{"created":"2023-06-19T07:19:20.934322+00:00","id":5393,"links":{},"metadata":{"_buckets":{"deposit":"c8ca398a-419f-44f2-919e-8bf35947406f"},"_deposit":{"created_by":4,"id":"5393","owners":[4],"pid":{"revision_id":0,"type":"depid","value":"5393"},"status":"published"},"_oai":{"id":"oai:az.repo.nii.ac.jp:00005393","sets":["370:15:392"]},"author_link":["23097"],"item_10006_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2020-02-25"}]},"item_10006_degree_grantor_9":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"麻布大学"}],"subitem_degreegrantor_identifier":[{"subitem_degreegrantor_identifier_name":"32701","subitem_degreegrantor_identifier_scheme":"kakenhi"}]}]},"item_10006_degree_name_8":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士(獣医学)"}]},"item_10006_description_22":{"attribute_name":"Abstract","attribute_value_mlt":[{"subitem_description":"To date, toxicity testing of pharmaceutical and industrial chemicals, as well as environmental agents, relies primarily on data derived from animal studies.\nWhile animal toxicity testing is still widely regarded as the most acceptable systems, it is costly and time-consuming and has for animal welfare concerns. Significant advances in stem cell engineering have now resulted in the availability of human induced pluripotent stem cell (iPSC)-derived cells, a physiologically relevant, organotypic in vitro model that promises to overcome these animal model key limitations. iPSC differentiates into almost any cell type, and the organotypic cells derived from iPSC are physiologically relevant and chemical toxicity testing using these organotypic cells promise to avoid problems for animal toxicity testing. Furthermore, iPSC genetic homogeneity and unlimited proliferation provide high quality per isolated batch and stable supply for in vitro toxicity testing. From these advantages, there is increasing demand that iPSC derived organotypic cells apply high-throughput and high content screening (HCS) using automated microscopy system. In terms of conflict with human ethical principles, iPSC-derived cells are easily available compared with primary cell culture models.\nThis study used iPSC derived organotypic cells to establish in vitro toxicity assays which are high-throughput and can measure multiple physiological functions. In the chapter 1, by using HCS, we developed functional vascularization and cellular toxicity evaluation in iPSC-endothelial cells (ECs) which fully has not been reported on applications to toxicity testing. In the chapter 2, we developed the high-throughput multiplex HCS systems to obtain effectively toxicity profiling, using iPSC cardiomyocytes and hepatocytes which has been reported on availability to prediction of cardio- and hepato-toxicity. In the chapter 3, we applied the HCS system developed in the chapter 2 to evaluation of toxicity profiling of 21 petroleum substances, as a case study.\n\nChapter 1: Establishment of a vascularization and cellular toxicity assay in induced pluripotent stem cell-derived endothelial cells\nIn the chapter 1, in order to test the efficiency of iPSC-ECs for toxicity evaluation of chemicals, we demonstrated a vascularization and cellular toxicity assay. We conducted a side-by-side performance comparison of Human Umbilical Vein Endothelial Cells (HUVECs) and iPSC-derived ECs with the hypothesis that the iPSC-derived ECs would be similar or better in performance.