High-Density Microporous Drainage-Integrating Sheath Flow Generator for Streamlining Microfluidic Cell Sorting Systems.

Tremendous efforts have been made to develop practical and efficient microfluidic cell and particle sorting systems; however, there are technological limitations in terms of system complexity and low operability. Here, we propose a sheath flow generator that can dramatically simplify operational procedures and enhance the usability of microfluidic cell sorters. The device utilizes an embedded polydimethylsiloxane (PDMS) sponge with interconnected micropores, which is in direct contact with microchannels and seamlessly integrated into the microfluidic platform. The high-density micropores on the sponge surface facilitated fluid drainage, and the drained fluid was used as the sheath flow for downstream cell sorting processes. To fabricate the integrated device, a new process for sponge-embedded substrates was developed through the accumulation, incorporation, and dissolution of PMMA microparticles as sacrificial porogens. The effects of the microchannel geometry and flow velocity on the sheath flow generation were investigated. Furthermore, an asymmetric lattice-shaped microchannel network for cell/particle sorting was connected to the sheath flow generator in series, and the sorting performances of model particles, blood cells, and spiked tumor cells were investigated. The sheath flow generation technique developed in this study is expected to streamline conventional microfluidic cell-sorting systems as it dramatically improves versatility and operability.

Process simplification and structure design of parallelized microslit isolator for physical property-based capture of tumor cells.

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.

Laborless, Automated Microfluidic Tandem Cell Processor for Visualizing Intracellular Molecules of Mammalian Cells.

Visualization and quantification of intracellular molecules of mammalian cells are crucial steps in clinical diagnosis, drug development, and basic biological research. However, conventional methods rely mostly on labor-intensive, centrifugation-based manual operations for exchanging the cell carrier medium and have limited reproducibility and recovery efficiency. Here we present a microfluidic cell processor that can perform four-step exchange of carrier medium, simply by introducing a cell suspension and fluid reagents into the device. The reaction time period for each reaction step, including fixation, membrane permeabilization, and staining, was tunable in the range of 2 to 15 min by adjusting the volume of the reaction tube connecting the neighboring exchanger modules. We double-stained the cell nucleus and cytoskeleton (F-actin) using the presented device with an overall reaction period of ∼30 min, achieving a high recovery ratio and high staining efficiency. Additionally, intracellular cytokine (IL-2) was visualized for T cells to demonstrate the feasibility of the device as a pretreatment system for downstream flow-cytometric analysis. The presented approach would facilitate the development of laborless, automated microfluidic systems that integrate cell processing and analysis operations and would pave a new path to high-throughput biological experiments.

Adult hepatocytes direct liver organogenesis through non-parenchymal cell recruitment in the kidney.

BACKGROUND & AIMS: Since the first account of the myth of Prometheus, the amazing regenerative capacity of the liver has fascinated researchers because of its enormous medical potential. Liver regeneration is promoted by multiple types of liver cells, including hepatocytes and liver non-parenchymal cells (NPCs), through complex intercellular signaling. However, the mechanism of liver organogenesis, especially the role of adult hepatocytes at ectopic sites, remains unknown. In this study, we demonstrate that hepatocytes alone spurred liver organogenesis to form an organ-sized complex 3D liver that exhibited native liver architecture and functions in the kidneys of mice. METHODS: Isolated hepatocytes were transplanted under the kidney capsule of monocrotaline (MCT) and partial hepatectomy (PHx)-treated mice. To determine the origin of NPCs in neo-livers, hepatocytes were transplanted into MCT/PHx-treated green fluorescent protein transgenic mice or wild-type mice transplanted with bone marrow cells isolated from green fluorescent protein-mice. RESULTS: Hepatocytes engrafted at the subrenal space of mice underwent continuous growth in response to a chronic hepatic injury in the native liver. More than 1.5 years later, whole organ-sized liver tissues with greater mass than those of the injured native liver had formed. Most remarkably, we revealed that at least three types of NPCs with similar phenotypic features to the liver NPCs were recruited from the host tissues including bone marrow. The neo-livers in the kidney exhibited liver-specific functions and architectures, including sinusoidal vascular systems, zonal heterogeneity, and emergence of bile duct cells. Furthermore, the neo-livers successfully rescued the mice with lethal liver injury. CONCLUSION: Our data clearly show that adult hepatocytes play a leading role as organizer cells in liver organogenesis at ectopic sites via NPC recruitment. LAY SUMMARY: The role of adult hepatocytes at ectopic locations has not been clarified. In this study, we demonstrated that engrafted hepatocytes in the kidney proliferated, recruited non-parenchymal cells from host tissues including bone marrow, and finally created an organ-sized, complex liver system that exhibited liver-specific architectures and functions. Our results revealed previously undescribed functions of hepatocytes to direct liver organogenesis through non-parenchymal cell recruitment and organize multiple cell types into a complex 3D liver at ectopic sites. Transcript profiling: Microarray data are deposited in GEO (GEO accession: GSE99141).

