Kyushu University Academic Staff Educational and Research Activities Database
List of Presentations
Hiroyuki Ijima Last modified date:2022.05.19

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


Presentations
1. Application development of decellularized tissue.
2. Uehara Uyeda Mario Kokichi、Wu Fanqi、Fukuda Yukako、Sakai Yusuke、Shirakigawa Nana、Miyata Tatsunori、Nakao Yosuke、Yamao Takanobu、Aishima Shinichi、Yamashita Yo-ichi、Baba Hideo、Ijima Hiroyuki, Development and Functional Evaluation of a Miniature Liver Model
(ミニチュア肝臓モデルの開発と機能評価), 化学工学会第87年会, 2022.03.
3. Medical engineering deployment based on ECM-mimetic material and its shaping.
4. New technology of Gel-in-Oil emulsion delivery for inducing angiogenesis.
5. Development of nervous ECM nanofibers and research for peripheral nerve regeneration.
6. Fabrication of bile excretion system by co-culturing hepatocyte spheroids with bile duct-like tissue.
7. Development of a smart folding and expanding cell sheet delivery device.
8. Effect of tobacco smoke exposure on the visual function.
9. A new technology of bFGF-contained Gel-in-Oil emulsion delivery for inducing angiogenesis.
10. Cyst formation from chinese hamster kidney-derived stem cells using organ specific ECM gel.
11. Jannatul Fardous, Yuji Omoso, Emiko Yamamoto, Kozue Yoshida, Fumiyasu Ono, Hiroyuki Ijima, Development and Characterization of Gel-in-Water Nanodispersion as a Novel Drug Delivery System, 2019 Japan/Taiwan/Korea Chemical Engineering Conference, 2019.11, [URL], .
12. Kozue Yoshida, Fumiyasu Ono, Takehiro Chouno, Nana Shirakigawa, Yusuke Sakai, Hiroyuki Ijima, Development of Cell Cryoprotectant Based on Trehalose, 2019 Japan/Taiwan/Korea Chemical Engineering Conference, 2019.11, [URL].
13. Yue Yue, Kohji Sasaki, Yuki Naruo, Hiroshi Mizumoto, Hiroyuki Ijima, Toshihisa Kajiwara, Preparation of Heparin-Conjugated Collagen Gel and Its Application to a Scaffold for Formation of a Hepatic Tissue, 2019 Japan/Taiwan/Korea Chemical Engineering Conference, 2019.11, [URL].
14. Jannatul Fardous, Yuji Omoso, Emiko Yamamoto, Kozue Yoshida, Fumiyasu Ono, Hiroyuki Ijima, Development and Characterization of gel-in-water nanoemulsion as hydrophobic drug carrier, The 6th SKY International Joint Symposium on Chemical Engineering, 2019.11.
15. Stable delivery of functional molecules by development of Gel-in-Oil nanoemulsion.
16. Tuyajargal Iimaa, Takahiro Hirayama, Yukako Fukuda, Hiroyuki Ijima, The Effects of Antibacterial Agent with Functional Biomaterials: in Vivo and In Vitro Studies, 第56回化学関連支部合同九州大会, 2019.07.
17. Tuyajargal Iimaa, Takahiro Hirayama, Yukako Fukuda, Hiroyuki Ijima , Heparin conjugated gelatin hydrogel immobilized growth factors: A potential candidate for treatment of pressure ulcer, The 14th Asian Congress on Biotechnology, 2019.07.
18. R. Bual, H. Sakamoto, T. Miyata, T. Yamao, Y. Yamashita, H. Baba, H. Ijima, Development of implantable liver-derived extracellular matrix gel sheet for liver tissue engineering, 6th Nano Today Conference, 2019.06.
19. Yasuhiro Ikegami, Hiroyuki Ijima, Alignment and immobilized neurotrophic factors of the heparin-functionalized nanofibrous scaffold provide synergistic effects on the peripheral nerve regeneration, 6th Nano Today Conference, 2019.06.
20. Development of Gel-in-Oil emulsion for transdermal delivery of hydrophilic drug and its functional evaluation using cultured dermis model

Hiroyuki Ijima, Emiko Yamamoto, Seiya Nagao, Fumiyasu Ono, Nana Shirakigawa.
21. Basic Study for Construction of Miniature Human Liver as Screening Model

