記述言語：その他   掲載種別：研究論文（学術雑誌）  

Motor nerve organoids could be generated by culturing a spheroid of motor neurons differentiated from human induced pluripotent stem (iPS) cells within a polydimethylsiloxane (PDMS) chip which guides direction and fasciculation of axons extended from the spheroid. To isolate axon bundles from motor nerve organoids, we developed a rapid laser dissection method based on localized photothermal combustion. By illuminating a blue laser on a black mark on the culture device using a dry-erase marker, we induced highly localized heating near the axon bundles. Moving the laser enabled spatial control over the local heating and severing of axon bundles. This laser dissection requires a black mark, as other colors did not produce the same localized heating effect. A CO₂ laser destroyed the tissue and the device and could not be used. With this simple, economical laser dissection technique, we could rapidly collect abundant pure axon samples from motor nerve organoids for biochemical analysis. Extracted axonal proteins and RNA were indistinguishable from manual dissection. This method facilitates efficient axon isolation for further analyses.

DOI： 10.3389/fbioe.2024.1259138

記述言語：その他   掲載種別：研究論文（学術雑誌）  

<jats:title>Abstract</jats:title><jats:p>Mitochondria possess their own genome that encodes components of oxidative phosphorylation (OXPHOS) complexes, and mitochondrial ribosomes within the organelle translate the mRNAs expressed from mitochondrial genome. Given the differential OXPHOS activity observed in diverse cell types, cell growth conditions, and other circumstances, cellular heterogeneity in mitochondrial translation can be expected. Although individual protein products translated in mitochondria have been monitored, the lack of techniques that address the variation in overall mitochondrial protein synthesis in cell populations poses analytic challenges. Here, we adapted mitochondrial-specific fluorescent noncanonical amino acid tagging (FUNCAT) for use with fluorescence-activated cell sorting (FACS) and developed mito-FUNCAT-FACS. The click chemistry-compatible methionine analog L-homopropargylglycine (HPG) enabled the metabolic labeling of newly synthesized proteins. In the presence of cytosolic translation inhibitors, HPG was selectively incorporated into mitochondrial nascent proteins and conjugated to fluorophores via the click reaction (mito-FUNCAT). The application of <jats:italic>in situ</jats:italic> mito-FUNCAT to flow cytometry allowed us to disentangle changes in net mitochondrial translation activity from those of the organelle mass and detect variations in mitochondrial translation in cancer cells. Our approach provides a useful methodology for examining mitochondrial protein synthesis in individual cells.</jats:p>

DOI： 10.1101/2022.01.03.474764

記述言語：英語   掲載種別：研究論文（学術雑誌）  

PURPOSE: At present, ≥ 20&#37; of patients experience clinically relevant postoperative pancreatic fistula (POPF) after distal pancreatectomy (DP). METHODS: We developed a new bioabsorbable pancreatic clip (BioPaC) made of polycaprolactone that does not crush the pancreatic parenchyma during occlusion of the pancreatic stump. We confirmed the efficacy of this BioPac in a porcine DP model and compared it to a linear stapling device (Reinforce®). RESULTS: Pigs were killed at 1 month after DP. In the BioPaC group, all swine (n = 3) survived well without POPF. In the Reinforce® group (n = 2), one pig died early at postoperative day 7 with Grade C POPF (amylase 43 700 U/l), and the other survived until 1 month at scarification with biochemical leakage of POPF (amylase 3 725 U/l). Pathologically, the main pancreatic duct and pancreatic parenchyma were well closed by BioPaC. CONCLUSION: The newly developed BioPaC is effective in a porcine DP model.

DOI： 10.1007/s00595-021-02435-x

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Growth factors (GFs) are indispensable in regenerative medicine because of their high effectiveness. However, as GFs degenerate easily, the development of a suitable carrier with improved stability for GFs is necessary. In this study, we developed a gel-in-oil (G/O) emulsion technology for the transdermal delivery of growth factors. Nanogel particles prepared with heparin-immobilized gelatin that can bind growth factors were dispersed in isopropyl myristate. The particle size of the G/O emulsion could be controlled by changing the surfactant concentration, volume ratio of the water phase to the oil phase, and gelatin concentration. In vitro skin penetration studies showed better penetration through the stratum corneum of fluorescent proteins containing G/O emulsions than of the aqueous solution of GF. Similarly, an in vivo study showed an angiogenesis-inducing effect after transdermal application of GF-immobilized G/O emulsion. Angiogenesis in mice was confirmed owing to both an increased blood vessel network and higher hemoglobin content in the blood. Therefore, the G/O emulsion could be a promising carrier for GFs with better stability and can effectively deliver GFs at the target site.

