シンポジウム
56 人工神経線維組織モデルの発展と、工学系から医学系への応用
56 Novel development of artificial nerve fiber tissue model, and application from engineering to medicine
座長：芝田 晋介（新潟大学大学院医歯学研究科 組織学分野）・池内 与志穂（東京大学生産技術研究所） |
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| 2022年6月30日 9:00～9:20 沖縄コンベンションセンター 会議場B5～7 第4会場
1S04m-01
創薬スクリーニングおよび移植治療のための神経細胞ファイバ
Neural cell fiber for drug screening and transplantation therapy
*根岸 みどり(1)、竹内 昌治(2,3,4)
1. 武蔵野大学、2. 東京大学大学院情報理工学系研究科、3. 東京大学大学生産技術研究所、4. 東京大学ニューロインテリジェンス国際研究機構
*Negishi Midori(1), Shoji Takeuchi(2,3,4)
1. Musashino University, 2. Department of Mechano-Informatics, Graduate School of Information Science and Technology, The University of Tokyo, 3. IIS, The University of Tokyo, 4. WPI-IRCN, The University of Tokyo
Keyword: drug screening, microdevice, tissue engineering
In recent years, devices based on Micro Electro Mechanical Systems (MEMS) technology have been used to construct and model diseased tissues for drug screening and contributing to the field of regenerative medicine. We have developed technologies for forming neural networks by arraying neuronal spheroids in microwells and assembling microplates with a neuron created by microfabrication techniques. We have also fabricated 100 μm diameter, meter-sized neural tissues "Neural cell fibers" by applying the laminar flow method using microfluidic devices. This symposium will introduce the characteristics of neural cell fibers, which are core-shell hydrogel fibers encapsulating neurons, glial cells, and neural stem cells, and their application in drug screening and transplantation. Cell fiber is a core-shell hydrogel fiber with the core formed by cells and ECM and the shell composed of calcium alginate. One of the significant features of cell fibers is that the encapsulated cells come into contact and form a fiber-shaped tissue in the extracellular matrix. Cell fiber can be formed from various types of cells such as cancer cells, stem cells, muscle cells, adipocytes, and nerve cells. We have succeeded in creating an array for drug screening using these cell fibers. Although two-dimensional (2D) culture has been the mainstream for drug screening, drug efficacy differs substantially between 2D culture systems and 3D tissues. Therefore, 3D tissues such as spheroids and organoids are already attracting attention for screening anticancer drugs. They mimic the intracellular environment and communication much more accurately, missing from 2D culture systems. We developed a device for arraying cell fiber a 96 well plate and showed that it could be applied to drug screening. In addition, we transplanted bundles of neural cell fibers produced from mouse neural stem cells into mice with spinal cord injuries and confirmed their differentiation into neurons and glial cells. The fabrication techniques used to produce standardized fiber-shaped tissues have the potential to be applied to high-throughput screening of drugs and transplantation therapy in the future. | | 2022年6月30日 9:20～9:40 沖縄コンベンションセンター 会議場B5～7 第4会場
1S04m-02
運動ニューロンオルガノイド培養を用いた筋萎縮性側索硬化症の病態解明
Elucidation of the mechanism of amyotrophic lateral sclerosis using a motor nerve organoid device
*鈴木 直輝(1)
1. 東北大学
*Naoki Suzuki(1)
1. Tohoku University
Keyword: motor nerve organoid device, amyotrophic lateral sclerosis (ALS), Human-induced pluripotent stem cell (hiPSC), axon
Amyotrophic lateral sclerosis (ALS) is an intractable adult-onset neurodegenerative disease that leads to the loss of upper and lower motor neurons (MNs). The long axons of MNs become damaged during the early stages of ALS. Genetic and pathological analyses of ALS patients have revealed dysfunction in the MN axon homeostasis. However, the molecular pathomechanism for the degeneration of axons in ALS, including the neuronal cytoskeleton, cargo transport within axons, axonal energy supply, clearance of junk protein, neuromuscular junctions (NMJs), and aberrant axonal branching has not been fully elucidated. Using sets of isogenic human-induced pluripotent stem cell (hiPSCs)-derived MNs possessing the single amino acid difference in the fused in sarcoma (FUS) and TARDBP (coding TDP-43) were constructed. We identified aberrant increasing of axon branching and shortened neurite length in mutant hiPSCs-derived MN axons compared with isogenic controls. RNA profiling of isolated axons was conducted by applying the microfluidic devices that enable axon bundles to be produced for omics analysis. The relationship between the target gene, which was identified as a pathological candidate in ALS with RNA-sequencing, and the MN phenotype was confirmed by intervention with si-RNA or overexpression to hiPSCs-derived MNs and even in vivo model. The commonality was further confirmed with other ALS-causative mutant hiPSCs-derived MNs and human pathology. The development of novel strategies to analyze axon fractions provides a new approach to establishing a detailed understanding of resilience of long MN and MN pathology in ALS. | | 2022年6月30日 9:40～10:00 沖縄コンベンションセンター 会議場B5～7 第4会場