\nBoth iPSC-ECs and HUVECs were exposed to selected angiogenesis inhibitors and cytotoxic agents in response for either 18 or 24 hours for the purpose of establishing vascularization and cellular toxicity assay. Both type of cells formed tube-like structures in vascularization assay. Exposure to a known chemical inhibitor of angiogenesis, nocodazole and suramin, resulted in decrease or disruption of angiogenic growth in both cell types. 100 nM nocodazole treatment showed 90% inhibition of angiogenic growth in HUVEC and 50 % inhibition in iPSC-ECs, compared with vehicle treatment. A cytotoxic agent treatment, 50 µM Tetrabutylammonium Bromide (TAB), revealed a decrease in the number of nuclei in both cell types (HUVEC: 80%, iPSC-ECs: 50 %).\nAltogether, we established an HCS approach to evaluate chemical effects on the angiogenesis potential and general cytotoxicity of ECs in a high throughput compatible format, using iPSC-ECs and HUVECs. In terms of a comparison of the cell model, HUVECs performed better particularly for the angiogenesis assays, because the reactivity of the nocodazole-treated samples was higher for HUVECs as compared with iPSC-ECs. In the cellular toxicity assay, the variability of the TAB-treated samples was lower for HUVECs as compared with iPSC-ECs. However, HUVECs have problems that the number of passages keeping cellular functions is limited, and batch-to-batch variations. Therefore, once these problems of iPSC-ECs are improved, they should be a model that promises to overcome the key limitations associated with traditional primary cell culture systems.\n\nChapter 2: Establishment of multiplex high-content assay for toxicity screening in induced pluripotent stem cell-derived cardiomyocytes and Hepatocytes\nIn the rodent risk assessment of chemicals, cardiotoxicity evaluation is insufficient and hepatotoxicity is one of the most prevalent toxicity. Therefore, in the chapter 2, we used two human iPSC types, cardiomyocytes and hepatocytes, to test various high-content assay combinations for cardiotoxicity and hepatotoxicity prediction in a multiparametric format. It has been reported that conventional in vitro hepatotoxicity assays which measure only one parameter have poor prediction to in vivo toxicity. A greater predictive power could be achieved when several parameters regarding hepatotoxicity are combined. Thus, simultaneous detection of various parameters through combination of HCS is useful for toxicity prediction. We tested the combination of cardiomyocyte beating assay and measurement of intracellular cAMP in iPSC cardiomyocytes to determine if the effects on cardiac physiology were β-adrenergic action dependent. Effects on cardiomyocyte beating were characterized by calcium flux measurements. Subsequently, intracellular cAMP levels were measured by ELISA. In addition, we utilized high-content cell imaging to simultaneously determine reactive oxygen species (ROS) formation, mitochondrial integrity, and cytotoxicity which are related to