated hierarchically-engineered alginate-based hydrogel sheets using multi-laminated microfluidic devices, and clearly demonstrated that embedded cancer cells invasion was guided to the collagen layer. By counting the number of cells reaching the collagen gel, the evaluation of cell invasion would become possible. The proposed system would be useful because it realizes facile and precise cell migration assays in 3D platforms.

Here we present microfluidic systems to produce micrometer-sized particles made of proteins, using monodisperse droplets in a non-equilibrium state. Droplets of an aqueous solution of protein were formed in the continuous phase of a water-dissolving polar organic solvent, and then the droplets were shrunk because of the dissolution of water molecules. Protein molecules were precipitated, and then stable protein microparticles were obtained after the crosslinking reaction. We obtained spherical/non-spherical protein microparticles using several types of proteins, and examined factors affecting the particle morphology. In particular, highly unique tear-drop-shaped particles and hemispherical particles were obtained when incompletely dehydrated droplets were crosslinked. In addition, we cultured mammalian cells on the particles made of collagen, demonstrating the applicability of the particles as cell-culture scaffolds. The presented protein microparticles would be useful as carriers or scaffolds for various biological/medical applications.