Kyushu University [Nana Shirakigawa (Assistant Professor) Faculty of Engineering, Department of Chemical Engineering]

Nana Shirakigawa

Last modified date：2024.04.23

Assistant Professor / Molecular and Biochemical Systems Engineering / Department of Chemical Engineering / Faculty of Engineering

Papers

1.	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..
2.	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..
3.	Hiroki Sakamoto, Nana Shirakigawa, Ronald Perocho Bual, Yukako Fukuda, Shunsuke Nakamura, Tatsunori Miyata, Takanobu Yamao, Yo ichi Yamashita, Hideo Baba, Hiroyuki Ijima, A novel evaluation system for whole-organ-engineered liver graft by ex vivo application to a highly reproducible hepatic failure rat model, Journal of Artificial Organs, 10.1007/s10047-019-01106-6, 22, 3, 222-229, 2019.09, © 2019, The Japanese Society for Artificial Organs. In recent years, studies on liver graft construction using the decellularized liver as a template for transplantation therapy have attracted much attention. However, the therapeutic effect of constructed liver grafts in hepatic failure has not been evaluated. Therefore, we aimed to develop a novel evaluation system demonstrating the curative effect of a constructed liver graft in animals with hepatic failure. First, we developed a highly reproducible rat model of hepatic failure by combining 80% partial hepatectomy with warm ischemia. In this model, severity could be controlled by the warm ischemic period. We also constructed a liver graft by recellularization of decellularized liver, and confirmed the ammonia metabolic function in the graft in vitro as one of the most important functions for recovery from hepatic failure. The graft was then applied to our developed hepatic failure rat model using a blood extracorporeal circulation system. In this application, the graft metabolized the ammonia in the blood of animals with hepatic failure and was thus suggested to be effective for the treatment of hepatic failure. In summary, a novel evaluation system for whole-organ-engineered liver graft as a preliminary stage of transplantation was developed. This system was expected to provide much information about the curative effect of a constructed liver graft..
4.	Safrina Dyah Hardiningtyas, Seiya Nagao, Emiko Yamamoto, Nana Shirakigawa, Rie Wakabayashi, Masahiro Goto, Hiroyuki Ijima, Noriho Kamiya, A nano-sized gel-in-oil suspension for transcutaneous protein delivery, International Journal of Pharmaceutics, 10.1016/j.ijpharm.2019.118495, 567, 118495-118495, 2019.08, © 2019 Elsevier B.V. We developed a new oil-based delivery system for transdermal protein delivery, a gel-in-oil (G/O) nanosuspension, where gelatin-based hydrogel was coated with hydrophobic surfactants. The high entrapment efficiency of a model protein, phycocyanin (PC), into nano-sized gelatin hydrogel particles was achieved. Spectroscopic evaluation of PC suggested that the G/O nanosuspension could retain the functional form of PC in isopropyl myristate. In vitro skin permeation studies showed that the G/O nanosuspension facilitated the delivery of PC through the stratum corneum of Yucatan micropig skin..
5.	Tuyajargal Iimaa, Takaaki Hirayama, Nana Shirakigawa, Daisuke Imai, Takanobu Yamao, Yo Ichi Yamashita, Hideo Baba, Hiroyuki Ijima, Antibacterial-agent-immobilized gelatin hydrogel as a 3d scaffold for natural and bioengineered tissues, Gels, 10.3390/gels5020032, 5, 2, 2019.06, © 2019 by the authors. Licensee MDPI, Basel, Switzerland. Hydrogels and their medical applications in tissue engineering have been widely studied due to their three-dimensional network structure, biocompatibility, and cell adhesion. However, the development of an artificial bile duct to replace the recipient’s tissue is still desired. Some challenges remain in the tissue engineering field, such as infection due to residual artifacts. In other words, at present, there are no established technologies for bile duct reconstruction as strength and biocompatibility problems. Therefore, this study investigated hydrogel as an artificial bile duct base material that can replace tissue without any risk of infectious diseases. First, an antibacterial agent (ABA), Finibax (an ABA used for the clinical treatment of biliary tract infection), was immobilized in gelatin using a crosslinking agent, and the antibacterial properties of the gel and its sustainability were tested. Furthermore, the immobilized amount and the improvement of the proliferation of the human umbilical vein endothelial cells (HUVECs) were cultured as the ABA-Gelatin hydrogel was introduced to prepare a 3D scaffold. Finally, we performed hematoxylin and eosin (H&E) staining after subcutaneous implantation in the rat. Overall, the ABA-Gelatin hydrogel was found to be viable for use in hydrogel applications for tissue engineering due to its good bactericidal ability, cell adhesion, and proliferation, as well as having no cytotoxicity to cells..
6.	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, © 2018 Acta Materialia Inc. 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..