DOI： 10.1016/j.jbiosc.2021.03.015

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cryopreservation is important for enabling long-term cell preservation. However, physical damage due to ice crystal formation and membrane permeation by dimethyl sulfoxide (DMSO) severely affects cryopreserved cell viability. To ensure cell survival and functional maintenance after cryopreservation, it is important to protect the cell membrane, the most vulnerable cell component, from freeze-thaw damage. This study aimed to create a glycolipid derivative having a positive interaction with the cell membrane and cytoprotective effects. As a result, we synthesized a novel trehalose derivative, oleyl-trehalose (Oleyl-Treh), composed of trehalose and oleyl groups. Its use led to increased viable cell counts when used with DMSO in a non-cytotoxic concentration range (1.6 nM-16 μM). Oleyl-Treh significantly improved viability and liver-specific functions of hepatocytes after cryopreservation, including albumin secretion, ethoxyresorufin-O-deethylase activity (an indicator of cytochrome P450 family 1 subfamily A member 1 activity), and ammonia metabolism. Oleyl-Treh could localize trehalose to the cell membrane; furthermore, the oleyl group affected cell membrane fluidity and exerted cryoprotective effects. This novel cryoprotective agent, which shows a positive interaction with the cell membrane, provides a unique approach toward cell protection during cryopreservation.

DOI： 10.1016/j.jbiosc.2021.03.010

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Liver transplantation plays an important role in the medical field. To improve the quality of a donor liver, there is a need to establish a preservation system to prevent damage and maintain liver function. In response to this demand, machine perfusion (MP) has been proposed as a new liver preservation method instead of the conventional static cold storage. There is controversy about the optimal MP temperature of the donor liver. Since the oxygen consumption of the liver differs depending on the temperature, construction of a system that satisfies the oxygen demand of the liver is crucial for optimizing the preservation temperature. In this study, an MP system, which satisfies the oxygen demand of liver at each temperature, was constructed using an index of oxygen supply; the overall volumetric oxygen transfer coefficient, the amount of oxygen retention of perfusate and oxygen saturation. Both subnormothermic MP (SNMP, 20-25 degrees C) and normothermic MP (NMP, 37 degrees C) could maintain liver viability at a high level (94&#37;). However, lactate metabolism of the liver during NMP was more active than that during SNMP. Furthermore, the ammonia metabolism of liver after NMP was superior to that after SNMP. Hence, NMP, which maintains the metabolic activity of the liver, is more suitable for preservation of the donor liver than SNMP, which suppresses the metabolic activity. In summary, normothermia is the optimal temperature for liver preservation, and we succeeded in constructing an NMP system that could suppress liver damage and maintain function. (C) 2020, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2020.08.011

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Introduction: Cells have various applications in biomedical research. Cryopreservation is a cell-preservation technique that provides cells for such applications. After cryopreservation, sensitive cells, such as primary hepatocytes, suffer from low viability due to the physical damage caused by ice crystals, highlighting the need for better methods of cryopreservation to improve cell viability. Given the importance of effectively suppressing ice crystal formation to protect cellular structure, trehalose has attracted attention as cryoprotectant based on its ability to inhibit ice crystal formation; however, trehalose induces osmotic stress. Therefore, to establish a cell-cryopreservation technique, it is necessary to provide an optimal balance between the protective and damaging effects of trehalose. Methods: In this study, we evaluated the effects of osmotic stress and ice crystal formation on the viability and function of primary rat hepatocytes at wide range of trehalose concentration. Results: There was no osmotic stress at very low concentrations (2.6 μM) of trehalose, and 2.6 μM trehalose drives the formation of finer ice crystals, which are less damaging to the cell membrane. Furthermore, we found that the number of viable hepatocytes after cryopreservation were 70&#37; higher under the 2.6 μM trehalose-supplemented conditions than under the dimethyl sulfoxide-supplemented conditions. Moreover, non-cryopreserved cells and cells cryopreserved with trehalose showed comparable intracellular dehydrogenase activity. Conclusions: We showed that trehalose at very low concentrations (2.6 μM) improved dramatically viability and liver function of hepatocyte after cryopreservation.

