Immunotherapy has emerged as a current and future paradigm of cancer treatment, which utilizes the body’s immune system to eradicate cancer. Natural Killer (NK) cells as part of the innate immune system have immense potential in their anti-tumor cytotoxic activities and host cell surveillance properties. NK cells comprise approximately five to fifteen percent of peripheral blood lymphocytes and can be proliferated in vitro using recently developed methods with co-cultures with feeder cells (derived from engineered tumor cells) or plasma membrane (PM) particles, produced from the fore mentioned feeder cells, in combination with soluble cytokines. For efficient growth and maintenance of these NK cells, Interleukin-2 (IL-2) is utilized. IL-2 in solution, through receptor mediated signaling, stimulates proliferation of T-cells and NK cells. NK cells have lower responsiveness to IL-2 and consequently require a larger systemic dose to stimulate them as opposed to competing cell populations that have higher expression of receptors for IL-2, such as T-cells, which can have the effect of lower effective stimulation of NK cell growth. Such difference in the stimulatory capability of IL-2 toward NK cells and the short circulation lifetime of soluble IL-2 require higher dosages of soluble IL-2 for effective in vivo NK cell proliferation for therapeutic application against cancer, but is toxic. Therefore establishing another form of IL-2 delivery that improves its specific targeting to NK cells would be beneficial and may be crucial for novel therapeutic improvement. The Copik Laboratory has made an IL-2 fusion protein construct having a membrane anchor for expression of membrane-bound IL-2 on K562-41bbl-21 cells (K562-IL21). K562-IL21 cells are selectively recognized by NK cells and stimulate their proliferation and cytotoxicity. Hence, a K562-IL21 membrane–bound IL-2 form should be targeted to NK cells with IL-2 delivery. K562-IL21-2 cells were then used to prepare PM21-2 particles which have the potential to provide NK cell targeted, long-lived form of IL-2 for use as an injectable drug for in vivo adjuvant stimulation of NK cells. The presence of IL-2 on the in the PM21-2 particle product was verified by Western blot, and ELISA. Particle preparations from the modified K562 cells should possess characteristics that allow them to possibly replace soluble IL-2 and more specifically increase the numbers or anti-tumor activity of NK cell populations. The effect of PM21-2 particles was studied in in vitro culture based experiments, which tested the effectiveness the PM21-2 particles to induce selective NK cells expansion as compared to PM21 particles in the presence or absence of soluble IL-2.

Cytokines are small, secreted proteins that are important for cell signalling in theimmune system. Interferon gamma (IFN-γ) is one of the most potent cytokines thatnatural killer (NK) cells of the innate immune system secrete with both antiviral,antibacterial, and antitumoral activity. Analysis of NK cells, such as that of secretionof IFN-γ, is important for studying the immune response to cancer and for developingeffective immunotherapies. In this master thesis project, a method was developedfor determining the amount of IFN-γ secreted by NK cells when being confinedwith cancer cells in deep microwells. Antibody-coated microbeads was used tocapture secreted IFN-γ, which was fluorescently labeled and detected by imaging usingfluorescence microscopy. Microbead seeding into small microwells for single cellassays and into large microwells for embedding of beads into 3D tumor spheroidswas investigated. An analytical model based on experimental standard curves wasdeveloped for straightforward quantification of the amount of bound IFN-γ, with ademonstrated detection down to 2.10−18 moles per bead. The detection of IFN-γ wasevaluated for primary NK cells stimulated by PMA/ionomycin for different incubationtimes. The secretion rate of IFN-γ by IL-2 activated NK cells under PMA/ionomycinstimulation was estimated at 184 molecules per second. IFN-γ detection was alsoevaluated in cell cytotoxicity assays where NK cells were confined over time togetherwith cancer cells in microwells. Both assays showed a successful detection of IFN-γ secretion, demonstrating the potential of the developed method for immune cellanalysis. / Cytokiner är små proteiner som är viktiga för cellsignalering inom immunförsvaret.Interferon gamma (IFN-γ) är en av de mest potenta cytokinerna som naturligamördarceller (NK) i det medfödda immunsystemet utsöndrar med både antiviral,antibakteriell och antitumoral aktivitet. Analys av NK-celler, av till exempelutsöndring av IFN-γ, är viktigt för att studera immunsvaret vid cancer och för attutveckla effektiva immunterapier. I detta examensarbete har en metod utvecklatsför att bestämma mängden IFN-γ som utsöndras av NK-celler när de är tillsammansmed cancerceller i djupa mikrobrunnar. Antikroppsbelagda mikrokulor användesför att fånga utsöndrat IFN-γ, som sedan fluorescensinmärktes och detekteradesgenom fluorescensmikroskopi. Distributionen av dessa kulor studerades i småmikrobrunnar för encellsanalyser och i stora mikrobrunnar för inbäddning av kulornai 3D-tumörsfäroider. En analytisk modell baserad på experimentella standardkurvorutvecklades för enkel kvantifiering av mängden bunden IFN-γ, med en påvisaddetektion ner till 2.10−18 mol per kula. Detektionen av IFN-γ utvärderades för primäraNK-celler stimulerade med PMA/ionomycin för olika inkubationstider. Sekretionenav IFN-γ från IL-2-aktiverade NK-celler vid stimulering med PMA/ionomycinuppskattades till 184 molekyler per sekund. IFN-γ-detektion utvärderades ocksåför analyser av cell-cytotoxicitet där NK-celler var placerade tillsammans medcancerceller i mikrobrunnar över tid. Båda analyserna visade en framgångsrikdetektering av utsöndrad IFN-γ, vilket visar potentialen hos den utvecklade metodenför immuncellsanalys.

Given the rising demand for diverse cell types in regenerative and transfusion medicines, such as human induced pluripotent stem cell-derived neurons (iPSC-Ns), human T/chimeric antigen receptor (CAR) T cells, and human natural killer (NK) cells, the ability to cryopreserve cells has become increasingly important. In regenerative medicine, iPSC-Ns are powerful tools for treating and modelling neurodegenerative diseases. Moreover, transplants/transfusions of T/CAR T cells or NK cells offer promising treatment for numerous types of tumors, such as leukemia and multiple myeloma. Cryopreservation of cells at sub-zero temperatures (-80 to -196 °C) allows for the development of master cell banks that can be used for clinical applications. Conventional cryoprotective agents (CPAs), such as dimethylsulfoxide (DMSO) and glycerol, are utilized to protect cells from cryoinjuries associated with the freezing process. However, the use of high concentrations of DMSO (i.e., 10 to 20%) has been shown to be accompanied with toxic effects on patients receiving cell therapies if it is not removed or diluted prior to transfusion. Moreover, DMSO does not prevent the occurrence of the cryoinjury associated with ice recrystallization, which is one of the major causes of cell death/damage during cryopreservation. As a result, there is a surge of attention toward developing new non-toxic cryo-additives that inhibit ice recrystallization during cryopreservation to permit future advancement in regenerative and transfusion medicines. Moreover, the use of ice recrystallization inhibitors (IRIs) as novel CPAs has become a promising strategy to improve cell viability and function post-thaw. The Ben laboratory heavily invested in synthesizing several classes of carbohydrate-based small molecule IRIs (i.e., O-linked alkyl and aryl glycosides, and N-aryl-D-gluconamides), and studying the correlation between their IRI activity and molecular properties, such as polar surface area to molecular surface area (PSA/MSA) ratio. Moreover, compounds that belong to the O-linked aryl glycosides and N-aryl-D-gluconamides classes of IRIs have been shown to enhance the viability and functionality of red blood cells (RBCs), hematopoietic