| 1. |
Tatsuya Osaki, Tomoya Duenki, Siu Yu A. Chow, Yasuhiro Ikegami, Romain Beaubois, Timothée Levi, Nao Nakagawa-Tamagawa, Yoji Hirano, Yoshiho Ikeuchi, Complex activity and short-term plasticity of human cerebral organoids reciprocally connected with axons, Nature Communications, 10.1038/s41467-024-46787-7, 15, 2945, 2024.04. |
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Takayuki Nagai, Yasuhiro Ikegami, Hideyuki Mizumachi, Nana Shirakigawa, Hiroyuki Ijima, Development of an in situ evaluation system for neural cells using extracellular matrix-modeled gel culture, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2017.04.018, 124, 4, 430-438, 2017.10, 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.. |
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Ronald Bual, Haruna Kimura, Yasuhiro Ikegami, Nana Shirakigawa, Hiroyuki Ijima, Fabrication of liver-derived extracellular matrix nanofibers and functional evaluation in in vitro culture using primary hepatocytes, Materialia, 10.1016/j.mtla.2018.11.014, 4, 518-528, 2018.12, 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.. |
| 4. |
Tuyajargal Iimaa, Yasuhiro Ikegami, Ronald Bual, Nana Shirakigawa, Hiroyuki Ijima, Analysis of Sulfated Glycosaminoglycans in ECM Scaffolds for Tissue Engineering Applications: Modified Alcian Blue Method Development and Validation, Journal of Functional Biomaterials, 10.3390/jfb10020019, 10, 2, 19-19, 2019.04, 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% and 3.16–7.02% (n = 9), respectively. Overall recovery for three concentration levels varied between 97 ± 3.5%, 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. . |
| 5. |
Yasuhiro Ikegami, Hideyuki Mizumachi, Kozue Yoshida, Hiroyuki Ijima, Heparin-conjugated collagen as a potent growth factor-localizing and stabilizing scaffold for regenerative medicine, Regenerative Therapy, 10.1016/j.reth.2020.10.002, 15, 236-242, 2020.12. |
| 6. |
Yasuhiro Ikegami, Hiroyuki Ijima, Development of heparin-conjugated nanofibers and a novel biological signal by immobilized growth factors for peripheral nerve regeneration, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2019.09.004, 129, 3, 354-362, 2020.03. |
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Kozue Yoshida, Fumiyasu Ono, Takehiro Chouno, Bual Ronald Perocho, Yasuhiro Ikegami, Nana Shirakigawa, Hiroyuki Ijima, Cryoprotective enhancing effect of very low concentration of trehalose on the functions of primary rat hepatocytes, Regenerative Therapy, 10.1016/j.reth.2020.08.003, 15, 173-179, 2020.12, 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% 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.. |
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Akshat Joshi, Zhe Xu, Yasuhiro Ikegami, Soichiro Yamane, Masanori Tsurashima, Hiroyuki Ijima, Co-culture of mesenchymal stem cells and human umbilical vein endothelial cells on heparinized polycaprolactone/gelatin co-spun nanofibers for improved endothelium remodeling, International Journal of Biological Macromolecules, 10.1016/j.ijbiomac.2020.02.163, 151, 186-192, 2020.05. |
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Kozue Yoshida, Shunsuke Nakamura, Hiroki Sakamoto, Mika Kondo, Takehiro Chouno, Yasuhiro Ikegami, Nana Shirakigawa, Hiroshi Mizumoto, Yo-ichi Yamashita, Hideo Baba, Hiroyuki Ijima, Normothermic machine perfusion system satisfying oxygen demand of liver could maintain liver function more than subnormothermic machine perfusion, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2020.08.011, 131, 1, 107-113, 2021.01, 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%). 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.. |
