Details of a Researcher - Tsukada, Kosuke

pH-taxis drives aerobic bacteria in duodenum to migrate into the pancreas with tumors.

Hiroaki Shirai, Cocoro Ito, Kosuke Tsukada

Scientific reports (Scientific Reports) 12 ( 1 ) 1783 - 1783 2022.12

ISSN 2045-2322

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As oral or intestinal bacteria have been found in pancreatic cystic fluid and tumors, understanding bacterial migration from the duodenum into the pancreas via hepato-pancreatic duct is critical. Mathematical models of migration of aerobic bacteria from the duodenum to the pancreas with tumors were developed. Additionally, the bacterial distributions under the pH gradient and those under flow were measured in double-layer flow based microfluidic device and T-shaped cylinders. Migration of aerobic bacteria from the duodenum into pancreas is counteracted by bile and pancreatic juice flow but facilitated by pH-taxis from acidic duodenum fluid toward more favorable slightly alkaline pH in pancreatic juice. Additionally, the reduced flow velocity in cancer patients, due to compressed pancreatic duct by solid tumor, facilitates migration. Moreover, measured distribution of GFP E. coli under the pH gradient in a microfluidic device validated pH-tactic behaviors. Furthermore, Pseudomonas fluorescens in hydrochloride solution, but not in bicarbonate solution, migrated upstream against bicarbonate flow of > 20 μm/s, with an advancement at approximately 50 μm/s.
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Ex vivo sensing of primordial follicles in ovarian tissues by spectral-domain optical coherence tomography

Masaki Hosoda, Daisuke Oida, Koichiro Ito, Seido Takae, Nao Suzuki, Kosuke Tsukada

Medical Imaging 2022: Biomedical Applications in Molecular, Structural, and Functional Imaging (SPIE) 12036 2022

ISSN 0277-786X
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Convolutional neural network-based automatic detection of follicle cells in ovarian tissue using optical coherence tomography

Kasumi Saito, Yuki Motani, Seido Takae, Nao Suzuki, Kosuke Tsukada

Biomedical Physics & Engineering Express ({IOP} Publishing) 6 ( 6 ) 065026 - 065026 2020.09

Research paper (scientific journal), Joint Work, ISSN 2057-1976

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To preserve the fertility of young female cancer patients, ovarian tissue cryopreservation and transplantation have been investigated as next-generation reproductive medical technologies. Noninvasive visualization of follicles in ovarian tissue and cryopreservation of higher density tissue is essential for effective transplantation. We proposed the use of optical coherence tomography (OCT) that can noninvasively visualize the internal structure of the ovarian tissue. However, a method for quantifying cell density has not yet been established because of the lack of available techniques to visualize follicles noninvasively. We proposed the use of a convolutional neural network (CNN) to extract small features from medical images as an image analysis method to automatically detect follicles from the obtained OCT images. First, we collected a total of 13 ovarian tissues from four-day-old mice and acquired OCT images using a full-field-type OCT. Then, the acquired images were analyzed using three detection methods: filter processing, filter processing combined with the CNN, and only CNN. Finally, to verify the detection accuracy of each method, the detection rate and precision were calculated by taking the doctor's detection as the correct result. The results showed that the detection method only using CNN achieved a detection rate of 0.81 and precision of 0.67; this indicated that follicles could be effectively detected using our proposed method. Furthermore, it is quantitatively evident that the density of follicles from the surface layer to the deep region differs depending on the tissue. In the future, these results could be used to detect follicles in tissues of different maturation stages and quantify follicles three-dimensionally, further accelerating next-generation reproductive medicine.
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Bacterial proteolytic activity improves drug delivery in tumors in a size, pharmacokinetic, and binding affinity dependent manner – A mechanistic understanding

Shirai H, Tsukada K

Journal of Controlled Release (Journal of Controlled Release) 321 348 - 362 2020.05