Chimeric mice with hepatocyte-humanized liver as an appropriate model to study human peroxisome proliferator-activated receptor-α.

Peroxisome proliferator (PP)-activated receptor-α (PPARα) agonists exhibit species-specific effects on livers of the rodent and human (h), which has been considered to reside in the difference of PPARα gene structures. However, the contribution of h-hepatocytes (heps) to the species-specificity remains to be clarified. In this study, the effects of fenofibrate were investigated using a hepatocyte-humanized chimeric mouse (m) model whose livers were replaced with h-heps at >70%. Fenofibrate induced hepatocellular hypertrophy, cell proliferation, and peroxisome proliferation in livers of severe combined immunodeficiency (SCID) mice, but not in the h-hep of chimeric mouse livers. Fenofibrate increased the expression of the enzymes of ß- and ω-hydroxylation and deoxygenation of lipids at both gene and protein levels in SCID mouse livers, but not in the h-heps of chimeric mouse livers, supporting the studies with h-PPARα-transgenic mice, a hitherto reliable model for studying the regulation of h-PPARα in the h-liver in most respects, except the induction of the peroxisome proliferation. This study indicates the importance of not only h-PPARα gene but also h-heps themselves to correctly predict effects of fibrates on h-livers, and, therefore, suggests that the chimeric mouse is a currently available, consistent, and reliable model to obtain pharmaceutical data concerning the effects of fibrates on h-livers.

bFGF-releasing biodegradable nanoparticles for effectively engrafting transplanted hepatocyte sheet.

Hepatic tissue engineering has been applied for the treatment of intractable liver diseases, and hepatocyte sheets are promising for this purpose. However, hepatocyte sheets have poor survival after transplantation because of their high metabolic activity. In this study, we aimed to develop basic fibroblast growth factor (bFGF)-releasing nanoparticles to prolong the survival of hepatocyte sheets after transplantation. The nanoparticles were prepared by electrospraying a bFGF-dispersed poly(D,l-lactide-co-glycolide) emulsion. bFGF-loaded PLGA nanoparticles can be developed by optimizing the applied electrospray voltage and the oil:water ratio of the emulsion. The prepared nanoparticles exhibited prompt release at the initial duration and continuous gradual release at the subsequent duration. Hepatocyte sheet engraftment was evaluated by transplanting hepatocyte sheets containing the prepared nanoparticles into rats. The hepatocyte sheets with the prepared nanoparticles exhibited longer survival than those without the bFGF nanoparticles or solution owing to the local and continuous release of bFGF from the nanoparticles and the subsequent enhanced angiogenesis at the transplantation site. These results indicated that the prepared bFGF-releasing nanoparticles can enhance the efficiency of hepatocyte sheet transplantation. The developed bFGF-releasing nanoparticles would be useful for the transplantation of cellular tissue with post-transplantation survival challenges.

Tissue factor triggers procoagulation in transplanted mesenchymal stem cells leading to thromboembolism.

Mesenchymal stem cells (MSCs) have shown extreme clinical promise as a therapeutic regenerative system in the treatment of numerous types of diseases. A recent report, however, documented lethal pulmonary thromboembolism in a patient following the administration of adipose-derived MSCs (ADSCs). In our study, we designed experiments to examine the role of tissue factor (TF), which is highly expressed at the level of mRNA and localized to the cell surface of cultured MSCs, as a triggering factor in the procoagulative cascade activated by infused MSCs. A high mortality rate of ~85% in mice was documented following intravenous infusion of mouse ADSCs within 24 h due to the observation of pulmonary embolism. Rotation thromboelastometry and plasma clotting assay demonstrated significant procoagulation by the cultured mouse ADSCs, and preconditioning of ADSCs with an anti-TF antibody or usage of factor VII deficient plasma in the assay successfully suppressed the procoagulant properties. These properties were also observed in human ADSCs, and could be suppressed by recombinant human thrombomodulin. In uncultured mouse adipose-derived cells (ADCs), the TF-triggered procoagulant activity was not observed and all mice infused with these uncultured ADCs survived after 24 h. This clearly demonstrated that the process of culturing cells plays a critical role in sensitizing these cells as a procoagulator through the induction of TF expression. Our results would recommend that clinical applications of MSCs to inhibit TF activity using anti-coagulant agents or genetic approaches to maximize clinical benefit to the patients.