stem cells (HSCs), and induced pluripotent stem cells (iPSCs) after thawing. Part of the research presented throughout this thesis focuses on structure-activity relationship (SAR) studies of alkyl pyranoses with modified alkyl chain lengths to explore any correlations between the IRI activity and the net polarity (i.e., PSA/MSA ratio) of the IRI candidates. O- and C-linked alkyl pyranose derivatives with different alkyl chain lengths were synthesized and their IRI activity was assessed using the modified splat cooling assay. While the IRI activity of the O- and C-linked alkyl glucosides did differ as the length of the alkyl chain increased, no correlation between the PSA/MSA ratios and their IRI activity was observed. In addition, this work allowed for investigation into the effect of the type of the glycosidic bond (i.e., C-O and C-C bonds) at the anomeric position, on the IRI activity of the different compounds. The O-linked alkyl glucosides appeared to be more IRI active than the C-linked compounds, suggesting the nature of the glycosidic bond is important for IRI activity. The second part of the research presented in this thesis focuses on examining the potential for IRIs to cryopreserve iPSC-Ns, T/CAR T cells, and NK cells. 2-fluorophenyl-D-gluconamides (2FA), which is one of the most active IRIs from the N-aryl-Dgluconamides, has shown promising results in maintaining a high number of viable and functional HSCs and iPSCs post-thaw, and therefore it was employed in the cryopreservation protocol of iPSC-Ns, human-derived T/CAR T cells, and human-derived NK cells. The efficacy of the cryopreservation protocol being constructed was evaluated by assessing the post-thaw viability and recovery rate, as well as the functionality of iPSCNs, T/CAR T cells, and NK cells post-thaw. These studies showed that protecting against ice recrystallization during cryopreservation with IRIs increases the number of viable and functional iPSC-Ns, and T/CAR T cells. It was also observed that employing IRI technology in the cryopreservation protocol of NK cells does not compromise their functionality compared to fresh, non-frozen NK cells. Overall, inhibition of ice recrystallization using IRIs appeared to enhance the cryopreservation outcomes of the different cell types, which will allow for the development of off-the-shelf cell therapy products and improvement of the delivery of efficacious cell products to clinics and hospitals.

Effectively treating infectious diseases often requires a multi-step approach to target different components involved in disease pathogenesis. Similarly, the COVID-19 pandemic has become a global health crisis that requires a comprehensive understanding of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) infection to develop effective therapeutics. One potential strategy to instill greater immune protection against COVID-19 is boosting the innate immune system. This boosting, termed trained immunity, employs immune system modulators to train innate immune cells to produce an enhanced, non-specific immune response upon reactivation following exposure to pathogens, a process that has been studied in the context of and clinical studies prior to the COVID-19 pandemic. Evaluation of the underlying pathways that are essential to inducing protective trained immunity will provide insight into identifying potential therapeutic targets that may alleviate the COVID-19 crisis. Here we review multiple immune training agents, including Bacillus Calmette-Guérin (BCG), β-glucan, and lipopolysaccharide (LPS), and the two most popular cell types involved in trained immunity, monocytes and natural killer (NK) cells, and compare the signaling pathways involved in innate immunity. Additionally, we discuss COVID-19 trained immunity clinical trials, emphasizing the potential of trained immunity to fight SARS-CoV-2 infection. Understanding the mechanisms by which training agents activate innate immune cells to reprogram immune responses may prove beneficial in developing preventive and therapeutic targets against COVID-19.