7.	Hiroyuki Ijima, Shintaro Nakamura, Ronald Bual, Nana Shirakigawa, Shuichi Tanoue, Physical Properties of the Extracellular Matrix of Decellularized Porcine Liver, GELS, 10.3390/gels4020039, 4, 2, 39-39, 2018.06, The decellularization of organs has attracted attention as a new functional methodology for regenerative medicine based on tissue engineering. In previous work we developed an L-ECM (Extracellular Matrix) as a substrate-solubilized decellularized liver and demonstrated its effectiveness as a substrate for culturing and transplantation. Importantly, the physical properties of the substrate constitute important factors that control cell behavior. In this study, we aimed to quantify the physical properties of L-ECM and L-ECM gels. L-ECM was prepared as a liver-specific matrix substrate from solubilized decellularized porcine liver. In comparison to type I collagen, L-ECM yielded a lower elasticity and exhibited an abrupt decrease in its elastic modulus at 37 degrees C. Its elastic modulus increased at increased temperatures, and the storage elastic modulus value never fell below the loss modulus value. An increase in the gel concentration of L-ECM resulted in a decrease in the biodegradation rate and in an increase in mechanical strength. The reported properties of L-ECM gel (10 mg/mL) were equivalent to those of collagen gel (3 mg/mL), which is commonly used in regenerative medicine and gel cultures. Based on reported findings, the physical properties of the novel functional substrate for culturing and regenerative medicine L-ECM were quantified..
8.	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..
9.	Ashok Kumar, Apeksha Damania, Mohsin Hassan, Nana Shirakigawa, Hiroshi Mizumoto, Anupam Kumar, Shiv K. Sarin, Hiroyuki Ijima, Masamichi Kamihira, Alleviating liver failure conditions using an integrated hybrid cryogel based cellular bioreactor as a bioartificial liver support, Scientific Reports, 10.1038/srep40323, 7, 2017.01, Conventionally, some bioartificial liver devices are used with separate plasmapheresis unit to separate out plasma from whole blood and adsorbent column to detoxify plasma before it passes through a hepatocytes-laden bioreactor. We aim to develop a hybrid bioreactor that integrates the separate modules in one compact design improving the efficacy of the cryogel based bioreactor as a bioartificial liver support. A plasma separation membrane and an activated carbon cloth are placed over a HepG2-loaded cryogel scaffold in a three-chambered bioreactor design. This bioreactor is consequently connected extracorporeally to a rat model of acute liver failure for 3 h and major biochemical parameters studied. Bilirubin and aspartate transaminase showed a percentage decrease of 20-60% in the integrated bioreactor as opposed to 5-15% in the conventional setup. Urea and ammonia levels which showed negligible change in the conventional setup increase (40%) and decrease (18%), respectively in the integrated system. Also, an overall increase of 5% in human albumin in rat plasma indicated bioreactor functionality in terms of synthetic functions. These results were corroborated by offline evaluation of patient plasma. Hence, integrating the plasmapheresis and adsorbent units with the bioreactor module in one compact design improves the efficacy of the bioartificial liver device..
10.	Nana Shirakigawa, Yuta Hara, Hiroyuki Ijima, Tissue-Engineered Bioreactors with Flow Channels Molded by Polypod Particles Advanced Biomedical Engineering, Advanced Biomedical Engineering, DOI:10.14326/abe.5.105, 5, 105-110, 2016.07, [URL].
11.	Jingjia Ye, Nana Shirakigawa, Hiroyuki Ijima, Fetal liver cell-containing hybrid organoids improve cell viability and albumin production upon transplantation, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2015.11.006, 121, 6, 701-708, 2016.06, Cell transplantation is a potential alternative for orthotopic liver transplantation because of the chronic donor shortage. Functional liver tissue is needed for cell transplantations. However, large functional liver tissue is difficult to construct because of the high oxygen consumption of hepatocytes. In our previous study, we developed a novel method to generate hybrid organoids. In this study, we used fetal liver cells (FLCs) to construct a hybrid organoid. Nucleus numbers, angiogenesis, and albumin production were measured in transplanted samples. Higher cell viability and larger liver tissue was found in FLC-containing samples than in hepatocyte-containing samples. Furthermore, the therapeutic efficiency of FLC-containing samples was evaluated by transplantation into Nagase analbuminemia rats. As a result, an increase in albumin concentration was found in rat blood. In summary, transplantation of a FLC-containing hybrid organoid is a potential approach for cell transplantation..
12.	Nana Shirakigawa, Tadamitsu IDEGUCHI, Kazuyuki ICHIKAWA, Takahisa IZUMI, Michiko HIGASHI, Shizunari YAMAMOTO, Hiroyuki Ijima, Quantitative Analysis of Vascular Structure in Decellularized Liver Using 3D Computed Tomography, Advanced Biomedical Engineering, 10.14326/abe.4.179, 4, 179-185, 2015.08.