A multiscale, vertical-flow perfusion system with integrated porous microchambers for upgrading multicellular spheroid culture.

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.

Expansion of Chinese hamster ovary cells via a loose cluster-assisted suspension culture using cell-sized gelatin microcarriers.

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.

Formation of 3D tissues of primary hepatocytes using fibrillized collagen microparticles as intercellular binders.

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 ∼200 µm simply by mixing F-CMPs with isolated primary rat hepatocytes and culturing them in cell culture inserts. Owing to the incorporated F-CMPs, 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.

Hepatic hyperplasia associated with discordant xenogeneic parenchymal-nonparenchymal interactions in human hepatocyte-repopulated mice.

Liver mass is optimized in relation to body mass. Rat (r) and human (h) hepatocytes were transplanted into liver-injured immunodeficient mice and allowed to proliferate for 3 or 11 weeks, respectively, when the transplants stopped proliferating. Liver/body weight ratio was normal throughout in r-hepatocyte-bearing mice (r-hep-mice), but increased continuously in h-hepatocyte-bearing mice (h-hep-mice), until reaching approximately three times the normal m-liver size, which was considered to be hyperplasia of h-hepatocytes because there were no significant differences in cell size among host (mouse [m-]) and donor (r- and h-) hepatocytes. Transforming growth factor-beta (TGF-beta) type I receptor, TGF-beta type II receptor, and activin A type IIA receptor mRNAs in proliferating r-hepatocytes of r-hep-mice were lower than in resting r-hepatocytes (normal levels) and increased to normal levels during the termination phase. Concomitantly, m-hepatic stellate cells began to express TGF-beta proteins. In stark contrast, TGF-beta type II receptor and activin A type IIA receptor mRNAs in h-hepatocytes remained low throughout and m-hepatic stellate cells did not express TGF-beta in h-hep-mice. As expected, Smad2 and 3 translocated into nuclei in r-hep-mice but not in h-hep-mice. Histological analysis showed a paucity of m-stellate cells in h-hepatocyte colonies of h-hep-mouse liver. We conclude that m-stellate cells are able to normally interact with concordant r-hepatocytes but not with discordant h-hepatocytes, which seems to be at least partly responsible for the failure of the liver size optimization in h-hep-mice.

Polyanion-induced, microfluidic engineering of fragmented collagen microfibers for reconstituting extracellular environments of 3D hepatocyte culture.

Artificial biological scaffolds made of extracellular matrix (ECM) components, such as type I collagen, provide ideal physicochemical cues to various cell culture platforms. However, it remains a challenge to fabricate micrometer-sized ECM materials with precisely controlled morphologies that could reconstitute the 3-dimensional (3D) microenvironments surrounding cells. In the present study, we proposed a unique process to fabricate fragmented collagen microfibers using a microfluidic laminar-flow system. The continuous flow of an acidic collagen solution was neutralized to generate solid fibers, which were subsequently fragmented by applying a gentle shear stress in a polyanion-containing phosphate buffer. The morphology of the fiber fragment was controllable in a wide range by changing the type and/or concentration of the polyanion and by tuning the applied shear stress. The biological benefits of the fragmented fibers were investigated through the formation of multicellular spheroids composed of primary rat hepatocytes and microfibers on non-cell-adhesive micro-vessels. The microfibers enhanced the survival and functions of the hepatocytes and reproduced proper cell polarity, because the fibers facilitated the formation of cell-cell and cell-matrix interactions while modulating the close packing of cells. These results clearly indicated that the microengineered fragmented collagen fibers have great potential to reconstitute extracellular microenvironments for hepatocytes in 3D culture, which will be of significant benefit for cell-based drug testing and bottom-up tissue engineering.

Sacrificial Alginate-Assisted Microfluidic Engineering of Cell-Supportive Protein Microfibers for Hydrogel-Based Cell Encapsulation.

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.

Susceptibility of chimeric mice with livers repopulated by serially subcultured human hepatocytes to hepatitis B virus.