<p dir="ltr">Hematopoietic stem cells (HSCs) originating from aorta-gonad-mesonephros (AGM) could self-renew and develop into various immune cells, such as T cells, neutrophil and natural killer (NK) cells, rendering them as a promising cell source for immunotherapy. NK cells belong to the family of the innate lymphoid cells, and are employed as one of immunotherapy to cure solid and hematological malignancies including leukemia. Neutrophils are one of the granulocytes, and they are emerging as a new therapeutic target in various cancers. Due to the lack of reliable sources for the amounts of HSCs and immune cells required for clinical infusions (~10⁹ cells/patient), it remains as a major challenge to realize their full potential in targeted cell and immunotherapy. While substantial efforts have been made to generate native cell-like HSPCs and immune cells from human pluripotent stem cells (hPSCs), intricate molecular process governing the differentiation of HSCs and immune cells remain elusive, preventing the development of robust strategies for HSC and immune cell productions.</p><p dir="ltr">In this study, we first demonstrated that critical role of temporally regulating Wnt signaling in initiating AGM-like hematopoiesis across 11 hPSC lines. By inhibiting TGFβ at the stage of aorta-like CD34+SOX17⁺ hemogenic endothelium, which led to the downregulation of Wnt signaling, we established a chemically defined, feeder-free culture system that efficiently produced robust AGM-like hematopoietic cells. Furthermore, we investigated how hypoxia affects the <i>in vitro</i> hPSC differentiation into HSPCs, which resulted in a hypoxia-enhanced HSPC differentiation platform.</p><p dir="ltr">Next, the temporal roles of transcription factors (TFs), including <i>NFIL3</i>, <i>ID2</i>,<i> </i>and <i>SPI1</i>, in regulating and promoting NK cell differentiation from hPSCs are determined. <i>NFIL3</i> and <i>SPI1</i> have been reported to influence the early stages of NK cell development, while <i>ID2</i> has an impact on the generation of NK cells throughout the early and intermediate stage. We genetically modified hPSCs with doxycycline-inducible expression of <i>NFIL3</i>, <i>ID2</i>,<i> </i>and <i>SPI1</i>, and investigated their roles in NK cell induction from hPSCs. Among these three TFs, forced expression of <i>ID2</i> yielded the highest percentage of NK cells under a chemically defined, feeder-free monolayer culture condition, demonstrating that forced expression of NK-specific TFs improves the efficiency of NK cell differentiation from hPSCs.</p><p dir="ltr">Chimeric antigen receptor (CAR) is an artificial cell receptor expressed on immune T or NK cells that has been engineered to allow T or NK cells to re-target cancer cells by exclusively binding to a cancer-specific protein. CAR engineering has significantly improved the anti-tumor efficacy of NK cell therapy, resulting in 6 FDA-approved CAR-T therapies and many other ongoing clinical trials. Recently, a chlorotoxin (CLTX)-based CAR was developed and shown to specifically bind to a variety of heterogenous glioblastoma (GBM) cell lines. To test whether CLTX-CAR could improve the anti-tumor cytotoxicity of hPSC-derived NK cells, hPSCs were engineered with CLTX-CAR for stable and homogenous CAR expression via Cas9-mediated homologous recombination. The expression of CLTX-CAR did not affect the pluripotency and NK cell differentiation potential of hPSCs, and CLTX-CAR significantly improved the cytotoxicity of hPSC-derived NK cells against GBM cells.</p><p dir="ltr">Finally, we implemented a GBM-on-a-chip microfluidic model to interrogate the tumor microenvironment (TME). Microfluidics are an emerging device for investigating cancer biology with spatiotemporal control over signaling modulators by using a small volume. The interaction between hPSC-drived neutrophils and GBM was explored in this microfluidic device. GBM TME is very complex and involves many cell types, including neurons, microglia, immune T and NK cells. In the future, microfluidic models with isogenic cell components will be designed and implemented to better model GBM TME.</p><p dir="ltr">In summary, these discoveries confirm the pivotal role of Wnt signaling in guiding hPSCs towards hematopoietic lineages, while also highlighting <i>ID2</i> as a potent enhancer of NK cell differentiation from hPSC-derived hematopoietic progenitor cells. Additionally, CAR engineering enhances the anti-tumor capabilities of hPSC-derived NK cells. Furthermore, microfluidic models are employed to interrogate GBM TME.</p>

No description available.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23775号 / 医博第4821号 / 新制||医||1057(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 永井 純正, 教授 濵﨑 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Metastatic breast cancer treatment has seen few advances in recent years, yet treatment resistance continues to rise, causing disease recurrence. A pilot study was performed to determine the efficacy of ex vivo expansion and reprogramming of tumor-reactive immune cells from experimental metastatic tumor-sensitized mice. Also, phenotypic changes in tumors due to metastasis or tumor microenvironment influences were characterized. Metastatic neu+ mouse mammary carcinoma (mMMC) and its distant relapsing neu-antigen-negative variant (mANV) were investigated in FVBN202 mice. Tumor-reactive central memory CD8+ T cells and activated NK/NKT cells were successfully reprogrammed and expanded during 6-day expansion from mMMC- and/or mANV-sensitized mice, resulting in tumor-specific cytotoxicity. mMMC exhibited a flexible neu-expression pattern and acquired stem-like, tumorigenic phenotype following metastasis while mANV remained stable except decreased tumorigenicity. Myeloid-derived suppressor cell (MDSC) levels were not increased. Adoptive cellular therapy (ACT) with reprogrammed tumor-reactive immune cells may prove effective prophylaxis against metastatic or recurrent breast cancer.