1S04m-03
神経オルガノイドチップを活用した脳白質損傷モデルの可能性
Neuronal organoid in a chip for studying human antenatal white matter injury
*阿久津 英憲(1)、菅原 亨(1)、川崎 友之(1)、平岩 幹(1,2)
1. 国立成育医療研究センター研究所、2. 福島県立医科大学
*Hidenori Akutsu(1), Tohru Sugawara(1), Tomoyuki Kawasaki(1), Tsuyoshi Hiraiwa(1,2)
1. Natl Ctr Child Health Dev, Tokyo, Japan, 2. Sch Med, Fukushima Med Univ, Fukushima, Japan
Keyword: neuronal chip, organoid, iPS, periventricular cerebral white matter injury
Periventricular cerebral white matter injury (PWMI) is the major form of preterm brain injury, which is defined by selective disruption of oligodendrocyte linage maturation and myelination. PWMI and maternal intrauterine infections in preterm fetuses result in Cerebral Palsy. Premature oligodendrocytes are vulnerable to perinatal hypoxic injury. However, biomedical systems for studying fetal brain conditions are extremely limited and so we hardly to approach the mechanism of human antenatal white matter injury relevant to the development of the human fetal cerebral cortex. We aim to develop an in vitro model of PWMI by applying the microfluidic device in conjunction with nerve organoids that enables to recapitulate myelinated axon bundles in vitro. We first made neuronal spheres of cortical neurons and oligodendrocyte progenitor cells using human induced pluripotent stem cells. To evaluate its tissue properties in the neuronal axon fascicles, a cross-section of the tissue was analyzed using transmission electron microscopy and also immunohistochemical staining. Furthermore, we evaluated the nerve organoids to model neuronal damage and degeneration. We confirmed that numerous axons were contained within the fascicle, and the axons were well-organized cytoskeletal fibers as antenatal axon bundles in the neural chip model. We have successfully developed a novel in vitro model of human antenatal white matter injury, and this system could be applied to further studies to gain insight into the mechanism of PWMI. | | 2022年6月30日 10:00～10:20 沖縄コンベンションセンター 会議場B5～7 第4会場
1S04m-04
特殊培養デバイスによるヒトiPS細胞由来神経オルガノイドを使った末梢神経障害治療法開発
Development of novel medical material for peripheral neuropathy using human iPS cell-derived nerve organoids using a special culture device
*芝田 晋介(1,2)、西島 貴之(2)、木村 洋朗(2)、奥山 健太郎(1,2)、早津 学(1)、信藤 知子(2)
1. 新潟大学 大学院医歯学総合研究科、2. 慶應義塾大学 医学部
*Shinsuke Shibata(1,2), Takayuki Nishijima(2), Hiroo Kimura(2), Kentaro Okuyama(1,2), Manabu Hayatsu(1), Tomoko Shindo(2)
1. Grad Sch Med Dent, Niigata Univ, Niigata, Japan, 2. Keio Univ, Sch Med, Tokyo, Japan
Keyword: nerve injury, human iPS cell, nerve organoid, special culture device
It is necessary to perform surgical operation when the peripheral nerve fibers were damaged due to cancer surgery or traumatic injury. At present, the most common treatment is to carry out the self-nerve transplantation with a part of patient's own peripheral sensory nerve. The self-nerve removal leads to the various defects, such as loss of the sensory perception carried by the removed donor sensory nerve and expansion of the surgery periods. Our nerve regeneration project team has been conducting collaborative research for many years, trying the several challenging tasks by taking advantage of each other's advanced technologies as surgeon and imaging specialists. In recent years, we have been working on developing a new treatment for peripheral nerve deficits that promotes functional recovery by transplantation. The novel nerve organoid culture device with the excellent patented technology was utilized in this project, and we tried to develop a novel artificial nerve for nerve transplantation, as a substitution of the self-nerve. Human iPS cells were induced to differentiate into neurons and cultured using a patented special culture device. The novel artificial nerve was transplanted into the sciatic nerve defect of rats with optimized transplantation surgery, the motor function was evaluated over time, and the condition of peripheral nerve regeneration by transplantation was evaluated at several weeks after transplantation. The histological evaluation is performed in detail using immunostaining and an electron microscope to evaluate the effect of this novel transplantation therapy. We hope new artificial nerve prepared in advance will be transplanted if a major peripheral nerve is damaged due to traumatic injury or surgical operation of cancer, without any loss of patient's own peripheral nerve in the near future. | | 2022年6月30日 10:20～10:40 沖縄コンベンションセンター 会議場B5～7 第4会場
1S04m-05
相互に接続された脳オルガノイドの複雑な神経活動と光刺激による短期記憶誘導
Advanced complexity and plasticity of neural activity in reciprocally connected human brain organoids