DOI： 10.1016/j.reth.2020.08.003

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.ijbiomac.2020.02.163

記述言語：英語   掲載種別：研究論文（学術雑誌）  

<jats:p>Accurate determination of the amount of glycosaminoglycans (GAGs) in a complex mixture of extracellular matrix (ECM) is important for tissue morphogenesis and homeostasis. The aim of the present study was to investigate an accurate, simple and sensitive alcian blue (AB) method for quantifying heparin in biological samples. A method for analyzing heparin was developed and parameters such as volume, precipitation time, solvent component, and solubility time were evaluated. The AB dye and heparin samples were allowed to react at 4 ℃ for 24 h. The heparin-AB complex was dissolved in 25 N NaOH and 2-Aminoethanol (1:24 v/v). The optical density of the solution was analyzed by UV-Vis spectrometry at 620 nm. The modified AB method was validated in accordance with U.S. Food and Drug Administration guidelines. The limit of detection was found to be 2.95 µg/mL. Intraday and interday precision ranged between 2.14–4.83&#37; and 3.16–7.02&#37; (n = 9), respectively. Overall recovery for three concentration levels varied between 97 ± 3.5&#37;, confirming good accuracy. In addition, this study has discovered the interdisciplinary nature of protein detection using the AB method. The basis for this investigation was that the fibrous protein inhibits heparin-AB complex whereas globular protein does not. Further, we measured the content of sulfated GAGs (sGAGs; expressed as heparin equivalent) in the ECM of decellularized porcine liver. In conclusion, the AB method may be used for the quantitative analysis of heparin in ECM scaffolds for tissue engineering applications.</jats:p>

DOI： 10.3390/jfb10020019

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Fabrication of functional scaffolds is one of the main goals of tissue engineering, particularly scaffolds mimicking the native extracellular matrix (ECM) and stimulating a microenvironment that supports cell growth and function. In this study, a scaffold was developed using liver-derived ECM (L-ECM), gelatin, and polycaprolactone (PCL) by conventional electrospinning. This study aimed to improve the gelatin-PCL nanofiber blend by incorporating L-ECM. The morphology of the nanofibrous scaffold was investigated by scanning electron microscopy. Characterizations such as Fourier transform infrared spectroscopy (FTIR), tensile strength test, and water contact angle analysis revealed the favorable characteristics of the scaffold. Particularly, the FTIR spectra confirmed the presence of L-ECM, gelatin, and PCL, even after crosslinking treatment, while tensile strength analysis revealed suitable mechanical properties and water contact angle measurement showed the improved hydrophilic characteristics of the scaffold. The biocompatibility of the composite nanofibers was evaluated by in vitro culture of primary hepatocytes. Phase-contrast microscope images verified that the L-ECM nanofibers improved hepatocyte adhesion and facilitated the formation of a tissue-like structure. Moreover, high liver-specific functions were obtained in L-ECM nanofibers compared to gelatin/PCL nanofibers. Thus, L-ECM nanofibers can be used in tissue engineering, particularly in liver regeneration.

DOI： 10.1016/j.mtla.2018.11.014

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Two-dimensional monolayer culture is the most popular cell culture method. However, the cells may not respond as they do in vivo because the culture conditions are different from in vivo conditions. However, hydrogel-embedding culture, which cultures cells in a biocompatible culture substrate, can produce in vivo-like cell responses, but in situ evaluation of cells in a gel is difficult. In this study, we realized an in vivo-like environment in vitro to produce cell responses similar to those in vivo and established an in situ evaluation system for hydrogel-embedded cell responses. The extracellular matrix (ECM)-modeled gel consisted of collagen and heparin (Hep-col) to mimic an in vivo-like environment. The Hep-col gel could immobilize growth factors, which is important for ECM functions. Neural stem/progenitor cells cultured in the Hep-col gel grew and differentiated more actively than in collagen, indicating an in vivo-like environment in the Hep-col gel. Second, a thin-layered gel culture system was developed to realize in situ evaluation of the gel-embedded cells. Cells in a 200-μm-thick gel could be evaluated clearly by a phase-contrast microscope and immunofluorescence staining through reduced optical and diffusional effects. Finally, we found that the neural cells cultured in this system had synaptic connections and neuronal action potentials by immunofluorescence staining and Ca2+ imaging. In conclusion, this culture method may be a valuable evaluation system for neurotoxicity testing.

DOI： 10.1016/j.jbiosc.2017.04.018

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B205 The novel culture technology of neural stem cells consist of the easy observable/evaluable device with in vivo-like environment

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Fabrication of artificial blood vessel using gelatin/PCL core-shell nanofiber

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Quantitative determination of sGAG in natural ECM Scaffold for Liver Tissue Engineering

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3D culture of primary hepatocytes on L-ECM/gelatin/PCL electrospun nanofibers for tissue engineering applications

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Development of L-ECM/gelatin/PCL electrospun nanofibers as a potential substrate for primary hepatocytes in vitro

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Artificial Nerve Guidance Conduits based onPolycaprolactone/Gelatin-based Core/Shell typeAligned Electrospun Fibers

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Hyaluronic Acid Double-Network Hydrogels Immobilized with bFGF for Wound Healing

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Study on preparation and formulation of ceramide nanoemulsions

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Development of a functional miniature liver based on the right lobe of mice