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Akshat Joshi, Zhe Xu, Yasuhiro Ikegami, Kozue Yoshida, Yusuke Sakai, Akshay Joshi, Tejinder Kaur, Yosuke Nakao, Yo-ichi Yamashita, Hideo Baba, Shinichi Aishima, Neetu Singh, Hiroyuki Ijima, Exploiting synergistic effect of externally loaded bFGF and endogenous growth factors for accelerated wound healing using heparin functionalized PCL/gelatin co-spun nanofibrous patches, Chemical Engineering Journal, 10.1016/j.cej.2020.126518, 404, 126518-126518, 2021.01, Growth factors (GFs) are signaling molecules that are principle mediators in tissue regeneration. Biomaterial scaffolds employed as wound dressings are often hampered by their limitations to deliver GFs exogenously due to their instability and low half-life. The key to overcome this challenge lies in the better organization and use of endogenous pro-regenerative GFs released at regenerative site, with an aim to minimize the sole dependency on exogenous factors. Considering such challenges, this research utilizes the exogenous and endogenous GFs sequestering capability of heparin functionalized PCL/gelatin co-spun nanofabrics to mediate synergistically driven tissue regeneration by utilizing combined therapeutic effect of exogenous and endogenous GFs, and thereby minimizing the sole dependency on exogenous GFs for tissue regeneration. Basic fibroblast growth factor (bFGF) was chosen as GF for exogenous loading whereas vascular endothelial growth factor (VEGF) was chosen as a representative example to demonstrate the endogenous pro-regenerative GF sequestration capability of fabricated nanofabrics. From our results, the fabricated nanofabrics showed loading efficiency of 80% for exogenous bFGF and can sequester 15-fold more amount of endogenous VEGF compared to non-heparin functionalized nanofibrous dressings. When applied as wound dressings, heparin functionalized nanofibers showed better therapeutic capability compared to control groups that were treated using patches without heparin functionalization, indicating endogenously driven tissue regeneration. This was indicated by significant higher number of newly formed skin appendages, lesser scarring and lower inflammatory levels in newly formed granulation. Additionally, further improvements in therapeutic effect was observed when exogenous bFGF was employed indicating effectiveness of synergistically mediated tissue regeneration.. |
| 11. |
Jannatul Fardous, Emiko Yamamoto, Yuji Omoso, Seiya Nagao, Yuuta Inoue, Kozue Yoshida, Yasuhiro Ikegami, Yi Zhang, Nana Shirakigawa, Fumiyasu Ono, Hiroyuki Ijima, Development of a gel-in-oil emulsion as a transdermal drug delivery system for successful delivery of growth factors, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2021.03.015, 132, 1, 95-101, 2021.07, 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.. |
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Kozue Yoshida, Fumiyasu Ono, Takehiro Chouno, Shota Nakada, Yasuhiro Ikegami, Nana Shirakigawa, Yusuke Sakai, Hiroyuki Ijima, Creation of a novel lipid-trehalose derivative showing positive interaction with the cell membrane and verification of its cytoprotective effect during cryopreservation, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2021.03.010, 132, 1, 71-80, 2021.07, 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.. |
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Yo-ichi Yamashita, Takanobu Yamao, Yosuke Nakao, Tatsunori Miyata, Yasuhiro Ikegami, Soichiro Yamane, Taiga Ito, Taku Furukawa, Jaeyong Cho, Fanqi Wu, Yasumitsu Fujie, Mitsuru Arima, Shinichi Aishima, Hiroyuki Ijima, Hideo Baba, Efficacy of a newly developed bioabsorbable pancreatic clip for distal pancreatectomy in swine, Surgery Today, 10.1007/s00595-021-02435-x, 52, 7, 1109-1114, 2022.01, PURPOSE: At present, ≥ 20% 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.. |
| 14. |
Yasuhiro Ikegami, Muhammad Shafiq, Shinichi Aishima, Hiroyuki Ijima, Correction to: Heparin/Growth Factors-Immobilized Aligned Electrospun Nanofibers Promote Nerve Regeneration in Polycaprolactone/Gelatin-Based Nerve Guidance Conduits, Advanced Fiber Materials, 10.1007/s42765-023-00261-z, 5, 2, 696-696, 2023.02. |