Streptococcus du Groupe B (GBS) et Streptococcus suis sont deux pathogènes encapsulés qui induisent des pathologies similaires dont la méningite et la septicémie chez les animaux et/ou les humains. Les sérotypes III et V du GBS et les sérotypes 2 et 14 du S. suis (utilisés dans cette étude) sont parmi les plus prévalents et/ou les plus virulents. La capsule polysaccharidique (CPS) définit le sérotype et est considérée comme un facteur de virulence essentiel pour les deux espèces bactériennes. Malgré que plusieurs études aient été réalisées au niveau des interactions entre ces streptocoques et les cellules de l’immunité innée, aucune information n’est disponible sur la régulation de la réponse immunitaire contre ces pathogènes par les cellules dendritiques (DCs) et leur interactions avec d’autres cellules, notamment les cellules ‘natural killer’ (NK). Dans cette étude, différentes approches (in vitro, ex vivo et in vivo) chez la souris ont été développées pour caractériser les interactions entre les DCs, les cellules NK et GBS ou S. suis. L’utilisation de mutants non encapsulés a permis d’évaluer l’importance de la CPS dans ces interactions. Les résultats in vitro avec les DCs infectées par GBS ou S. suis ont démontré que ces deux pathogènes interagissent différemment avec ces cellules. GBS est grandement internalisé par les DCs, et ce, via de multiples mécanismes impliquant notamment les radeaux lipidiques et la clathrine. Le mécanisme d’endocytose utilisé aurait un effet sur la capacité du GBS à survivre intracellulairement. Quant au S. suis, ce dernier est très faiblement internalisé et, si le cas, rapidement éliminé à l’intérieur des DCs. GBS et S. suis activent les DCs via différents récepteurs et favorisent la production de cytokines et chimiokines ainsi que l’augmentation de l’expression de molécules de co-stimulation. Cette activation permet la production d’interferon-gamma (IFN-y) par les cellules NK. Cependant, GBS semble plus efficient à activer les DCs, et par conséquent, les cellules NK que S. suis. La production d’IFN-y, en réponse à la stimulation bactérienne, est principalement assurée par un contact direct entre les DCs et les cellules NK et ne dépend qu’en partie de facteurs solubles. De plus, nos résultats in vivo ont démontré que ces deux streptocoques induisent rapidement la libération d'IFN-y par les cellules NK lors de la phase aiguë de l'infection. Ceci suggère que les interactions entre les DCs et les cellules NK pourraient jouer un rôle dans le développement d’une réponse immune T auxiliaire de type 1 (T ‘helper’ 1 en anglais; Th1). Cependant, la capacité de S. suis à activer la réponse immunitaire in vivo est également plus faible que celle observée pour GBS. En effet, les CPSs de GBS et de S. suis jouent des rôles différents dans cette réponse. La CPS de S. suis empêche une activation optimale des DCs et des cellules NK alors que c’est l’opposé pour la CPS de GBS, indépendamment du sérotype évalué. En résumé, cette étude adresse pour la première fois la contribution des DCs et des cellules NK dans la réponse immunitaire innée lors d’une infection à GBS ou à S. suis et, par extension, dans le développement d’une réponse Th1. Nos résultats renforcent davantage le rôle central des DCs dans le contrôle efficace des infections causées par des bactéries encapsulées. / Group B Streptococcus (GBS) and Streptococcus suis are two encapsulated pathogens that induce similar pathologies, including septicemia and meningitis in animals and/or humans. Serotypes III and V of GBS and serotypes 2 and 14 of S. suis (evaluated in this study) are the most prevalent and/or virulent types. The capsular polysaccharide (CPS) defines the serotype and is considered as a key virulence factor for both bacterial species. Although several studies have addressed the interactions of these streptococci and various cells of the innate immune system, no information is available on the regulation of the