This dissertation evaluated the feasibility of using commercially available immortalized cell lines in building a tissue engineered in vitro blood-brain barrier (BBB) co-culture model for preliminary drug development studies. Mouse endothelial cell line and rat astrocyte cell lines purchased from American Type Culture Collections (ATCC) were the building blocks of the co-culture model. An astrocyte derived acellular extracellular matrix (aECM) was introduced in the co-culture model to provide a novel in vitro biomimetic basement membrane for the endothelial cells to form endothelial tight junctions. Trans-endothelial electrical resistance (TEER) and solute mass transport studies were engaged to quantitatively evaluate the tight junction formation on the in-vitro BBB models. Immuno-fluorescence microscopy and Western Blot analysis were used to qualitatively verify the in vitro expression of occludin, one of the earliest discovered tight junction proteins. Experimental data from a total of 12 experiments conclusively showed that the novel BBB in vitro co-culture model with the astrocyte derived aECM (CO+aECM) was promising in terms of establishing tight junction formation represented by TEER values, transport profiles and tight junction protein expression when compared with traditional co-culture (CO) model setups and endothelial cells cultured alone. Experimental data were also found to be comparable with several existing in vitro BBB models built from various methods. In vitro colorimetric sulforhodamine B (SRB) assay revealed that the co-cultured samples with aECM resulted in less cell loss on the basal sides of the insert membranes than that from traditional co-culture samples. The novel tissue engineering approach using immortalized cell lines with the addition of aECM was proven to be a relevant alternative to the traditional BBB in vitro modeling.

AML is a cancer of the blood and bone marrow characterized by the presence of highly proliferative and abnormally differentiated myeloblasts. Previous work from the Bhatia lab utilized the orthotopic xenograft model in order to isolate a population of leukemic regenerating cells (LRC) that exists prior to relapse. Affymatrix analysis of LRCs revealed up-regulation of 248 genes that can act as unique targets to prevent relapse. In order to screen compounds against all 248 targets, it is important to develop an in vitro model that is able to appropriately recapture the functional and molecular markers of LRCs. Primary AML samples were treated with 5-doses of 0.15 μM, 1 μM AraC, or DMSO control and several outcomes were measured. In vitro AraC treatment was not able to recapitulate the progenitor frequency curve and CD34 expression curve observed in vivo. Additionally, we were not able to see a consistent increase in select LRC targets DRD2, GLUT2, FUT3, and FASL via flow cytometry. Despite an increase in the mRNA levels of LRC genes 24h after treatment with 0.15 μM AraC, long term analysis could not be completed due to poor RNA quality and low expression of LRC-targets. Primary AML cells were co-culture with mouse MS-5 stromal cell line order to study the effects of mesenchymal stromal cells on AML response to AraC. Co-culture with MS-5 cells had different effects on select primary AML cells. AML 14939 showed an increase in CD34 and LRC targets DRD2 and FUT3 following AraC treatment when co-cultured with MS-5 cells; while A374 showed no differences between DMSO and AraC treated groups. Overall, these findings suggest the LRC signature is not induced by treatment with AraC alone. Complex interactions between AML cells and their bone marrow niche during AraC treatment plays an important role in the development of LRCs prior to AML relapse. / Thesis / Master of Science (MSc) / AML is a cancer of blood cells characterized by the presence of rapidly dividing cancer cells termed myeloblasts. AML has a high rate of disease relapse. The Bhatia lab modelled AML relapse in a mouse and discovered an unique population of cells that exist prior to relapse termed LRCs. LRCs express distinctive genes that can act as targets for the development of new therapies to prevent relapse. In order to screen potential relapse preventing compounds, we set out to recapture AML relapse using cells in a dish. AML cells from patients were treated with chemotherapy reagent AraC and the number of cancer progenitors and the expression of specific LRC proteins were measured. AraC did not increase the level of 3 out of 4 LRC proteins studied. We determined the LRCs were not caused by AraC treatment, and the physiology of the bone marrow environment plays an important role in inducing relapse.