heaptotoxicity in iPSC hepatocytes. As a result, cardiac concentration–response beating changes were observed in isoproterenol treatment for 90 min. The changes were statically and consistent with previous observations reported before (e.g. beat frequency increase induced by isoproterenol, a β-adrenergic agonist, at more than 0.1 μM). Isoproterenol treatment at 10 μM increased intracellular cAMP levels nine fold. This result indicate that a combination of the calcium flux and intracellular cAMP assays is applicable in a same screening format. However, beat frequency increase was seen at more than 0.1 μM, whereas intracellular cAMP increase was only confirmed at 10 μM. For example, in the experiment using mouse cardiomyocytes, it has been reported that intracellular cAMP increase reach a peak within a few minutes after isoproterenol stimulation. Therefore, we need to examine the earlier time point at which both beat frequency and intracellular cAMP increase is detactable at the same concentration. The feasibility of combining ROS formation, mitochondria integrity and cytotoxicity, for high-content imaging of iPSC hepatocytes was evaluated by incubating cells for 30 min and 24 hrs in the presence of TAB and menadione. ROS formation was observed after 30min in cells treated with 100 μM menadione, increasing the number of cells with detectable ROS formation to above 60%. After 30 minutes TAB treatment at 10 μM, mitochondria injury and cytotoxicity was observed. Thus, it was found that ROS formation, mitochondria integrity and cytotoxicity was simultaneously detectable. Although ROS formation in cells treated with menadione had decreased to basal levels after 24 hrs, Mitochondria injury and cytotoxicity in the treatment with TAB at 50 µM was more apparent than after 30 minutes. Altogether, we established a multiplex assay which detects simultaneously ROS formation, mitochondria integrity, and cytotoxicity in iPSC hepatocyte HCS format. This three parameters are considered to be important for hepatotoxicity prediction. Therefore the assay we developed potentially shows high predictability for hepatotoxicity.\n\nChapter 3: An application of the developed multiplex high-content assay to evaluation of petroleum substances\nIn the chapter 3, we applied the HCS system we developed to the evaluation of petroleum substances. The aim of this study was to test the applicability of our combinatorial HCS approaches. The risks of petroleum substance exposure to human need to be concerned in their refining process and daily life of consumer. However, most petroleum substances lack traditional animal study-derived data that can be used to evaluate their safety. \nTherefore, it is important to conduct animal testing effectively after the selection of substances and parameters considered to be evaluate by using alternative methods including in vitro toxicity testing. Especially, we need to predict severe and prevalent type of toxicity, such as, cardiotoxicity potentially resulting in death, and hepatotoxicity often reported as toxicity of chemicals.