UNLABELLED: We previously identified a small population of replicative hepatocytes in long-term cultures of human adult parenchymal hepatocytes (PHs) at a frequency of 0.01%-0.09%. These hepatocytes were able to grow continuously through serial subcultures as colony-forming parenchymal hepatocytes (CFPHs). In the present study, we generated gene expression profiles for cultured CFPHs and found that they expressed cytokeratin 19, CD90 (Thy-1), and CD44, but not mature hepatocyte markers such as tryptophan-2,3-dioxygenase (TO) and glucose-6-phosphatase (G6P), confirming that these cells are hepatic progenitor-like cells. The cultured CFPHs were resistant to infection with human hepatitis B virus (HBV). To examine the growth and differentiation capacity of the cells in vivo, serially subcultured CFPHs were transplanted into the progeny of a cross between albumin promoter/enhancer-driven urokinase plasminogen activator-transgenic mice and severe combined immunodeficient (SCID) mice. The cells were engrafted into the liver and were able to grow for at least 10 weeks, ultimately reaching a maximum occupancy rate of 27%. The CFPHs in the host liver expressed differentiation markers such as TO, G6P, and cytochrome P450 subtypes and could be infected with HBV. CFPH-chimeric mice with a relatively high replacement rate exhibited viremia and had high serum levels of hepatitis B surface antigen. CONCLUSION: Serially subcultured human hepatic progenitor-like cells from postnatal livers successfully repopulated injured livers and exhibited several phenotypes of mature hepatocytes, including susceptibility to HBV. In vitro-expanded CFPHs can be used to characterize the differentiation state of human hepatic progenitor-like cells.

One-Step Formation of Microporous Hydrogel Sponges Encapsulating Living Cells by Utilizing Bicontinuous Dispersion of Aqueous Polymer Solutions.

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.

Formation of pressurizable hydrogel-based vascular tissue models by selective gelation in composite PDMS channels.

Vascular tissue models created in vitro are of great utility in the biomedical research field, but versatile, facile strategies are still under development. In this study, we proposed a new approach to prepare vascular tissue models in PDMS-based composite channel structures embedded with barium salt powders. When a cell-containing hydrogel precursor solution was continuously pumped in the channel, the precursor solution in the vicinity of the channel wall was selectively gelled because of the barium ions as the gelation agent supplied to the flow. Based on this concept, we were able to prepare vascular tissue models, with diameters of 1-2 mm and with tunable morphologies, composed of smooth muscle cells in the hydrogel matrix and endothelial cells on the lumen. Perfusion culture was successfully performed under a pressurized condition of ∼120 mmHg. The presented platform is potentially useful for creating vascular tissue models that reproduce the physical and morphological characteristics similar to those of vascular tissues in vivo.

Suitable reference genes for the analysis of direct hyperplasia in mice.

The liver is capable of undergoing a proliferative growth, known as direct hyperplasia, in which the naïve liver increases in size due to stimulation with primary mitogens. To produce accurate gene expression data, housekeeping genes (HKGs) that are stably expressed need to be determined. In the present study, liver regeneration was promoted via the direct hyperplasia mode by inducing mice with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene. Gene expression levels of nine commonly used HKGs were analyzed in the liver of different timing during the regeneration. The stability of gene expression was assessed using two different analysis programs, geNorm and NormFinder. Using these analyses, we identified that PPIA and RPL4 showed the most stable expression regardless of the status of the liver regeneration. In conclusion, the present study demonstrated that the use of PPIA and RPL4 were the most optimal in providing reliable normalization of gene expression when assessing liver regeneration attributed to direct hyperplasia.

Engraftment of human hepatocytes in the livers of rats bearing bone marrow reconstructed with immunodeficient mouse bone marrow cells.