13.	Jingjia Ye, Nana Shirakigawa, Hiroyuki Ijima, Hybrid organoids consisting of extracellular matrix gel particles and hepatocytes for transplantation, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2015.01.004, 120, 2, 231-237, 2015.08, Hepatocyte transplantation is a potential therapy for treating various liver diseases. However, oxygen shortage leading to loss of hepatocyte function becomes a limitation following hepatocyte transplantation. To overcome this problem, we developed a hybrid organoid, consisting of growth factor (GF)-immobilizable gel particles combined with hepatocytes. Benefits of the hybrid organoid were evaluated in three groups: (i) hybrid organoid consisting of cells and GF-immobilizable gel particles (HG-C); (ii) hybrid organoid consisting of cells and gel particles (G-C); and (iii) cells suspended in collagen (C-C). We found liver-specific functions of HG-C were maintained longer than in the other conditions during in vitro culture. Furthermore, after transplantation, HG-C was effective in maintaining viability of transplanted hepatocytes and promoting angiogenesis around the hepatocytes. In summary, transplantation of HG-C is a potential method for future liver tissue engineering..
14.	Nana Shirakigawa, Takayuki Takei, Hiroyuki Ijima, Base structure consisting of an endothelialized vascular-tree network and hepatocytes for whole liver engineering, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2013.05.020, 116, 6, 740-745, 2013.12, Reconstructed liver has been desired as a liver substitute for transplantation. However, reconstruction of a whole liver has not been achieved because construction of a vascular network at an organ scale is very difficult. We focused on decellularized liver (DC-liver) as an artificial scaffold for the construction of a hierarchical vascular network. In this study, we obtained DC-liver and the tubular network structure in which both portal vein and hepatic vein systems remained intact. Furthermore, endothelialization of the tubular structure in DC-liver was achieved, which prevented blood leakage from the tubular structure. In addition, hepatocytes suspended in a collagen sol were injected from the surroundings using a syringe as a suitable procedure for liver cell inoculation. In summary, we developed a base structure consisting of an endothelialized vascular-tree network and hepatocytes for whole liver engineering..
15.	Nana Shirakigawa, Hiroyuki Ijima, Nucleus number in clusters of transplanted fetal liver cells increases by partial hepatectomy of recipient rats, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2012.11.015, 115, 5, 568-570, 2013.05, The growth of transplanted hepatocytes is required for the construction of tissue-engineered liver. In this study, cell-embedded hydrogel-filled polyurethane foam plates were subcutaneously transplanted into rat. A liver tissue-like structure was formed by transplanted fetal liver cells in 70% partial hepatectomy treated rat..
16.	Yung Te Hou, Hiroyuki Ijima, Nana Shirakigawa, Takayuki Takei, Koei Kawakami, Development of growth factor-immobilizable material for hepatocyte transplantation, Biochemical Engineering Journal, 10.1016/j.bej.2012.09.007, 69, 172-181, 2012.12, Growth factor (GF)-immobilizable materials were developed as a practical hepatocyte transplantation method for reconstructing a tissue-like structure in liver tissue engineering. Two GF-immobilizable scaffolds, namely single hepatocyte-embedded, heparin-immobilized, collagen-gel-filled polyurethane foam, and hepatocyte spheroid-embedded, heparin-immobilized, collagen-gel-filled polyurethane foam were developed by covalently incorporating heparin into collagen gel, using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/. N-hydroxysuccinimide for hepatocyte transplantation. Seventy percent partial hepatectomy (PH) was performed at the same time after hepatocyte transplantation. Angiogenesis efficiency and viability of transplanted cells are discussed in terms of normalized hemoglobin content, nuclear density and histological observations after transplantation. In summary, the normalized hemoglobin content and viability of transplanted cells were higher in GF-immobilized scaffolds with PH pretreatment than in the other scaffolds with/without PH pretreatment. These materials have the potential for in vivo hepatocyte transplantation, as GFs released from remnant liver were easily incorporated into the heparin-immobilized collagen gel system. These GF-heparin complexes may promote the survival of embedded cells. Furthermore, the transplantation of spheroids promoted increased angiogenesis compared with hepatocytes, and resulted in sufficient vascularization for cell survival..
17.	Nana Shirakigawa, Hiroyuki Ijima, Takayuki Takei, Decellularized liver as a practical scaffold with a vascular network template for liver tissue engineering, Journal of Bioscience and Bioengineering, 10.1016/j.jbiosc.2012.05.022, 114, 5, 546-551, 2012.11, The construction of a functional liver-tissue equivalent using tissue engineering is a very important goal because the liver is a central organ in the body. However, the construction of functional organ-scale liver tissue is impossible because it requires a high-density blood vessel network. In this study, we focused on decellularization technology to solve this problem. Decellularized liver tissue with a fine vascular tree network template was obtained using Triton X-100. The distance between each vascular structure was less than 1 mm. Endothelialization of the blood vessel network with human umbilical vein endothelial cells (HUVECs) was successfully performed without any leakage of HUVECs to the outside of the vessel structure. Furthermore, hepatocytes/spheroids could be located around the blood vessel structure. This study indicates that decellularized liver tissue is a potential scaffold for creating a practical liver tissue using tissue engineering technology..

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