\nIn the chapter 3, we assessed 21 petroleum substances from three product groups (gas oils, heavy fuel oils, residues) in iPSC cardiomyocytes and hepatocytes using HCS systems established in the chapter 2. As a result, gas oils and heavy fuel oils contributed the most bioactive petroleum substances in the cardiomyocyte beating assay. Gas oils and heavy fuel oils increased cardiomyocyte beat frequency at lower concentrations, resulting in a biphasic appearance of the respective concentration-response plots, i.e. initial increases in the beat rate at low concentrations, followed by decreasing beat rate at high concentrations. In contrast to gas oils and heavy fuel oils, residues did not show these bioactivities in cardiomyocytes. Bioactivity trends observed in hepatocytes correlated well within cardiomyocytes. Gas oils constituted the most bioactive trend of tested petroleum substances. However, Treatment of hepatocytes with heavy fuel oils and residues resulted in moderate to low bioactivity. It has been reported that gas oils contain more highly toxic substances than heavy fuel oils and residues. In this study, gas oils showed higher bioactivity than heavy fuel oils and residues. Hence, there is a relationship between the bioactivities of in our HCS and in vivo toxicity, and the multiplex HCS potentially is applicable to the prediction of petroleum substance toxicity.\nIn this study, in in the chapter 1, in order to examine the applicability of iPS-derived organotypic cells to assess toxicity of chemicals, we established HCS using iPSC-derived ECs. In the chapter 2, we used two human iPSC types, cardiomyocytes and hepatocytes, to test several HCS combinations for their applicability to in vitro toxicity screening. In iPSC heaptocytes, we developed multiplex HCS for prediction of hepatotoxicity. In the chapter 3, to test the applicability of the developed system, we evaluate toxicity of petroleum substances. In addition, the assays presented here could be further enhanced by incorporating other type of iPSC-derived organotypic cells to increase data generated and further refine the toxicity profile of the chemicals including petroleum substances.  In the future, toxicity prediction by our multiplex HCS system will be expected to effectively select chemicals which have unknown toxicity profiles for complementation of toxicity testing using animals."}]},"item_10006_description_7":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"医薬品、工業用および環境中の化学物質の毒性評価は主に動物を用いた試験により行われている。化学物質の毒性評価において動物試験は最も信頼性の高い評価法とされる一方、コストや時間がかかり効率が悪い点、動物愛護の観点で問題がある。これらの問題を解決するために、近年、ヒト人工多能性幹細胞（iPS細胞）から分化誘導した細胞を用いた毒性評価系への期待が高まっている。iPS細胞は生体を構成する全ての細胞に分化可能であるとされる。ヒトiPS細胞から分化誘導した細胞は生体の各種臓器細胞に近い生理学的機能を保持しており、これら臓器別細胞を化学物質評価に用いることで、動物試験の問題点を回避することが期待されている。\n　遺伝的背景が同じ細胞を繰り返し作製できるため、ロット間差が少なく、細胞の供給が安定的である点もiPS細胞の利点と考えられている。これらの利点から、再現性の高い評価が安定的に実施できると考えられ自動顕微鏡を用いたスループット性の高いハイコンテントスクリーニング（HCS）への応用も期待されている。また心筋細胞など、倫理的な問題から初代培養による入手が難しいヒト由来細胞を安定的に入手できる点も大きな利点である。\n　本研究ではヒトiPS細胞由来の臓器別細胞を用い、高いスループット性を有し、複数の生理学的機能の測定ができる毒性評価系を構築するため、第一章では、これまで化学物質の毒性評価への応用報告のないヒトiPS細胞由来内皮細胞を用いてHCSによるアッセイを行い、ヒトiPS細胞由来内皮細胞の細胞モデルとしての有用性を検討した。第二章ではすでに毒性評価に一部応用され、その有用性が報告されているiPS細胞由来心筋および肝細胞のHCSによる評価系を組み合わせ、ハイスループットな評価系の構築を行った。第三章では第二章で構築したHCSの応用例として、第二章で構築した評価系を利用し、21個の石油化学物質のiPS細胞由来心筋および肝細胞への反応性を評価した。\n\n第一章: iPS細胞由来内皮細胞を用いた血管新生アッセイおよび細胞毒性評価系の構築\n　iPS細胞由来内皮細胞の化学物質評価への有用性を検討するため、HCSによる血管新生アッセイおよび細胞毒性評価系の構築を試みた。