BACKGROUND: Previously, we created, a chimeric mouse (humanized mouse), a severe combined immunodeficiency (SCID) mouse whose liver was >90% repopulated with human (h)-hepatocytes, which are useful for the testing of drug metabolism and toxicity, as well as a hepatitis B virus and hepatitis C virus-susceptible animal model. However, their small body size and small total blood volume limited the utilization for analytical purposes, which led us to develop a method to create a chimeric rat bearing h-hepatocyte-repopulated liver. METHODS: F344 nude rats devoid of T cells were irradiated with X-rays and injected with bone marrow cells (BMCs) from SCID mice (m(SCID)). The rate of replacement with m(SCID)-BMCs was evaluated by two-color flow cytometry analysis of peripheral blood mononuclear cells (PBMCs). After m(SCID)-BMCs repopulated the host bone marrow (BM), the rats were treated with retrorsine, partially hepatectomized (PHx), and transplanted with 5 x 10(6) h-hepatocytes isolated from the chimeric mice. h-Albumin (h-Alb) concentrations in the host blood and the expression levels of protein and mRNA of hepatocyte differentiation markers in the h-hepatocytes were evaluated by ELISA, immunostaining, and reverse transcription-PCR, respectively. RESULTS: The m(SCID)-BMCs successfully repopulated the rats, the percentage of mouse cells reaching 94% among host (r(nudeF344)) PBMCs at 4 weeks after m-BMC transplantation. h-Hepatocytes isolated from the chimeric mice were transplanted to the liver of the m(SCID)-BMC-repopulated rats. The engrafted h-hepatocytes expressed h-Alb and h-cytochrome P450 (CYP) subtypes and survived showing normal phenotypes until at least 3 weeks post-h-hepatocytes transplantation (h-HPCT). However, the blood concentrations of h-Alb declined at 4 weeks post-HPCT, concomitant with the emergence of both r(nudeF344)- and m(SCID)-macrophages, suggesting the rejection of h-hepatocytes due to the activation of macrophages. CONCLUSION: We developed a novel method to create a rat that bears the liver engrafted with h-hepatocytes, utilizing a rat with the BM composed of m(SCID)-BMCs as a host. This h-hepatocyte-bearing rat will be a valuable model for studying the immunologic mechanisms involved in xenogeneic transplantation and for generating rats with higher rates of repopulation with h-hepatocytes.

Development of a perfusable 3D liver cell cultivation system via bundling-up assembly of cell-laden microfibers.

Although the reconstruction of functional 3D liver tissue models in vitro presents numerous challenges, it is in great demand for drug development, regenerative medicine, and physiological studies. Here we propose a new approach to perform perfusion cultivation of liver cells by assembling cell-laden hydrogel microfibers. HepG2 cells were densely packed into the core of sandwich-type anisotropic microfibers, which were produced using microfluidic devices. The obtained microfibers were bundled up and packed into a perfusion chamber, and perfusion cultivation was performed. We evaluated cell viability and functions, and also monitored the oxygen consumption. Furthermore, fibers covered with vascular endothelial cells were united during the perfusion culture, to form vascular network-like conduits between fibers. The presented technique can structurally mimic the hepatic lobule in vivo and could prove to be a useful model for various biomedical research applications.

The liver surface as a favorable site for islet cell sheet transplantation in type 1 diabetes model mice.

INTRODUCTION: Islet transplantation is one of the most promising therapeutic approaches for patients with severe type 1 diabetes mellitus (T1DM). Transplantation of engineered islet cell sheets holds great potential for treating T1DM as it enables the creation of stable neo-islet tissues. However, a large mass of islet cell sheets is required for the subcutaneous transplantation to reverse hyperglycemia in diabetic mice. Here, we investigated whether the liver surface could serve as an alternative site for islet cell sheet transplantation. METHODS: Dispersed rat islet cells (0.8 × 106 cells) were cultured on laminin-332-coated thermoresponsive culture dishes. After 2 days of cultivation, we harvested the islet cell sheets by lowering the culture temperature using a support membrane with a gelatin gel. We transplanted two recovered islet cell sheets into the subcutaneous space or onto the liver surface of severe combined immunodeficiency (SCID) mice with streptozocin-induced diabetes. RESULTS: In the liver surface group, the non-fasting blood glucose level decreased rapidly within several days after transplantation. In marked contrast, the hyperglycemia state was maintained in the subcutaneous space transplantation group. The levels of rat C-peptide and insulin in the liver surface group were significantly higher than those in the subcutaneous space group. An immunohistological analysis confirmed that most of the islet cells engrafted on the liver surface were insulin-positive. The CD31-positive endothelial cells formed vascular networks within the neo-islets and in the surrounding tissues. In contrast, viable islet cells were not found in the subcutaneous space group. CONCLUSIONS: Compared with the subcutaneous space, a relatively small mass of islet cell sheets was enough to achieve normoglycemia in diabetic mice when the liver surface was selected as the transplantation site. Our results demonstrate that the optimization of the transplantation site for islet cell sheets leads to significant improvements in the therapeutic efficiency for T1DM.