Hiroyuki Ijima, Yukako Fukuda, Hiroki Sakamoto, Nana Shirakigawa, Tatsunori Miyata, Nobuhiro Yamao, Yoichi Yamashita, Hideo Baba.
22. Akshat, Joshi Akshat, Zhu Xu, Nana Shirakigawa, Hiroyuki Ijima, Development of heparin immobilized PCL/gelatin core-shell type nanofibrous scaffolds: A potential candidate for Tissue Engineering Applications, 第54 回九大生体材料・力学研究会, 2018.11.
23. Hiroyuki Ijima, Akshat Joshi, Zhe Xu, Nana Shirakigawa, Development of Functional Core-Shell Type Nanofibrous Scaffolds, JAACT 2018 Tsukuba, 2018.11.
24. Development of organ-specific ECM nanofiber substratum as a scaffold for liver tissue engineering.
25. Joshi Akshat, Zhe Xu, Shirakigawa Nana, Ijima Hiroyuki, Development of biomimetic PCL/gelatin core-shell type nanofibrous scaffolds immobilized with Heparin Sulfate for Tissue Engineering applications, 化学工学会 第50回秋季大会, 2018.09.
26. Iimaa Tuyajargal, Ikegami Yasuhiro, Bual Ronald, Shirakigawa Nana, Ijima Hiroyuki, Quantitative determination of sGAG in natural ECM Scaffold for Liver Tissue Engineering, 化学工学会 第50回秋季大会, 2018.09.
27. Ronald Perocho Bual, Haruna Kimura, Yasuhiro Ikegami, Nana Shirakigawa, Hiroyuki Ijima, Development of liver-specific ECM electrospun nanofiber as a potential substrate for primary hepatocytes, 5th TERMIS World Congress 2018, 2018.09.
28. Kozue Yoshida, Mika Kondo, Shunsuke Nakamura, Hiroki Sakamoto, Nana Shirakigawa, Hiroyuki Ijima, Effect of temperature and oxygen supply to liver function expression in hepatocyte culture toward to organ culture, 5th TERMIS World Congress 2018, 2018.09.
29. Yukako Fukuda, Jaeyong Cho, Hiroki Sakamoto, Nana Shirakigawa, Takanobu Yamao, Tatsunori Miyata, Yo-ichi Yamashita, Hideo Baba, Hiroyuki Ijima, Development of miniature human liver with mouse decellularized liver, 5th TERMIS World Congress 2018, 2018.09,
.
30. Yue Yue, Yu Nakano, Kohji Sasaki, Yuki Naruo, Nana Shirakigawa, Hiroyuki Ijima, Hiroshi Mizumoto, Toshihisa Kajiwara, Functional analysis of heparin-conjugated collagen gel as a scaffold for regenerative medicine, 5th TERMIS World Congress 2018, 2018.09.
31. Yasuhiro Ikegami, Nana Shirakigawa, Hiroyuki Ijima, Development of heparin-conjugated ECM fibers for peripheral nerve regeneration across a long gap with controlled neurites outgrowth, 5th TERMIS World Congress 2018, 2018.09.
32. 山本惠美子, 長尾征哉, 小野文靖, 白木川奈菜, 井嶋博之 , Gel-in-Oil (G/O) エマルションの調製と培養真皮モデルによる機能性評価, 第55回 化学関連支部合同九州大会, 2018.06,
.
33. Xu Zhe, Joshi Akshat, Yasuhiro Ikegami, Nana Shirakigawa, Hiroyuki Ijima, Fabrication of artificial blood vessel using gelatin/PCL core-shell nanofiber, 第55回 化学関連支部合同九州大会, 2018.06.
34. Liver-specific extracellular matrix as a substrate for hepatocyte culture and hepatocyte transplantation.
35. Development of liver construction by using decellularized liver as a scaffold.
36. Ronald Bual, Haruna Kimura, Yasuhiro Ikegami, Nana Shirakigawa, Hiroyuki Ijima, 3D culture of primary hepatocytes on L-ECM/gelatin/PCL electrospun nanofibers for tissue engineering applications, 第7回日本バイオマテリアル学会九州ブロック講演会, 2017.12.
37. Ronald Bual, Haruna Kimura, Yasuhiro Ikegami, Nana Shirakigawa, Hiroyuki Ijima, Development of L-ECM/gelatin/PCL electrospun nanofibers as a potential substrate for primary hepatocytes in vitro, 第53回九大生体材料・力学研究会, 2017.10.
38. Construction of Whole Organ Engineering, and Regenerative Medicine - Liver -, [URL].
39. Hiroyuki Ijima, Practical hybrid artificial liver support system and whole liver engineering, 5th International Symposium & Exhibition of Aqua Science and Water Resources (ISASWR'17), 2017.08, The liver is the central organ of metabolism in our body. Therefore, failure of liver function is a serious disease faced death. Currently, only liver transplantation is a fundamental treatment for serious liver failure. However, a serious donor shortage is a global issue. Therefore, development of a novel therapeutic treatment is indispensable for the life-saving of severe liver failure patients.
In this study, a hybrid artificial liver support system consisting of a porous substrate packed bed type module in which hepatocytes spheroid was immobilized was developed [1]. In preclinical animal experiments using warm ischemic liver failure pigs, this system realized high expression of liver functions and remarkable prolongation of survival time [2-4].
I have advocated Whole Organ Engineering, which creates organs for transplantation by combining "cells", "functional ECM substrate" and "organ template with fined vascular network". Liver-specific ECM substrates with high affinity for growth factors such as HGF, bFGF, VEGF have been developed, and it was effective for constructing functional liver tissue with angiogenesis. Simple and effective decellularization procedure with detergent and subsequent DNase treatment has been developed [5, 6]. This method enables to achieve good decellularization while maintaining a fine vascular network compared to the previous report. Based on these technologies, a prototype of Whole Liver Engineered-Liver for transplantation was constructed using rats as model animals. Blood ammonia was well metabolized in blood circulation culture containing this graft. Furthermore, the survival time of liver failure rats was applied was markedly prolonged by applying this graft. From the above results, the effectiveness of the practical hybrid artificial liver support system and Whole Organ Engineered-Liver graft was demonstrated..
40. Shinya Ogawa, Nagayama Katsuya, Nana Shirakigawa, Hiroyuki Ijima, 3D Particle Simulation of Liver Cell Proliferation with Angiogenesis - Whole Hepatic Lobule Formation-, TSME-ICoME 2016 7th Thai Society of Mechanical Engineers-International Conference on Mechanical Engineering, 2016.12.
41. Jaeyong Cho, Nana Shirakigawa, Hiroyuki Ijima, Cell-based optimization method for the preparation of decellularized liver as a scaffold for tissue engineering, The 29th International Symposium on Chemical Engineering, 2016.12.
42. Yuki Naruo, Tatsuya Okudaira, Nana Shirakigawa, Hiroyuki Ijima, Hiroshi Mizumoto, Toshihisa Kajiwara, Effect of heparin-conjugated collagen gel on formation of a vascularized hepatic tissue, The 29th International Symposium on Chemical Engineering, 2016.12.
43. Development of an in situ evaluation system of ECM-modelized gel culture toward in vitro neurotoxicity tests
Takayuki NAGAI, Hideyuki MIZUMACHI, Nana SHIRAKIGAWA, Hiroyuki IJIMA.
44. Construction of organ engineered-liver which will be effective for clinical treatment.
45. Effectiveness of whole organ-engineered liver graft for the treatment of hepatic failure rat.
46. Liver tissue engineering based on decellularized organ