Joint Work, ISSN 01683659

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© 2020 Elsevier B.V. Motile bacteria are able to penetrate in the distal areas of blood vessel, which makes bacteria attractive to researchers as a drug delivery vehicle carrying anti-cancer drugs to tumors. Not only therapeutic bacteria show wide anti-tumor effect but also the combination of therapeutic bacteria and conventional chemotherapy leads to dramatically large synergetic effect. We provide a mechanistic understanding of enhanced drug delivery in tumors by co-administration of chemotherapeutic agents and therapeutic bacteria. In this work, simultaneous delivery of C. novyi-NT and chemotherapeutic agents in tumors is mathematically modeled. Simulated doxorubicin concentration in tumors after Doxil administration with or without bacteria agreed reasonably well with experimental literature. Simulated doxorubicin concentration in tumors by the combination of Doxil and C. novyi-NT is over twice higher than that of Doxil alone. This enhanced doxorubicin concentration in tumors is due to the degradation of extracellular matrix of collagen by bacterial proteolytic activity, which increases hydraulic conductivity of interstitium, reduces interstitial fluid pressure, and thus increases convection through vessel walls. Additionally, it alleviates solid stress, which decompresses blood vessels, and thus increases vessel density. On the other hand, simulated doxorubicin concentration in tumors for non-liposomal free-doxorubicin is not enhanced by C. novyi-NT because vascular permeability of free-doxorubicin is larger than Doxil, and thus increased but relatively small convection across vessel walls is offset by the efflux due to increased interstitial flow. A strategy to further enhance this combination therapy is discussed along with sensitivity analysis.
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High-throughput single-cell live imaging of photobiomodulation with multispectral near-infrared lasers in cultured T cells

Katagiri W., Lee G.H., Tanushi A., Tsukada K., Choi H.S., Kashiwagi S.

Journal of Biomedical Optics (Journal of Biomedical Optics) 25 ( 3 ) 1 - 18 2020.03

ISSN 10833668

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© 2020 by ASME. Significance: Photobiomodulation is a well-established therapeutic modality. However, the mechanism of action is poorly understood, due to lack of research in the causal relationship between the near-infrared (NIR) light irradiation and its specific biological effects, hindering broader applications of this technology. Aim: Since biological chromophores typically show several absorption peaks, we determined whether specific effects of photobiomodulation are induced with a combination of two wavelengths at a certain range of irradiance only, rather than a single wavelength of NIR light. Approach: In order to analyze a wide array of combinations of multispectral NIR light at various irradiances efficiently, we developed a new optical platform equipped with two distinct wavelengths of NIR lasers by high-throughput multiple dosing for single-cell live imaging. Two wavelengths of 1064 and 1270 nm were selected based on their photobiomodulatory effects reported in the literature. Results: A specific combination of wavelengths at low irradiances (250 to 400 mW/cm2 for 1064 nm and 55 to 65 mW/cm2 for 1270 nm) modulates mitochondrial retrograde signaling, including intracellular calcium and reactive oxygen species in T cells. The time-dependent density functional theory computation of binding of nitric oxide (NO) to cytochrome c oxidase indicates that the illumination with NIR light could result in the NO release, which might be involved in these changes. Conclusions: This optical platform is a powerful tool to study causal relationship between a specific parameter of NIR light and its biological effects. Such a platform is useful for a further mechanistic study on not only photobiomodulation but also other modalities in photomedicine.
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Papers, etc., Registered in KOARA 【 Display / hide 】

Super-resolution oxygen partial pressure imaging of single cells

Tsukada, Kosuke

科学研究費補助金研究成果報告書 2021
Development of microfluidic cell culture devices to artificially mimic the cancer microenvironment

Tsukada, Kosuke

学事振興資金研究成果実績報告書 (慶應義塾大学) 2020
Development of a microfluidic cell culture device that artificially mimics cancer microenvironment

Tsukada, Kosuke

学事振興資金研究成果実績報告書 (慶應義塾大学) 2019
Flexible film-type oxygen sensor for measurement of skin and organ surfaces