Les particules issues de la pollution sont responsables de millions de morts prématurées. Les nanoparticules (au diamètre inférieur à 100 nm) atteignent les alvéoles où elles rencontrent la barrière alvéolo-capillaire. Cette barrière est composée d'un épithélium alvéolaire et d'un endothélium, dos à dos contre une membrane ultrafine (environ 0.2 µM), soumis à une stimulation constante exercée par l'inflation cyclique des alvéoles et par le cisaillement dû à la circulation sanguine. Nous nous sommes appliqués à développer un modèle in vitro innovant de cette barrière alvéolo-capillaire afin d'observer les interactions des nanoparticules avec cette barrière. Dans un premier temps, nous avons développé un substrat micro-fabriqué qui reproduit les propriétés géométriques et physiques de la membrane alvéolo-capillaire. Sur cette membrane, nous avons mis en place une co-culture de cellules épithéliales alvéolaires (A549) et endothéliales (HUVEC). Grâce à une étude de microscopie confocale, nous avons observé le comportement de ce modèle en termes d'étanchéité et de fonctions biologiques. Finalement nous avons observé les interactions entre des nanoparticules de silice et notre modèle en termes de toxicité, d'internalisation et de translocation. Dans une seconde partie, nous avons développé une puce microfluidique à deux chambres qui permet de reproduire autour de notre modèle de co-culture le microenvironnement spécifique des alvéoles pulmonaires. Des études de conception mécanique et l'optimisation de méthodes de microfabrication nous ont permis de générer une puce réversible compatible avec de la culture à long-terme et de l'observation en live par microscopie confocale. Dans une troisième partie, nous avons commencé un travail préliminaire visant à intégrer des cellules pluripotentes induites différenciées dans notre modèle in vitro. Nous avons travaillé à optimiser deux protocoles de différentiation sur une lignée commerciale: vers un endothélium et vers un épithélium alvéolaire. Finalement, nous proposons ici un modèle in vitro offrant de nombreux avantages: une importante communication intercellulaire via leur co-culture sur une membrane ultrafine, une culture long-terme observable au quotidien, la reproduction des stimuli dynamiques de l'environnement alvéolo-capillaire in vivo et la possibilité d'effectuer des tests d'interaction et de translocation de nanoparticules. / Pollutions particles are responsible for millions of premature death. Nanoparticles (with a diameter below 100 nm) reach the alveolar sacs where they encounter the alveolar capillary barrier. This barrier is constituted of an alveolar epithelium and an endothelium back to back on an ultra-thin membrane (about 0.2 µm), submitted to constant stimuli due to cyclic alveolar inflation and blood flow shear stress. We focused here on developing an innovative in vitro model of the alveolar capillary barrier to study the interactions of the nanoparticles with this barrier. Firstly, we have developed a micro-engineered substrate reproducing the geometrical and physical properties of the alveolar capillary membrane. We implemented the co-culture of an alveolar epithelium (A549) and an endothelium (HUVEC) on this membrane. We used confocal microscopy to observe the behavior of our model regarding barrier integrity and specific phenotypes. Finally, we observed the interactions between Silica nanoparticles and our model in terms of toxicity, internalization and translocation. Secondly, we developed a two-chamber microfluidic chip reproducing the specific microenvironment of the alveoli around our co-culture model. Studies of mechanical design and fabrication processes optimization allowed for the generation of a reversible chip compatible with long-term culture and live observation with a confocal microscope. Thirdly, we launched preliminary experiments aiming at the integration of differentiated induced pluripotent stem cells in our in vitro model. We worked on optimizing two directed differentiation protocols: towards an endothelium and towards an alveolar epithelium.Finally, we present here an in vitro model with numerous features: a close-contact co-culture on an ultra-thin membrane enabling important intercellular communication, a long-term culture allowing for live monitoring, mimicking the in vivo dynamic stimuli of the alveolar capillary barrier microenvironment and the possibility for nanoparticles interaction and translocation studies.

京都大学 / 新制・論文博士 / 博士(医科学) / 乙第13611号 / 論医科博第12号 / 新制||医科||11(附属図書館) / 九州大学大学院薬学府創薬科学専攻 / (主査)教授 井上 治久, 教授 松田 秀一, 教授 萩原 正敏 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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