Three-dimensional tissue engineering for regenerative medicine is desired fo r serious organ diseases. Especially, liver is a central organ for metabolis m in our body and is complicated structure. Therefore, liver tissue engineer ing is one of the most important and difficult themes.
However, formation of thick tissue (>1mm) is still impossible, because oxygen consumption rate of hepatocytes is higher than the other organs'
cells. Organ-scale scaffold having a template of blood vessel network was obtained by decellularization with detergent. The fineness of the network was the same as original liver, evaluated by 3D-CT. Template of blood vessel network was endothelialized wi th HUVEC and blood leakage was prevented. On the other hand, decellularized liver was recellularized with hepatocytes. Furthermore, this recellularized liver expressed liver-specific functions of hepatocytes in organ culture, an d well metabolized ammonia during blood circulation. In conclusion, decellul arized organ is effective scaffold in three dimensional tissue engineering..
47. Evaluation of a recellularized liver by applying a blood extracorporeal circulation system to hepatic failure rats.
48. Whole organ engineering toward construction of transplantable liver.
49. Blood circulation system consist of a whole organ engineered-liver graft and the functional evaluation of it.
50. Decellularization of liver and the recellularization for transplantation.
51. The novel culture technology of neural stem cells consist of the easy observable/evaluable device with in vivo-like environment.
52. Development of biocompatible material combining antibacterial activity and regenerative promotion ability for tissue engineered-bile duct.
53. Culture system with blood circulation for the construction of liver based on whole organ engineering.
54. Nana Shirakigawa, Hiroki Sakamoto, Cho Jaeyong, Daisuke Imai, Yo-ichi Yamashita, Ken Shirabe, Yoshihiko Maehara, Hiroyuki Ijima, Fundamental technology for the creation of whole liver engineering, and functional evaluation of recellularized liver, 2015 4th TERMIS World Congress (Tissue Engineering and Regenerative Medicine International Society), 2015.09, Technology for regenerative medicine based on tissue engineering is desired earnestly as an effective medical treatment for serious organ diseases. Especially, liver is a central organ for metabolism in our body and is complicated structure. Therefore, liver tissue engineering is one of the most important and difficult themes. However, formation of tissue-like structure with the thickness more than 1mm is still impossible, because oxygen consumption rate of hepatocytes is higher than the other organs’ cells. Scale-up and easy process development are required. For the realization, creation of whole liver engineering (WLE) consisting of cells, functional ECM and fine organ template will be indispensable.
Heparin-collagen conjugate and solubilized liver ECM were developed as growth factor-immobilizable materials. VEGF and HGF were immobilized on these functional materials (>90%). Hepatocytes on these materials well expressed various liver-specific functions in vitro. Hepatocytes or fetal liver cells (FLCs)-embedded functional gel was subcutaneously transplanted into rat. Angiogenesis and viability of hepatocytes were enhanced in the gel. Furthermore, transplanted FLCs form liver tissue-like structure with vascular network.
Organ-scale scaffold having a template of blood vessel network was obtained by decellularization with detergent. The fineness of the network was the same as original liver, evaluated by 3D-CT. Furthermore, endothelialization and expression of liver-specific function of hepatocytes were confirmed. Furthermore, recellularized liver well metabolize ammonia during blood circulation.
Based on the above-mentioned results, we expected that fundamental technology for the creation of WLE was developed.
Keywords: Whole organ engineering, Decellularized liver, Liver tissue engineering, Functional ECM.
55. Jingjia Ye, Nana Shirakigawa, Hiroyuki Ijima, Hybrid organoid consisting of extracellular matrix gel particles and cells for functional liver tissue construction, 2015 4th TERMIS World Congress (Tissue Engineering and Regenerative Medicine International Society), 2015.09.
56. Effect of heparin-conjugated collagen gel on cell viability and functions of hepatocytes and endothelial cells within engineered tissue construct.
57. Development of substrate having antibacterial activity for tissue-engineered artificial bile duct.
58. Co-culture of hepatocyte and endothelial cell in functional gel.
59. Studies on decellularization and recellularization conditions of liver toward a creation clinical liver graft for transplantation.
60. Prototype of a blood circulatable liver graft based on whole organ engineering.
61. Development of a novel culture method enables both in vivo-like environment and easy observation/evaluation.
62. Solubilized matrix derived from decellularized liver as scaffold for liver tissue engineering.
63. Thin-layer gel culture system as a neurotoxic bioassay device.
64. Jingjia Ye, Nana Shirakigawa, Hiroyuki Ijima, Construction of functional liver tissue by hybrid organoid trasnplantation, JAACT 2014 (27th Annual and International Meeting of Japanese Association for Animal Cell Technology 2014), 2014.11, 1. Introduction
Hepatocyte transplantation is a potential therapy for treating various liver diseases. However, oxygen shortage and loss of hepatocyte function becomes a limitation following hepatocyte transplantation. In our study, we tried to establish a technology, termed ‘hybrid organoid’, using heparin-conjugated gel particles combined with cells. This technology allows structures to form with high local cell density and low total cell number, owing to the existence of gel particles. What’s more, if growth factor can be immobilized in gelatin gel it will promote the construction of functional liver tissue. Moreover, primary hepatocytes don’t have proliferation ability, so we used fetal liver cells (FLCs) for the construction of hybrid organoid. FLCs consisted hybrid organoid was transplanted into nagase analbuminemia rat (NAR), then albumin secretion and drug metabolism was evaluated.
2. Experimental
Activated heparin was added to 10% gelatin solution, then cross-linked by EDC/NHS to form the gel. This gel was crashed into small particles (diameter=160 m) and washed by culture medium. Cells and heparin gel particles-packed polyurethane foam (HG-C) and cells embedded polyurethane foam (C-C) was developed. Hepatocytes or FLCs was seeded in HG-C and C-C respectively. Samples were transplanted in hepatectomy (PH)-treated rat. Cell viability was evaluated by gene expression of albumin and H&E stain at 7 days after transplantation. Furthermore, FLCs-filled HG-C was transplanted in PH-treated NAR, and albumin concentration in serum was evaluated at 0,3,5,7 days. Gene expression of CYP was evaluated by reverse transcription PCR (RT-PCR).
3. Results and discussion
From the results of H&E stain, cell viability in HG-C was higher than that in C-C in both hepatocyte and FLCs transplantation. Calibration curve of albumin mRNA-cell number was obtained. Cell viability of transplanted samples was calculated by gene expression level of albumin (ALB). FLCs contained samples showed a higher viability than hepatocyte contained samples caused by the proliferation ability of FLCs. Moreover, in the NAR transplantation, albumin concentration in serum increased in 3 days. Gene expression of CYP7A1, ALB and CYP2E1 was also confirmed by RT-PCR. These results indicated that transplanted sample maintain the albumin synthesis and drug metabolism function. Therefore, functional liver tissue was formed by hybrid organoid transplantation.
4. Conclusions
Liver specific functions were confirmed by hybrid organoid transplantation. This hybrid organoid is potential for the establishment of functional liver tissue in vivo. .
65. Souta Nishimura, Shintaro Nakamura, Nana Shirakigawa, Hiroyuki Ijima, Gel Culture System for Hepatocytes Using Solubilized Matrix Derived from Decellulized Liver, JAACT 2014 (27th Annual and International Meeting of Japanese Association for Animal Cell Technology 2014), 2014.11, 1. Introduction
Tissue engineering consisting of cell, scaffold and growth factor is important as radical treatment of serious organ diseases. We focused on scaffold in tissue engineering field, and developed L-ECM as a functional material which will be able to reconstruct an organ-specific environment 1. A purpose of this study is to create a novel artificial liver support system which does not need blood extracorporeal circulation, and the final aim is to create functional liver. Enhancement of the expression of liver-specific functions of primary hepatocytes cultured on film of liver-specific ECM (L-ECM) film was reported 1. However, three-dimensional scaffold is indispensable for our purpose. Therefore, hepatocytes-embedded gel culture using L-ECM was developed and basic evaluation for a novel artificial liver was performed in this study.