Tsukada, Kōsuke

科学研究費補助金研究成果報告書 2018
A microfluidic cell culture device for mimicking tumor microenvironment in vitro

Tsukada, Kosuke

学事振興資金研究成果実績報告書 (慶應義塾大学) 2018

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Reviews, Commentaries, etc. 【 Display / hide 】

皮内投与型ワクチンの免疫賦活化に適した近赤外レーザーデバイスの開発

片桐渉, 君塚善文, 柏木哲, 塚田孝祐

日本レーザー医学会誌 ((NPO)日本レーザー医学会) 39 ( 3 ) 254 - 254 2018.09

ISSN 0288-6200
“標識しない”という新たなバイオイメージングの流れ

塚田孝祐

光学 (日本光学会) 44 ( 6 ) 215 2015.06

Article, review, commentary, editorial, etc. (scientific journal), Single Work
AFMを用いた生体試料弾性の評価法

川村友美, 塚田孝祐, 関塚永一, 大塩力, 南谷晴之

Molecular electronics and bioelectronics 11 ( 4 ) 265 - 270 2000.11
Analysis of elasticity and deformability of erythrocytes using micro-channel flow system and atomic force microscope

H Minamitani, K Tsukada, T Kawamura, E Sekizuka, C Oshio

1ST ANNUAL INTERNATIONAL IEEE-EMBS SPECIAL TOPIC CONFERENCE ON MICROTECHNOLOGIES IN MEDICINE & BIOLOGY, PROCEEDINGS (IEEE) 68 - 71 2000

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Mechanical properties of erythrocytes under diabetic and some other physico-chemical conditions were measured by using transparent micro-channel flow system for the analysis of erythrocyte deformability and also by using atomic force microscope (AFM) for the analysis of microscopic elasticity of the erythrocyte cell membrane respectively. Deformability of diabetic erythrocytes was significantly lower than that of healthy controls, especially at higher shear rate. The spring constant and the Young's modulus of diabetic erythrocytes were much higher than those of healthy controls. These erythrocytes were obviously stiffened to be hard to deform themselves in the capillary flow passage that may follow to the growth of microangiopathy.
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Presentations 【 Display / hide 】

共焦点光学系を用いた腫瘍血管の透過性及び酸素分圧の定量化

岩本侑一郎，小田慶太郎，塚田孝祐

第43回レーザ顕微鏡研究会＆シンポジウム,

2018.01
,
Oral presentation (general)
近赤外光を用いた非侵襲的ワクチンアジュバント

柏木哲，君塚善文，片桐渉，塚田孝祐

第38回日本レーザー医学会総会,

2017.11
,
Oral presentation (general)
近赤外半導体レーザーデバイスによる免疫活性化作用の検証

片桐渉，君塚善文，Kashiwagi Satoshi，塚田孝祐

第38回日本レーザー医学会総会,

2017.11
,
Poster presentation
Quantification and imaging of vascular permeability and partial pressure of oxygen in vivo

Iwamoto Y., Oda K., Tsukada K.

Annual Meeting of BMES 2017 (Phoenix, USA) ,

2017.10
,
Oral presentation (general), Biomedical Engineering Society
Three-dimensional in vivo analysis of histology for low-level laser therapy

Katagiri W., Tsukada K.

Annual Meeting of BMES 2017 (Phoenix, USA) ,

2017.10
,
Poster presentation, Biomedical Engineering Society

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Research Projects of Competitive Funds, etc. 【 Display / hide 】

Interpretation of regulatory system of fetal cardiovascular development with re-evaluation of substance transport characteristics in fetal circulation

2020.04

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2022.03

Grants-in-Aid for Scientific Research, Grant-in-Aid for Challenging Research (Pioneering), No Setting