*大崎 達哉(1)、池内 与志穂(1,2)
1. 東京大学 生産技術研究所、2. 東京大学 Beyond AI 研究推進機構
*Tatsuya Osaki(1), Yoshiho Ikeuchi(1,2)
1. Institute of Industrial Science, The University of Tokyo, 2. The Institute for AI and Beyond, The University of Tokyo
Keyword: Brain organoid, iPS cells, Optogenetics, Bioengineering
Cerebral organoids mimic not only structural and cellular characteristics of human cerebral cortex, but also complex oscillation activity patterns of human brains at developmentally early stages. Although brain organoids provide novel avenues to investigate human brain structures and functions by modeling small neuronal networks of the brain in vitro, the functionality of the macroscopic connections between distant regions has not been well investigated with organoids. Connections between distinct regions of the brain (e.g. cortex-cortex, cortex-thalamus, etc.) through axonal projections are necessary for generating coordinated neuronal activity and function of the brain. Here, in this symposium, we present the formation of reciprocally connected human cerebral organoids as a model tissue of a macroscopic cortical circuit. The organoids were connected by a bundle of axons in a microfluidic device which receives two organoids and let them grow axons toward each other in a narrow channel between the two organoids. The “connected” organoids produced significantly more intense and complex oscillatory activity than individual cerebral organoids. Furthermore, optogenetic manipulations revealed that the connected organoids could respond to external stimuli and maintain the elevated frequency of neuronal activity for a short term, indicating that the networked neural tissue exhibit plasticity at the circuit level. In addition, the connected organoids exhibited accelerated maturation of neurons which was characterized by single cell (sc) RNA-seq. These findings highlight the functional importance of inter-regional connections and suggest that connected organoids can be used as a powerful tool for investigating the development and functions of macroscopic circuits in the human brain. | | 2022年6月30日 10:40～11:00 沖縄コンベンションセンター 会議場B5～7 第4会場
1S04m-06
Thin-Film-Transistor Sensing Platform for Real-time Multi-modal Analyses of Excitable Cells Culture
*Agnes Tixier-Mita(1), Satoshi Ihida(1), Anne-Claire Eiler(2), Tieying Xu(1), Pierre-Marie Faure(3), Timothee Levi(3), Hiroshi Toshiyoshi(1)
1. IIS, Univ of Tokyo, Tokyo, Japan, 2. Grad Sch Eng, Univ of Tokyo, Tokyo, Japan, 3. Lab Syst to Mat Integration, Univ of Bordeaux, Bordeaux, France
Keyword: Thin-Film-Transistor Technology, Multimodal Sensing, Electrophysiology, Inter-organ Communication
Homeostasis in the body is fundamentally dependent on inter-organ communication, like in between brain, heart, liver and pancreas. Inter-organ communication is usually studied by means of behavioral changes and in-vivo measurements investigations on small animals, and might include surgical experimentation. However, the detailed mechanisms of communication has not been elucidated yet, and there is a need to develop methods for systematic evaluation in a more controlled environment, like in-vitro environment, which could be also applied to in-vivo environment.
Our group has been developing multi-modal sensing platforms, based on micro-fabricated electrical sensors, for electrophysiology and bio-chemical sensing of cells, as well as for electrical biomimetic stimulation that could bypass signals transmitted from brain or nervous system. The platforms are made by Thin-Film-Transistor technology, which is the same technology as for liquid crystal displays (LCD). Compared to usual commercially available sensing devices, which remain for most dedicated to single-modal sensing on limited area, these platforms offer a large surface (several cm size) covered by high-resolution of thousands of microelectrodes array, with dimensions until single cell (10um), which can be individually selected and used either for sensing or for stimulation. In addition the devices are transparent, so compatible for optical observation and optogenetics stimulation.
In our group, various electrophysiology studies are under investigation on neurons, cardiomyocytes and pancreatic beta cells, as well as simultaneous cell culture monitoring by impedance measurements and biochemical sensing. We are targeting in-vitro neuro-cardiac interactions investigations, comprising as well multi-modal sensing as biomimetic stimulation of cells by integrating neuromorphic technologies. The large scale surface covered with sensors makes possible large culture studies, like multi co-cultures. In-vitro multi-organoid communication investigation is also among our targets. In the future, this platform could be adapted to in-vivo environment. | |
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