2. Experimental
Rat liver was decellularized using 1% Triton X-100. The decellularized liver was solubilized by pepsin in 0.1N HCl, and obtained L-ECM. L-ECM and type I collagen were mixed in predetermined ratio, and L-ECM/collagen mixture gel was formed by pH adjustment. Cytotoxicity of L-ECM and influence of L-ECM gel on the expression of liver-specific functions were studied by culturing of primary rat hepatocytes. Hepatocytes-embedded L-ECM gel was subcutaneously transplanted into partial hepatectomy-treated rat. The sample was retrieved 7 days after transplantation and histological analysis was performed.

3. Results and discussion
Mitochondrial activity and albumin secretion rate of hepatocytes in L-ECM mixture gel were the same as those in collagen gel on 1 day of culture. However, hepatocytes cultured in L-ECM mixture gel expressed higher liver-specific function (EROD activity) than that in collagen gel for 2 weeks. Furthermore, tissue-like structure (aggregate) formation of hepatocytes and angiogenesis in transplanted L-ECM mixture gel were enhanced than those in collagen gel. Therefore, L-ECM-containing gel will be a potential material for the creation of a novel artificial liver support system.

4. Conclusions
L-ECM-containing gel culture system was developed. This system is effective for not only the expression of liver functions in vitro, but also the formation of liver tissue-like structure in vivo.

References
1. S. Nakamura and H. Ijima .J Biosci Bioeng, (2013)..
66. Hiroyuki Ijima, Hideyuki Mizumachi, Takayuki Nagai, Nana Shirakigawa, Biological Assay Device for Neural Stem Cells with ECM-inspired Matrix Gel, JAACT 2014 (27th Annual and International Meeting of Japanese Association for Animal Cell Technology 2014), 2014.11, 1. Introduction
In recent years, neurologic cell culture is performed as an alternative of animal experiment which evaluates the influences on central nervous system (CNS) of various chemical agents. Culture system which reproduces CNS is desirable for the reliable in vitro evaluation, but neurologic cells are generally cultured in non-physiological monolayer condition. This causes the difference in cell behavior, and a tendency getting a false-negative result is strong. Therefore, we developed a heparin-collagen conjugate inspired by extracellular matrix (ECM) composition of CNS1, 2. Furthermore, we aimed to create a novel 3D culture system which enables in situ observation.

2. Experimental
Heparin was chemically cross-linked to collagen, and heparin-collagen conjugate was obtained. This conjugate forms gel structure only by pH adjustment. Basic fibroblast growth factor (bFGF) was added to this conjugate gel, and the density of immobilized bFGF by the affinity between bFGF and heparin was measured with ELISA. Next, neural stem cells (NSCs) were embedded into EM-gel and cultured with bFGF-containing medium. The response of cell to pentobarbital was studied. Furthermore, NSCs were embedded into the thin-layered conjugate gel of about 0.1 mm in thickness, and induced differentiation into neuron. The cell response to L-glutamic acid was observed by using Ca2+ imaging method.

3. Results and discussion
Immobilized bFGF in conjugate gel was at least 550 ng/ml-gel. NSCs in conjugate gel are more sensitive to pentobarbital than those in monolayer. This result indicated that conjugate gel reproduce in vivo-like culture environment3. Thin-layered EM-gel makes much easier to observe embedded cells and neural network than conventional gel culture of 2 mm in thickness. In addition, glutamate-induced intracellular Ca2+ increase was able to be evaluated without confocal laser microscope. These results suggest that NSCs cultured in EM-gel differentiated into glutamatergic neurons, and thin-layered gel culture system enabled in situ live cell imaging.

4. Conclusion
Development of a bioassay device with in situ observable 3D culture system composed of CNS-ECM-inspired matrix was expected.