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母親の胎内は生後に比べて非常に酸素濃度が低いが、母親と胎児をつなぐ胎児循環（胎盤や血液中で酸素を運ぶヘモグロビン分子）は効率的に酸素を運ぶためのシステムとして捉えられることが多い。しかし胎児のエネルギー消費は大きくなく、過剰な酸素は細胞分裂時に有害にさえなり得る。本研究では胎児循環を胎児細胞の為に最適な低酸素環境を維持し、体を構成するための基質（アミノ酸や脂肪酸）を選択的に運搬する循環システムであると捉え、研究対象として出生後すぐに細胞分裂を止める心筋細胞を扱う。何億年もかけ培ってきた細胞制御システムを分子・細胞・臓器レベルで解明することは、再生医療にも役立つ情報の取得に繋がると期待される。
胎生期の栄養環境が心臓に及ぼす影響を明らかにするため、活発に分裂を繰り返して原始心臓を形成する胎児心筋細胞とアミノ酸環境の関係をラットの培養心筋細胞で検討した。ラットを用いて、胎生20日目、出生後２日目、５日目もしくは６日目、８週齢において、血中および心筋組織中の各種アミノ酸濃度を測定したところ、血中アミノ酸濃度は、Lys，Met，Phe，Trpなどが心筋分裂が盛んな胎生20日目で濃度が高く、出生後には減少した。心筋組織中のアミノ酸濃度は、出生前後で血中ほどの大きな変化はみられなかったが、Leu，Lys，Phe，Valは、出生後にやや減少していた。これらの結果より、ラット胎児の培養心筋細胞を用い、細胞増殖マーカー（Ki67）を指標として心筋細胞分裂能とアミノ酸の関係を検討した。まず胎生20日目の心筋細胞において、単独のアミノ酸での培養を行なった時の分裂能を検討し、分裂能に促進効果のありそうなアミノ酸を探索した。次いで、胎生17日目と胎生20日目の心筋細胞において、心筋分裂に影響のありそうな複数のアミノ酸を除去して培養を行ない、分裂能を検討した。単独アミノ酸培養では、Cys，Gly，His，Lys，Valでの培養が、単独でも、通常培地と同程度の細胞増殖マーカーの陽性率を示した。アミノ酸除去実験では、胎生17日目の心筋細胞においては一度に10種のアミノ酸を除去すると陽性率は有意に減少したが、その他の組み合わせ（３～９種）でははっきりとした陽性率の減少はみられなかった。胎生20日目の心筋細胞では、10種のアミノ酸の除去で陽性率が有意に減少し、Cys・Gly・Ser、His・Ile・Trpの３種、Gly・Lys・Tyr・Valの４種、必須アミノ酸９種の除去で、有意ではなかったが、陽性率が減少する傾向にあった。
胎盤における選択的物質輸送の具体例としてアミノ酸に着目し、成体と胎児血液におけるアミノ酸濃度の違いを確認出来た。心筋細胞培養実験では、アミノ酸の種類によって心筋分裂能の維持に影響の強いものもあったが、特に影響の強いアミノ酸を認めなかった。胎児循環の酸素環境が心臓形成に及ぼす影響に関しては、ポルフィリン燐光を用いた生体微小循環酸素計測システムを構築して臍帯動静脈の酸素分圧を同時に計測しながら母体側の酸素環境を変化させて胎児循環の酸素運搬特性が大量の酸素輸送を目的とするものではなく、成体と比べて著しく低い酸素濃度を維持するための緩衝機構であることを証明する予定であったが、社会的要因から共同実験の進捗が滞り課題が残されたため。
胎児循環のアミノ酸選択的輸送と心筋細胞分裂に関する研究に関しては、アミノ酸配合の様々な組み合わせと心筋細胞分裂能の関係を検証していく。また、胎生期の低栄養環境が心臓に及ぼす影響を解析するため、妊娠15日目から妊娠21日目まで、母ラットに50％摂食制限を施して胎生期低栄養ラットを作成し、自由摂食の母ラットから生まれた仔と出生前後（胎生20日目、出生後1日目、14日目、21日目、30週齢）において、体重、心重量の測定や、心筋組織における心機能関連遺伝子と細胞周期関連遺伝子の発現を比較する。また、胎生期低栄養ラット４週齢において、モノクロタリン（Mct）30mg/kgを皮下投与して肺高血圧症を発症させて、心エコーによる心機能評価や血中ANP濃度測定を行う。また、ポルフィリン燐光を用いた生体微小循環酸素計測システムの構築を進め、胎児循環の酸素環境と胎児心臓形成に関する情報を分子・細胞・個体循環システムでベルで統合的に検討する。
単一細胞の超解像酸素分圧イメージング