また既存の内皮細胞モデルであるヒト臍帯静脈内皮細胞（HUVEC）との比較を行った。血管新生阻害および細胞毒性物質を18-24時間細胞に反応させ、血管新生阻害作用を評価するための血管新生アッセイと細胞毒性評価系の構築を行った。その結果、血管新生アッセイではiPS細胞由来内皮細胞およびHUVECともに播種後18時間で、細胞外基質上に血管様の網目管様構造を形成した。これらの構造形成は血管新生阻害作用を持つノコダゾールとスラミン処置によって阻害された。ノコダゾールでは100 nM処置によってiPS細胞由来内皮細胞で50%、HUVECで90 %の管様構造形成が阻害された。また細胞毒性評価系においても細胞毒性物質であるTetrabutylammonium Bromide（TAB）50 µMの24時間処置によってiPS細胞由来内皮細胞で70%、HUVECで80 %の細胞数の減少（細胞毒性）がみられた。これらの結果から、iPS細胞由来内皮細胞を用いて内皮細胞に対する化合物の作用を評価できるHCS評価系の構築ができたと考えられた。細胞の比較という観点では血管新生アッセイにおいてHUVECの方がノコダゾール処置による反応性がより高い傾向があり、細胞毒性評価系においてもiPS細胞由来内皮細胞よりHUVECの方がTAB処置時のサンプル間のばらつきが少ないことがわかった。しかしながら、HUVECは初代培養細胞であるため、機能保持できる継代数が限られる、ロット間差が存在するといった欠点がある。したがってiPS細胞由来細胞で試験系のさらなる改良を行い反応性やサンプル間のばらつきを改善できれば、初代培養細胞の持つこれらの欠点を解決する評価系になると考えられた。\n\n第二章: iPS細胞由来心筋細胞および肝細胞を用いたHCSによる複合的毒性評価系の構築\n　従来のげっ歯類を用いた化学物質の評価では心毒性のリスク評価は不十分である。また一般的に化学物質は肝毒性を多く示すことが知られている。したがって本章では効率的に心・肝毒性を予測するために、iPS細胞由来心筋および肝細胞を用い、心および肝毒性に関する毒性予測では評価項目を組み合わせて測定する評価系の構築を目指した。肝毒性に関する評価項目を単一で測定するだけでは生体の毒性発現を十分に予測できず、数個の評価項目を組み合わせることが予測に重要とされており、評価項目の複合測定は有用性があると考えられる。iPS細胞由来心筋細胞では心毒性予測に有用とされる心筋細胞拍動変化とその作用発現経路（β作用による細胞内cAMP増加）の組み合わせ測定を検討するため、細胞内カルシウム濃度測定による心筋細胞拍動アッセイ後、cAMP濃度測定をELISAによって行い、この2項目を組み合わせて測定できるか検討した。iPS細胞由来肝細胞では肝障害に関与しているとされる評価項目を組み合わせるため、HCSによるROS産生、ミトコンドリア障害性、細胞毒性の3項目の複合測定を行った。その結果、心筋細胞拍動アッセイではβ作用を持つイソプロテレノール90分処置において0.1 µMの濃度から濃度依存的に溶媒対照群と比較して統計的に有意な拍動増加がみられた。細胞内cAMPに関してはイソプロテレノール10 µM処置時に溶媒対照群と比べて9倍程度の増加を確認でき、組み合わせ測定ができることがわかった。しかしながら、拍動数増加は0.1 µM処置からみられたのに対し、cAMPの増加がみられたのは10 µM処置であった。マウス心筋細胞などではイソプロテレノール処置によるcAMPの増加は処置後数分内で増加のピークを迎え、その後は減少することが知られている。したがって、今後の評価系の構築には測定時点を早期にする変更を行い、cAMP測定の反応濃度が心筋細胞拍動アッセイと同程度になるような検討をする必要があると考えられた。iPS細胞由来肝細胞のROS産生、ミトコンドリア障害性、細胞毒性の3つの複合測定では、ROS産生物質であるメナジオン100 µMの30分処置で60 %程度の細胞がROS産生陽性になり、また細胞毒性物質であるTAB 50 µMの30分処置でミトコンドリア障害性および細胞毒性が確認できたことから、ROS産生、ミトコンドリア障害性、細胞毒性の複合測定は可能であると考えられた。24時間処置ではメナジオン100 µM処置によるROS産生はみられなかったが、TABの50 µM処置によるミトコンドリア障害性および細胞毒性は30分処置時より反応が強く、ミトコンドリア障害性と細胞毒性の測定には24時間処置の方が適していることがわかった。したがってiPS細胞由来肝細胞を用いたHCSによるROS産生、ミトコンドリア障害性、細胞毒性の3つの項目を複合的に測定できる評価系の構築ができたと考えられた。ROS産生、ミトコンドリア障害性、細胞毒性は生体の肝毒性予測に重要な評価項目とされていることから、効率的に複数の毒性情報を取得できる本評価系によって肝毒性の予測ができる可能性を示した。\n\n第三章:構築した複合ハイコンテント毒性スクリーニング系による石油化学物質の安全性評価への応用\n　第二章で構築した評価系の有用性を示す応用例として既知の21個の石油化学物質の毒性を調べた。石油化学物質は消費者の身近で使用される製品が多く、また、その精製過程において作業従事者への暴露が懸念されるが、動物試験による有害性情報が明らかでない物質が数多く存在している。したがって動物試験の前段階で代替法により、生命に直接関係する重篤な毒性である心毒性や、化学物質の毒性として報告の多い肝毒性などの毒性予測を行い、評価すべきと考えられる物質および評価項目を絞り込んだ後に効果的かつ効率的に動物試験を実施することが重要である。第三章では、iPS細胞由来心筋細胞の心筋細胞拍動アッセイと、 iPS細胞由来肝細胞のミトコンドリア障害性と細胞毒性の複合HCS測定を利用し、石油の精製過程による分類で軽油、重油、残渣とされる21個の石油化学物質を処置した。その結果、軽油と重油では心筋細胞拍動アッセイにおいて低、中濃度処置での拍動増加、高濃度処置での拍動減少と停止の反応がみられ、残渣ではこれらの反応はみられなかった。肝細胞のミトコンドリア障害性と細胞毒性の複合測定では高濃度処置での反応が、軽油で最も強く、重油で中程度、残渣で最も弱かった。したがって利用した評価系での反応は軽油で最も強く、重油で中程度、残渣で最も弱い傾向を示すことがわかった。\n　軽油には重油や残渣より毒性物質が多く含まれることが知られており、生体への毒性がより強いと考えられている。今回の評価系の結果においても、実際の生体への毒性が強い可能性のある軽油で他の2分類の物質より強い反応がみられたことから、評価系の反応性と生体への毒性の強さの間には関連性があり、石油化学物質の毒性予測に構築した複合的HCSを利用できる可能性が考えられた。\n本研究ではiPS細胞由来臓器別細胞の化合物評価への有用性を検討するため、第一章ではiPS細胞由来内皮細胞を用いHCSによる毒性評価系の構築を行った。第二章ではヒトiPS細胞由来心筋・肝細胞を用い、HCSによる複合測定の評価系構築を試み、肝細胞では肝毒性予測への応用が可能な複合HCS評価系の構築ができた。第三章では第二章で構築した評価系の応用性を検討するために石油化学物質の評価を行った。将来的にはiPS細胞由来の様々な臓器別細胞を用いることで評価系をさらに拡充し、石油化学物質を含む種々の化学物質等、未知の毒性プロファイルを持つ物質の毒性予測を動物試験の前段階で構築した評価系を用いて行い、効果的に化学物質を絞り込むことで、動物試験を補完する役割が期待される。