References
1. CE. Bandtlow, DR. Zimmermann, Physiol. Rev. 80: 1267 (2000).
2. M. Inatani, Naturwissenschaften, 92, 549 (2005).
3. RH. Fitch, AL. Tatum, J. Pharmacol. Exp. Ther. 44: 325 (1932)..
67. Hideyuki Mizumachi, Hiroyuki Ijima, Biological assay device of neural stem cells with ECM-modelized matrix gel, YABEC 2014 (Young Asian Biochemical Engineers' Community (2014) ), 2014.11, In recent years, neurologic cell culture is performed as the alternative of animal experiments which evaluate influences on central nervous system (CNS) of various chemical agents. Culture system which mimics CNS is desirable for the reliable in vitro evaluation, but neurologic cells are generally cultured in non-physiological monolayer condition. This is likely to produce a false-negative result because of the difference of cell behavior. Therefore, we developed heparin-crosslinked collagen gel which was inspired by extracellular matrix (ECM) composition of CNS1, 2. Furthermore, we aimed to construct a novel 3D culture system which enables in situ observation.
Heparin was chemically cross-linked to collagen, and ECM-modelized matrix gel (EM-gel) was developed. Basic fibroblast growth factor (bFGF) was added to the EM-gel, and immobilization density of bFGF by cross-linked heparin was measured with ELISA. Next, neural stem cells (NSCs) were embedded into EM-gel and cultured with bFGF. The response of cell to pentobarbital was compared with that in monolayer. Furthermore, NSCs were embedded into the thin-layered EM-gel of about 0.1 mm in thickness, and induced differentiation into neuron. The cell response to L-glutamic acid was observed by using Ca2+ imaging method.
EM-gel immobilized bFGF at least 550 ng/ml-gel. NSCs in EM-gel are more sensitive to pentobarbital than those in monolayer (Fig. 1). This result indicated that EM-gel made in vivo-like culture environment3. Thin-layered EM-gel makes much easier to observe embedded cells and neural network than conventional gel culture of 2 mm in thickness. In addition, glutamate-induced intracellular Ca2+ increase was able to be evaluated without confocal laser microscopy (Fig. 2). These results suggest that NSCs cultured in EM-gel differentiated into glutamatergic neurons, and thin-layered gel culture system enabled in situ live cell imaging. In conclusion, constructing of bioassay device with in situ observable 3D culture system composed of ECM-modelized matrix was expected.
References
1. CE. Bandtlow, and DR. Zimmermann, Physiol. Rev., 80, 1267 (2000).
2. M. Inatani, Naturwissenschaften, 92, 549 (2005).
3. RH. Fitch, and AL. Tatum, J. Pharmacol. Exp. Ther., 44, 325 (1932)..
68. Nana Shirakigawa, Hiroyuki Ijima, Blood extracorporeal circulation system for rat consists of recellularized decellularized-liver, TERMIS-AP 2014 (Tissue Engineering and Regenerative Medicine International Society, Asia-Pacific Annual Conference 2014), 2014.09, [INTRODUCTION]
Many researchers including us are trying to create functional liver substitute by using decellularized liver (DC-liver). Mechanical and functional evaluation system of recellularized DC-liver is required for the optimization of the liver substitute prior to transplantation. In this study, we developed a blood extracorporeal circulation system consists of recellularized DC-liver for rat experiment.
[MATERIAL AND METHOD]
The decellularized liver was obtained by flowing 4% TritonX-100 solution via portal vein. The hepatocytes-suspended collagen sol was injected into decellularized liver by using syringe and incubated at 37 degrees C. Recellularized DC-liver was prepared. Carotid artery and jugular vein were canulated and blood extracorporeal circulation was performed.
[RESULTS AND DISCUSSION]
The blood extracorporeal circulation without DC-liver was successfully achieved via carotid artery and jugular vein in rats. However, many troubles were occurred in the circulation containing DC-liver. In other words, DC-liver was expanded, blood outflow via hepatic vein was hardly observed, and severe blood leakage from the DC-liver was observed. It was caused by the problem of pressure loss which depended on the length of blood extracorporeal circulation tube. Therefore, the tube length was shortened by removing a pump between the DC-liver and jugular vein. Based on the above-mentioned improvements, blood extracorporeal circulation containing recellularized DC-liver was achieved. In the future, the evaluation of liver-specific functions will be performed using this system.
[ACKNOWLEDGMENTS]
This work was supported by a Grant-in-Aid for Scientific Research (B): 22360348 from the MEXT of Japan..
69. Hiroyuki Ijima, Shintaro Nakamura, Jingia Ye, Nana Shirakigawa, Daisuke Imai, Yo-ichi Yamashita, Ken Shirabe, Yoshihiko Maehara, Fundamental technology for the creation of Whole Liver Engineering, TERMIS-AP 2014 (Tissue Engineering and Regenerative Medicine International Society, Asia-Pacific Annual Conference 2014), 2014.09, Technology for regenerative medicine based on tissue engineering is desired earnestly as an effective medical treatment for serious organ diseases. Especially, liver is a central organ for metabolism in our body and is complicated structure. Therefore, liver tissue engineering is one of the most important and difficult themes. However, formation of tissue-like structure with the thickness more than 1mm is still impossible, because oxygen consumption rate of hepatocytes is higher than the other organs’ cells. Development for upsizing and easy process is required. For the realization, creation of whole liver engineering (WLE) consisting of cells, functional ECM and fine organ template will be indispensable.
Heparin-collagen conjugate and solubilized liver ECM were developed as growth factor-immobilizable materials. VEGF and HGF were immobilized on these functional materials (>90%). Hepatocytes on these materials well expressed various liver-specific functions in vitro. Hepatocytes or fetal liver cells (FLCs)-embedded functional gel was subcutaneously transplanted into rat. Angiogenesis and viability of hepatocytes were enhanced in the gel. Furthermore, transplanted FLCs form liver tissue-like structure with vascular network.
Organ-scale scaffold having a template of blood vessel network was obtained by decellularization with detergent. The fineness of the network was the same as original liver, evaluated by 3D-CT. Furthermore, endothelialization and expression of liver-specific function of hepatocytes were confirmed. In other words, initial structure of WLE was successfully developed. Additionally, blood circulation system containing recellularized liver and functional evaluation system of the liver were developed.
Based on the above-mentioned results, fundamental technology for the creation of WLE was developed.