2019.04

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2022.03

MEXT,JSPS, Grant-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), Principal investigator
Development of the new technology of fertility preservation for the AYA cancer patients

2017.04

-

2020.03

Grants-in-Aid for Scientific Research, Nao Suzuki, Grant-in-Aid for Scientific Research (B), No Setting

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In recent years, the number of childhood and AYA (Adolescent and Young Adult is the age from 15 to 39 years old) generation cancer survivors who have overcome cancer has been increasing with the progress of multidisciplinary treatment in cancer treatment. On the other hand, cancer treatment for childhood and AYA (CAYA) cancer patients may cause gonadal dysfunction or loss of fertility depending on the treatment method. In Japan, fertility-preserving therapy before the start of cancer treatment for some female CAYA cancer patients has recently been introduced. Ovarian tissue cryopreservation, the only fertility-preserving therapy for children and adolescents with cancer, requires long-term storage for more than 10 years. As a result of this research, we succeeded in developing a new storage system that ensures the safety against human error in the long-term storage system and obtained the results showing the safety of antifreeze for long-term storage.
皮膚や臓器に貼付できるフレキシブル酸素イメージングフィルム

2016.04

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Present

日本学術振興会, Grants-in-Aid for Scientific Research, Tsukada Kosuke, Grant-in-Aid for Scientific Research (C), Principal investigator

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We have developed a new oxygen sensor device that can be attached to the surface of curved skin and organs. The oxygen sensor consisted of three layers, a sensing layer containing an oxygen sensitive dye, an excitation light source layer, and a light receiving layer. By encapsulating oxygen sensitive dye in polystyrene nanoparticles, we succeeded in improving the measurement accuracy. We adopted electrochemiluminescence as the excitation light source and determined its electrical condition. The calibration experiment showed the possibility of measuring oxygen concentration with high sensitivity. In the future, by arranging the elements at high density, application to two-dimensional imaging of oxygen concentration is expected.
近赤外光による新規ワクチンアジュバントのインフルエンザ集団予防接種への適用を目的とするポータブルレーザー医療機器の創出

2016.04

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Present

AMED, 橋渡し研究加速ネットワークプログラム, Principal investigator

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Awards 【 Display / hide 】

Best Poster Award

2015.09, Japanese Society for Microcirculation, A cell culture microdevice with a continuous oxygen gradient for microvascular research in vitro
Best Poster Award

2015.09, Japanese Society for Microcirculation, A cell culture microdevice with a continuous oxygen gradient for microvascular research in vitro
最優秀研究奨励賞

佐藤安沙子，内田英幸，宮山明，塚田孝祐, 2013.02, 日本微小循環学会, Development of a cell culture microdevice with oxygen gradient as a model for microvascular environment

Type of Award： Award from Japanese society, conference, symposium, etc.
最優秀研究奨励賞

佐藤安沙子, 内田英幸, 宮山明, 塚田孝祐, 2013.02, 日本微小循環学会, Development of a cell culture microdevice with oxygen gradient as a model for microvascular environment

Type of Award： Award from Japanese society, conference, symposium, etc.
第二回先端フォトニクスシンポジウムポスター賞

Tsukada Kosuke, 2011.10, 日本学術会議総合工学委員会ICO分科会, 生体分光計測が解き明かすがんの低酸素・代謝メカニズム

Type of Award： Award from Japanese society, conference, symposium, etc.