\n"}]},"item_10006_dissertation_number_12":{"attribute_name":"学位授与番号","attribute_value_mlt":[{"subitem_dissertationnumber":"乙第436号"}]},"item_10006_textarea_23":{"attribute_name":"Rights","attribute_value_mlt":[{"subitem_textarea_value":"本論文の一部は以下のとおり公表されている。(Part of this dissertation has been published as follows.)\n1. Grimm FA，Iwata Y (Co-first)，Sirenko O，Bittner M，Rusyn I:High-Content Assay Multiplexing for Toxicity Screening in Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Hepatocytes. Assay Drug Dev Technol. 2015; 13(9):529-46. \n\n2. Grimm FA，Iwata Y，Sirenko O，Chappell GA，Wright FA，Reif DM，Braisted J，Gerhold DL，Yeakley JM，Shepard P，Seligmann B，Roy T，Boogaard PJ，Ketelslegers HB，Rohde AM，Rusyn I. A Chemical-Biological Similarity-Based Grouping of Complex Substances as A Prototype Approach for Evaluating Chemical Alternatives. Green Chem. 2016; 18(16):4407-4419. \n\n3. Iwata Y，Klaren WD，Lebakken CS，Grimm FA，Rusyn I. High-Content Assay Multiplexing for Vascular Toxicity Screening in Induced Pluripotent Stem Cell-Derived Endothelial Cells and Human Umbilical Vein Endothelial Cells. Assay Drug Dev Technol. 2017 15(6):267-279."}]},"item_10006_version_type_18":{"attribute_name":"著者版フラグ","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_970fb48d4fbd8a85","subitem_version_type":"VoR"}]},"item_access_right":{"attribute_name":"アクセス権","attribute_value_mlt":[{"subitem_access_right":"open access","subitem_access_right_uri":"http://purl.org/coar/access_right/c_abf2"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"岩田, 康寛"}],"nameIdentifiers":[{}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2020-05-08"}],"displaytype":"detail","filename":"diss_dv_otsu0436.pdf","filesize":[{"value":"2.4 MB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"diss_dv_otsu0436","url":"https://az.repo.nii.ac.jp/record/5393/files/diss_dv_otsu0436.pdf"},"version_id":"f013fa6e-13bb-427e-b3ea-25e568ec1e46"},{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2020-05-08"}],"displaytype":"detail","filename":"diss_dv_otsu0436_jab&rev.pdf","filesize":[{"value":"214.7 kB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"diss_dv_otsu0436_jab&rev.pdf","url":"https://az.repo.nii.ac.jp/record/5393/files/diss_dv_otsu0436_jab&rev.pdf"},"version_id":"c1ac03b2-a5a6-4774-aa56-833a58335f3f"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"jpn"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"doctoral thesis"}]},"item_title":"iPS細胞由来心筋、肝および内皮細胞を用いた複合的ハイコンテント毒性スクリーニング系の構築","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"iPS細胞由来心筋、肝および内皮細胞を用いた複合的ハイコンテント毒性スクリーニング系の構築"},{"subitem_title":"Establishment of Multiplex High-Content Assay for Toxicity Screening in Induced Pluripotent Stem Cell-Derived Cardiomyocytes, Hepatocytes, and Endothelial Cells","subitem_title_language":"en"}]},"item_type_id":"10006","owner":"4","path":["392"],"pubdate":{"attribute_name":"公開日","attribute_value":"2020-05-11"},"publish_date":"2020-05-11","publish_status":"0","recid":"5393","relation_version_is_last":true,"title":["iPS細胞由来心筋、肝および内皮細胞を用いた複合的ハイコンテント毒性スクリーニング系の構築"],"weko_creator_id":"4","weko_shared_id":4},"updated":"2023-06-19T07:40:33.558280+00:00"}