Keywords: Whole organ engineering, Decellularized liver, Liver tissue engineering, Functional ECM.
70. Hiroyuki Ijima, Shintaro Nakamura, Souta Nishimura, Nana Shirakigawa, Solubilized liver-specific ECM for hepatocytes culture and Liver Tissue Engineering, TERMIS-AP 2014 (Tissue Engineering and Regenerative Medicine International Society, Asia-Pacific Annual Conference 2014), 2014.09, 1. Introduction
Functional scaffold is very important for the development of practical tissue engineering. Because, formation of tissue-like structure and expression of organ-specific phenotypes of functional cells were enhanced on ECM-derived culture substratum. In this study, solubilized ECM from decellularized liver was developed as a functional material for liver tissue engineering.
2. Experimental
Solubilized liver-specific ECM (L-ECM) was obtained from decellularized liver by TritonX-100 treatment. Furthermore, L-ECM from regenerating liver (RL-ECM) was also obtained. Air-dried ECM films were prepared, and HUVEC, primary hepatocyte and fetal liver cells (FLCs) were cultured on the films. Furthermore, cell-embedded ECM gels were used in culture and transplantation into rat.
3. Results and discussion
Major components of natural ECM in liver such as collagen and proteoglycan were well maintained in L-ECM. VEGF and HGF could be immobilized on the films of L-ECM even in BSA co-existence condition (>90%). Furthermore, mitochondrial activity of HUVEC on VEGF-immobilized L-ECM film increased in growth factor-free medium. Organoid formation and albumin production rate of hepatocytes were enhanced on HGF-immobilized L-ECM film. Furthermore, the effectiveness was amplified on RL-ECM film. On FLCs culture, similar effectiveness could be observed. On the other hand, L-ECM-containing gel was effective for the survival and tissue structure formation of transplanted cells. Therefore, L-ECM and RL-ECM will be a potential materials as not only functional culture substratum but also suitable scaffold for liver tissue engineering.
4. Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Research (B): 22360348 from the MEXT of Japan.
.
71. Keitaro Tokuyama, Nana Shirakigawa, Daisuke Imai, Yo-ichi Yamashita, Ken Shirabe, Yoshihiko Maehara, Hiroyuki Ijima, Functional hydrogel tube for artificial bile duct, TERMIS-AP 2014 (Tissue Engineering and Regenerative Medicine International Society, Asia-Pacific Annual Conference 2014), 2014.09, [INTRODUCTION]
The artificial bile duct is desired for repair or treatment of stricture of bile duct in laparoscopic cholecystectomy and living-donor liver transplantation. We have already reported the development of growth factor immobilizable gelatin. Our aim is the development of artificial bile duct which is replaceable to viable tissue by using this gelatin. In this study, we report the construction of hydrogel tube which has suitable mechanical strength for artificial bile duct.
[MATERIAL AND METHOD]
Gelatin was cross-linked with transglutaminase. The hydrogel tube was made using template of which size is 5 mm in internal diameter, 1 mm in thickness and 4 cm in length. Some tubes were including fibers for the improvement of the mechanical strength.
[RESULTS]
The hydrogel tube which is made of biocompatible materials was developed. Gelatin tube was too weak to use as an artificial bile duct. However, the mechanical strength was much improved by mixing fibers. The strength was controlled with the amount of mixed fibers and the condition. The developed gel tube could be stitched with medical suture. Furthermore, the transplantation to pig of our artificial bile duct with the ideal construction and size was successfully achieved. These results suggest that the creation of the artificial bile duct consist of functional hydrogel will be available. In the future, we want to optimize the amount of mixed fibers, and our methods will be connected to the development of biocompatible artificial bile duct.
Keyword: Bile duct, Artificial organ, Biomaterial.
72. Nana Shirakigawa, Jingjia Ye, Hiroyuki Ijima, Growth factor-immobilizable extracellular matrix gel particles for liver tissue engineering, TERMIS-AP 2014 (Tissue Engineering and Regenerative Medicine International Society, Asia-Pacific Annual Conference 2014), 2014.09, [INTRODUCTION]
The angiogenesis and cell growth is needed for liver tissue construction in vivo. We propound the hybrid organoid which is consists of packed cell aggregate and growth factor-immobilized gel. In this study, the effectiveness of hybrid organoid was studied about for in vitro cell culture and transplantation.
[MATERIAL AND METHOD]
Heparin was activated by using EDC/NHS. The heparin-conjugated gelatin was made by mixing activated heparin and gelatin. Heparin-conjugated gelatin gel particles and gelatin gel particles were used in this study. Mixture of hepatocytes and gel particles was packed by centrifugal force, and hybrid organoid was prepared. Hepatocytes-dispersed collagen gel sample was also prepared as a control experiment. These three types of samples were used for in vitro culture. Furthermore, they were subcutaneously transplanted in backs of partial hapatectomy-treated rats.
[RESULTS AND DISCUSSION]
The liver specific functions of hybrid organoid containing heparin-conjugated gelatin gel particles were maintained longer than those in the other conditions during culture period. In transplantation experiments, the viability was highest and the angiogenesis was promoted in hybrid organoid containing heparin-conjugated gelatin gel particles. This result was caused by the enhancement of immobilization of growth factors which were secreted by partial hepatectomy treatment of rats. These results suggest that this hybrid organoid will be useful for liver tissue engineering.
[ACKNOWLEDGMENTS]
This work was supported by a Grant-in-Aid for Scientific Research (B): 22360348 from the MEXT of Japan.
73. Construction procedure of liver substitute using decellularized liver based on whole organ engineering.
74. Nervous biological assay device composed of GF-immobilizable matrix.
75. Characterization of Solubilized matrix derived from decellularized liver.
76. Development of growth factor-immobilizable tissue-engineered tubular scaffold.
77. Decellularized brain-derived ECM aimed at neural tissue engineering.
78. Basic study on tissue-engineered vascular graft derived from heparin-conjugated material.
79. Liver tissue constructs with angiogenesis by hybrid organoid transplantation.
80. Circulation cell culture in decellularized liver.
81. Basic study on the evaluation of liver substitute by blood extracorporeal circulation system with fulminant hepatic failure rat.
82. Biocompatible solubilizer for hydrophobic substance in cell cultule.
83. Solubilized matrix derived from decellulized liver for the development of functional gel culture and the application to transplantable material.
84. Hideyuki Mizumachi, Hiroyuki Ijima, Biological assay device of neurologic cells with in situ observable gel culture system composed of ECM-modelized matrix, 9th World Congress on Alternatives and Animal Use in the Life Sciences (WC9), 2014.08, [URL].
85. Shintaro Nakamura, Souta Nishimura, Hiroyuki Ijima, Solubilized matrix from decellularized liver as a functional ECM to reproduce in vivo micro-environment in in vitro culture, 9th World Congress on Alternatives and Animal Use in the Life Sciences (WC9), 2014.08, [URL].
86. Molecular structure of matrices solubilized with HCl from decellularized liver and the production of albumin from hepatocytes cultured on the matrices.
87. Eric Jeffries, Shintaro Nakamura, Kee-Won Lee, Jimmy Clampffer, Chelsea Stowell, Hiroyuki Ijima, Yadong Wang, Fibrous Microvascular Scaffolds Made by Combined Electrospinning and Additive Manufacturing, 14th Biennial Meeting (International Society for Applied Cardiovascular Biology (ISACB)), 2014.04, [URL], Purpose: Sufficient vascularization is essential to sustaining viability of tissue engineered constructs after implantation. Artificial microvascular networks fabricated by lithography typically have thick solid layers separating “vasculature” from parenchyma, limiting diffusion. Cell/gel printing techniques have progressed, but remain mechanically weak. These limitations preclude current methods from use in vivo. We combine electrospinning and additive manufacturing (AM) to produce fibrous microvascular scaffolds which are biodegradable, porous, and easily handled.