immune response against these pathogens by dendritic cells (DCs), and their interactions with other cells, including natural killer (NK) cells. In this study, different approaches (in vitro, ex vivo and in vivo) in mice were developed to characterize the interactions between DCs, NK cells and GBS or S. suis. Non-encapsulated mutants were used to evaluate the importance of the CPS in these interactions. In vitro results with GBS- or S. suis-infected DCs showed that these two pathogens differently interact with these cells. GBS is largely internalized by DCs through multiple endocytosis mechanisms, mainly involving lipid rafts and clathrin. The use of a specific endocytosis pathway might help GBS to survive intracellularly. In contrast, S. suis is poorly internalized and, if the case, rapidly eliminated within the DCs. GBS and S. suis activate DCs through different receptors leading to the release of cytokines and chemokines and increased expression of co-stimulatory molecules. This activation allows the production of IFN- by NK cells. Yet, S. suis capacity to activate DCs and NK cells is lower than that observed for GBS. IFN- release in response to bacterial stimulation was mainly mediated by direct DC-NK cell contact and only partially dependant on soluble factors. In addition, our in vivo results showed that these two streptococcal species rapidly induce the release of IFN- by NK cells during the acute phase of the infection. This suggests that the DC-NK crosstalk might play a role in the development of a T helper 1 (Th1) response. Yet, S. suis capacity to activate the in vivo immune response was also lower than that observed for GBS. In fact, GBS and S. suis CPSs play different roles in this response. S. suis CPS prevents optimal activation of DCs and NK cells whereas it is the opposite for GBS, independently of the serotype tested. In summary, this study addresses for the first time the contribution of DCs and NK cells to the innate immune response against GBS and S. suis infections, and by extension, to the development of a Th1 response. Our results further highlight the central role of DCs in the effective control of infections caused by encapsulated bacteria.

During the last decade, ultrasonic manipulation has matured into an important tool with a wide range of applications, from fundamental cell biological research to clinical and industrial implementations. The contactless nature of ultrasound makes it possible to manipulate living cells in a gentle way, e.g., for positioning, sorting, and aggregation. However, when manipulating cells using ultrasound, especially using high acoustic amplitudes, a great deal of heat can be generated. This constitutes a challenge, since the viability of cells is dependent on a stable physiological temperature around 37°C.      In this Thesis we present applications of ultrasonic manipulation of fluids, particles, and cells in temperature-controlled micrometer-sized devices fabricated using well established etching techniques, directly compatible with high-resolution fluorescence microscopy. Furthermore, we present ultrasonic manipulation in larger up to centimeter-sized devices optimized for fluid mixing and cell lysis. In the present work, two new ultrasonic manipulation platforms have been developed implementing temperature control. These platforms are much improved with increased performance and usability compared to previous platforms. Also, two new ultrasonic platforms utilizing low-frequency ultrasound for solubilization and cell lysis of microliter-volumed and milliliter-volumed samples have been designed and implemented.      