Methods: Microvascular scaffolds were fabricated by electrospinning polydioxanone (PDO) fibers around extruded polyvinyl alcohol (PVA) templates (Fig. 1A) followed by template removal in water. Morphology and viability of human umbilical vein endothelial cells (HUVECs) cultured on PDO fibers were evaluated by scanning electron microscope (SEM) and Live/Dead assay. HUVEC distribution within microvascular channels was examined by immunofluorescent staining after culture with a flow bioreactor.

Results: SEM demonstrates open microvascular channels within fibrous PDO (Fig. 1B). HUVECs cultured on PDO fibers attached well (Fig. 1C) and demonstrated over 80% cell viability (Fig. 1D). HUVECs attached throughout the channel lumens (Fig. 1E-cross-sectional, 1F-top view).

Conclusions: We demonstrate in vitro endothelialization of a fibrous and degradable microvascular scaffold. Future study will expand this technology to provide intrinsic vascular supply with electrospun scaffolds in vivo.

Figure 1. Characterization of microvascular scaffolds. A) PVA template printed by fused deposition modeling (left). Electrospun PDO fibers encapsulate PVA template (right). B) SEM of fibrous PDO scaffold showing open channel after PVA dissolution. C) HUVEC attachment to PDO fibers (SEM). D) Live/Dead assay for HUVEC on PDO sheets. E/F) DAPI stained HUVEC within channels of microvascular structure (E-cross-sectional)(F-top). Cells were cultured 1 day..
88. Development of the organ-specific solubilized matrix and application to medical engineering, [URL].
89. Hepatocytes culture in decellularized liver and functional evaluation by ex vivo experiment, [URL].
90.
, [URL].
91. , [URL].
92. , [URL].
93. Title: Novel biological assay device of neurologic cells with thin-layer gel system composed of ECM-modelized matrix
Authors: Hideyuki Mizumachi, Hiroyuki Ijima
Abstract:
[Background, Purpose] In recent years, the culture of neurologic cells is carried out as alternative of animal experiments which evaluate influence o n Central Nervous System (CNS) of various chemical agents. Culture system wh ich mimics CNS is desirable for the evaluation, but neurologic cells are gen erally cultured in monolayer condition. Therefore, we developed an acidic po lysaccharide-crosslinked collagen gel which is inspired by ECM of the CNS (1,2). Furthermore, we aimed to construct a novel 3D culture system which enables in situ observation.
[Methods] Heparin, a type of acidic polysaccharide, was chemically cross-linked to collagen, and ECM-modelized matrix was developed. Neural stem cells (NSCs) were embedded into this gel and cul tured with basic fibroblast growth factor (bFGF). Furthermore, NSCs were emb edded into the thin-layered collagen gel of 0.1 mm in thickness. Immunohisto¬chemical analysis was performed.
[Results, Discussion] Embedded NSCs in ECM-modelized matrix showed greater proliferation and neurite outgrowth th an those in collagen gel. This indicated that ECM-modelized matrix had immob ilizability of bFGF like ECM of the CNS. Thin-layered gel makes much easier to observe embedded cells than gel of 2 mm in thickness which is commonly us ed. These results suggest constructing bioassay device with in situ observable 3D culture system composed of ECM modelized matrix.
[Conclusion] ECM-modelized matrix promote proliferation and neurite out-growth of NSCs by immob ilizing bFGF. Thin-layered gel enabled in situ observation of 3D cultured ce lls easily.[References]
1. Bandtlow CE, Zimmermann DR, Physiol Rev. 80:1267-1290 (2000)
2. Masaru Inatani, Naturwissenschaften, 92: 549-561 (2005), [URL].
94. Nana Shirakigawa, Hiroyuki Ijima, Albumin production of re-cellularized liver substitute based on whole organ engineering
, 2013 Joint of Japan/Taiwan/Korea Chemical Engineering Conference, 2013.11.
95. Jingjia Ye, Nana Shirakigawa, Hiroyuki Ijima, Hybrid Organoid Consists of ECM Gel Particles and Hepatocytes, 2013 Joint of Japan/Taiwan/Korea Chemical Engineering Conference, 2013.11.
96. Naoki Amimoto, Hiroshi Mizumoto, Kohji Nakazawa, Hiroyuki Ijima, Kazumori Funatsu, Toshihisa Kajiwara, In vitro and ex vivo functional evaluation of a hollow fiber type bioartificial liver module immobilizing ES cell-derived hepatic cells, 2013 Joint of Japan/Taiwan/Korea Chemical Engineering Conference, 2013.11.
97. Kaoru Morimura, Naoki Amimoto, Hiroshi Mizumoto, Kohji Nakazawa, Hiroyuki Ijima, Kazumori Funatsu, Toshihisa Kajiwara, Quantitative analysis of hepatic cells derived from ES cells in HF/organoid culture, 2013 Joint of Japan/Taiwan/Korea Chemical Engineering Conference, 2013.11.
98. Hideyuki Mizumachi, Hiroyuki Ijima, Charcoal/Dextran-treatedFBS enables reliable cellnumber evaluation ofHUVECs using WST-8, 生体医工学シンポジウム2013, 2013.09.
99. Hideyuki Mizumachi, Hiroyuki Ijima, Measuring stability ofvascular endothelial growthfactor using an immobilizationtechnique, 生体医工学シンポジウム2013, 2013.09.
100. Liver-specific extracellular matrix for cell culture of fetal liver cells

Shintaro Nakamura, Hiroyuki Ijima
.
101. Novel 3D culture technology based on thin-layer gel of neural ECM-mimetic material enables in situ observation

Hideyuki Mizumachi, Hiroyuki Ijima
.
102. Decellularized liver for a hepatocytes-embedded scaffold having vascular-tree network

Nana Shirakigawa, Hiroyuki Ijima
.
103. Numerical simulation of liver cell proliferation -basic model-.
104. Particle Simulation of Liver Regeneration – Basic model of Angiogenesis.
105. Non-Adhesive Single Cell Culture for Primary Rat Hepatocytes

Hiroyuki Ijima, Jun-ichi Sakai

[Background, Purpose] The creation of controlled and practical system for evaluation of cell function is highly desirable in many fields such as a drug development and regenerative medicine. Spherical multicellular aggregate “spheroid” is well-known as a functional and effective culture method for primary hepatocytes. However, spheroid has limited mass transfer because it has no vascular network. Here we report a single cell culture system for anchorage- dependent cells, which provides uniform morphology and excellent mass transfer.
[Methods] PEG-RGD consisting of polyethylene glycol and arginine-glycine-aspartic acid sequence was developed. Primary rat hepatocytes were suspended in PEG-RGD- or RGD-containing medium. Non-treated hepatocyte was used as a control experiment. Cytoskeleton and viability of the cells were evaluated by F-actin staining with Rhodamine-Phalloidin and Calcein-AM, respectively. Expressions of liver-specific functions were evaluated by EROD and albumin production activities.
[Results and Discussion] Integrin-mediated adhesion of primary rat hepatocytes was inhibited by pretreatment with 150 µM RGD. Adhesion and aggregation of primary rat hepatocytes were inhibited by pretreatment with PEG-RGD. All of the hepatocytes in control and RGD conditions were dead in non-adhesive single cell culture at day 1. Though hepatocytes in PEG-RGD condition were in a non-adhesive dispersion, integrin-binding mediated cytoskeleton organization led to excellent preservation of hepatocyte functions including EROD activity and albumin production. Therefore, this novel culture technology has great potential for well controlled and practical cell chip.
[Conclusion] We created functional non-adhesive single cell culture technology for primary rat hepatocytes using PEG-RGD. Hepatocytes in this system expressed the same liver-specific functions as spheroid culture..
106. Novel 3D culture technology of neurologic cells with in situ observable device composed of ECM modelized material

Hideyuki Mizumachi, Hiroyuki Ijima

[Background, Purpose] In recent years, the culture of neurologic cells is carried out as alternative of animal experiments which evaluate influence on Central Nervous System (CNS) of various chemical agents. Culture system which mimics CNS is desirable for the evaluation, but neurologic cells are generally cultured in monolayer condition. Therefore, we developed a functional molecules-immobilizable collagen gel which is inspired by ECM of the CNS1,2 .Furthermore, we aimed to construct a novel neurologic 3D culture system which enables in situ observation.
[Methods] NHS-activated heparin was made by mixing heparin, EDC, and NHS. It was mixed with collagen sol, and heparin-collagen conjugated gel was formed. Immobilizability of this matrix was evaluated by using VEGF and toluidine blue (TB). Furthermore, hippocampal cells were embedded into the thin-layered gel matrix of 100 m in thickness. Immunohisto¬chemical analysis was performed.
[Results and Discussion] The matrix which we developed was specifically stained blue by TB treatment. In addition, VEGF was specifically immobilized to the matrix. This indicates that various heparin-binding neurologic molecules such as netrin, slit, and semaphorin will be able to immobilize to this matrix. Thin-layered gel easily enabled in situ observation of embedded cells. By immunofluo¬rescence staining, the embedded cells were identified as the differentiating into astrocytes and neurons, and the neurites formed 3D networks. These results suggest constructing 3D culture system with in situ observable device composed of ECM modelized material.
[Conclusion] The heparin-collagen conjugate gel had immobilizability of functional molecules. Thin-layered gel enabled in situ observation of 3D cultured cells easily. Hippocampal cells embedded in this matrix constructed 3D nerve networks.
[References]
1. Bandtlow CE, Zimmermann DR, Physiol Rev. 80:1267-1290 (2000)
2. Masaru Inatani, Naturwissenschaften, 92: 549–561 (2005) .
107. Tissue Engineering based on growth factor-immobilizable substratum

Hiroyuki Ijima, Hideyuki Mizumachi, Nana Shirakigawa, Jingjia Ye.
108. Development of solubilized ECM from decellularized organ as a functional scaffold for tissue engineering

Shintaro Nakamura, Hiroyuki Ijima.
109. Development of functional molecule-immobilized hydrogel and the application for wound dressing

Yasuhiro Tomota, Shintaro Nakamura, Hiroyuki Ijima.
110. Novel chip for microsphere production based on the polarity change at the interface between solid and liquid phase

Yuta Hara, Hideyuki Mizumachi, Hiroyuki Ijima.
111. Basic Study for Liver Tissue Engineering by Using Decellularized Organ.
112. Development of growth factor immobilizable extracellular matrix culture substratum.
113. Quantitative evaluation of immobilized growth factor about functionality and the stability.
114. Cell-embedded functional gel-filled scaffold culture for liver tissue engineering
Hiroyuki Ijima, Nana Shirakigawa, Yung-Te Hou, Shintaro Nakamura, Takayuki Takei, Koei Kawakami .
115. Organoid formation and the function expression of primary rat hepatocytes are improved by culturing with hepatocyte growth factor-immobilized culture substratum.
116. Effect of Viscosity on Subsieve-sized capsule diameter.
117. Preparation of chitosan oligosaccharide-liposome complexes utilizing freeze-thawing method.
118. Development of culture medium and culture system for long term expression of liver specific functions.
119. Development of hybrid artificial kidney using cell adhesive molecule.
120. Development of biodegradable scaffold for hepatocyte culture.
121. Optimication of co-culture condition of hepatocyte and bone marrow cells for expression and maintenance of liver functions.
122. Induction of angiogenesis into biodegradable scaffold.
123. Enhancement and maintenance of liver functions by optimyzation of hepatocyte and bone marrow cells.
124. Optimization of co-culture condition of hepatocyte and bone marrow cells for expression and maintenance of liver functions.
125. Co-culture effect of hepatocyte and bone marrow cells for hepatocyte morphology and expression of liver functions.