We have applied ultrasound to synchronize the interaction between large numbers of immune, natural killer cells, and cancer cells to study the cytotoxic response, on a single cell level. A heterogeneity was found among the natural killer cell population, i.e., some cells displayed high cytotoxic response while others were dormant. Furthermore, we have used temperature-controlled ultrasound to form up to 100, in parallel, solid cancer HepG2 tumors in a glass-silicon multi-well microplate. Next, we investigated the immune cells cytotoxic response against the solid tumors. We found a correlation between the number of immune cells compared to the size of the tumor and the cytotoxic outcome, i.e., if the tumor could be defeated.             Finally, the effect of high acoustic pressure amplitudes in the MPa-range on cell viability has been studied in a newly developed platform optimized for long-term stable temperature control, independent on the applied ultrasound power. Lastly, we present two applications of ultrasonic fluid mixing and lysis of cells. One platform is optimized for small microliter-sized volumes in plastic disposable chips and another is optimized for large milliliter-sized volumes in plastic test tubes. The latter platform has been implemented for clinical sputum sample solubilization and cell lysis for genomic DNA extraction for subsequent pathogen detection / Ultraljudsmanipulering har under de senaste tio åren mognat och utvecklats till ett verktyg med ett brett användningsområde. Idag kan man finna applikationer inom allt från cellbiologisk grundforskning till industri samt sjukvård. Ultraljudsmanipuleringens kontaktlösa natur gör det till en varsam metod för att manipulera celler, till exempel inom positionering, sortering och aggregering. När ultraljud med hög amplitud används kan värmeutvecklingen, som är oundviklig, bli ett problem. För att kunna säkerställa hög cellviabilitet krävs temperaturkontroll som kan hålla en fysiologisk, stabil temperatur på 37°C.      I denna avhandling presenterar vi tillämpningar av temperaturkontrollerad ultraljudsmanipulering i mikrometerstora anordningar fabricerade med väletablerade etsningstekniker.  Dessa anordningar är optimerade för att vara fullt kompatibla med högupplöst fluorescensmikroskopi.  Vi demonstrerar även ultraljudsmanipulering i centimeterstora anordningar optimerade för omrörning och blandning av vätskor samt lysering av celler. Två nya plattformar för ultraljudsmanipulering med inbyggd temperaturkontroll har utvecklats. Dessa två plattformar erbjuder ökad prestanda, flexibilitet samt även användarvänlighet. Utöver dessa plattformar har ytterligare två anordningar för lågfrekvent ultraljudssolubilisering och cellysering av mikroliter- och milliliterstora prover konstruerats.      I denna avhandling har vi tillämpat ultraljud för att synkronisera interaktionen mellan populationer utav immunceller (natural killer-celler) och cancerceller för att på cellnivå studera det cytotoxiska gensvaret. Vi fann en heterogenitet hos immuncellspopulationen. Det manifesterade sig i en fördelning av immuncellerna, från celler med stort cytotoxiskt gensvar till inaktiva immunceller. Vi har dessutom använt temperaturkontrollerad ultrasljudsmanipulering för att skapa solida cancertumörer utav HepG2-cancerceller, upp till 100 stycken parallellt, i en multihåls-mikrotiterplatta bestående av glas och kisel. Med hjälp av dessa tumörer har vi studerat det cytotoxiska gensvaret från immuncellerna. Vi fann att förhållandet mellan antalet immunceller och storleken på tumören bestämde utfallet, det vill säga om tumören kunde bekämpas.      Vi presenterar dessutom effekten utav högamplitudsultraljudsexponering av cancerceller i en plattform speciellt designad för höga tryckamplituder med implementerad ultraljudseffektsoberoende temperaturkontroll. Slutligen presenterar vi två tillämpningar av ultraljud för vätskeblandning och cellysering. Den första tillämpningen är anpassad för små volymer i plastchip för engångsbruk och den andra är optimerad för större volymer i plastprovrör. Den senare tillämpningen är speciellt framtagen för ultraljudssolubilisering och cellysering utav kliniska sputumprover för att möjliggöra DNA-extrahering för detektion av smittämnen. / <p>QC 20150522</p>