The goal of this study was to create pre-formed neural spheroid networks (NSN) on a polydimethyl siloxane (PDMS) concave microwell array for eventual implantation into the rat brain. Recent studies have shown that stem cells have great potential in treating various neurological insults of the central nervous system, ranging from traumatic brain and spinal cord injury, to neurodegenerative disorders. However, the use of stem cell lines in research are controversial due to the method of obtaining cells, in their formation of teratomas and degeneration into cancer cells, their non-specific differentiation, and lastly in their inability to control the location of neural connections. A novel approach to address this issue utilizes pre-formed neural networks consisting of neural spheroids on polymer scaffolds for the implantation into the rat brain. Yet, it was observed that the cylindrical shape of the wells hindered the transfer process. This study aimed to overcome the lack of neural spheroid network detachment by utilizing concave well structures, using a simple method developed in this laboratory. Primary neurons were isolated from pregnant Sprague Dawley rats at 16 ~ 17 days of gestation. Isolated neurons were cultured in PDMS wells with a concave structure and interconnected by rounded micro channels. It was reported previously that a concave structure enabled an easier and more efficient formation of spheroids, not to mention the ease in extraction of spheroid cells. Various studies have demonstrated the effectiveness of guidance channels in promoting neurite growth. Therefore, micro channels were integrated in the micro array design, and served as a guidance conduit to enhance neurite growth, and by association, spheroid interconnection. The primary neurons formed a spheroid structure after 3 days, upon which they began to sprout new neurites. By day 8, neurite connections peaked. Spheroid diameter underwent an initial decrease then stabilized on day 2. Various well diameters (300~700 um) and channel lengths (1.5 x diameter ~ 3 x diameter) were evaluated, with a 300 um well diameter and 450 um center-to-center channel length found to be optimal. The completed network was assessed for interconnection using calcium imaging and showed coordinated calcium signals between the neural spheroids. The network was then successfully transferred to a collagen matrigel and cultured for a week. The methodology showed an improvement in the transfer of networks, with about a 90% extraction rate. The viability of the NSN on the matrigel was assessed using a Live/Dead assay, and cells were found to have greater than 95% viability. The optimal hydrophilicity was determined for neurite extension and transfer of NSNs onto the matrigel. It was found that an incubation time between 4~6 hours was optimal. Future studies will involve the implantation of the NSN into the rat brain. Additionally, the use of neural progenitor and stem cell lines may provide an autologous source of cells which are immunocompatible with the host. In particular, marrow stromal cells are interesting in that they may also address the ethical concerns. A long term goal is to refine the methodology and apply this research to enable studies in the treatment of patients suffering from spinal cord injury and other neurodegenerative disorders.

No description available.

<p>The aims of this study are to first immobilize positively charged gold nanorods to negatively charged cell culture surfaces. Second, to use polyelectrolytes for controlling the distance between gold nanorods and fluorophores. This is used to optimally determine the distance, of which maximum fluorescence enhancement is achieved, between gold nanorods and fluorophores. In order to approach these aims, we use UV/VIS absorption spectroscopy, fluorescence spectroscopy, atomic force microscopy, and ellipsometry. The results show that we could control the immobilization of gold nanorods on plastic microwell plates and create reproducible polyelectrolyte layers, in order to control the distance between the gold nanorods and fluorophores. In addition, the localized surface plasmon resonance wavelength red shifted as the PELs increased. In conclusion, we found that the maximum fluorescence enhancement of the fluorophores (Cy7) is about 2.3 times at a fluorophores-nanoparticles separation of approximately 9-12 nm. This work contributes some research information towards the design of optical biochip platforms based on plasmon-enhanced fluorescence.</p>

The aims of this study are to first immobilize positively charged gold nanorods to negatively charged cell culture surfaces. Second, to use polyelectrolytes for controlling the distance between gold nanorods and fluorophores. This is used to optimally determine the distance, of which maximum fluorescence enhancement is achieved, between gold nanorods and fluorophores. In order to approach these aims, we use UV/VIS absorption spectroscopy, fluorescence spectroscopy, atomic force microscopy, and ellipsometry. The results show that we could control the immobilization of gold nanorods on plastic microwell plates and create reproducible polyelectrolyte layers, in order to control the distance between the gold nanorods and fluorophores. In addition, the localized surface plasmon resonance wavelength red shifted as the PELs increased. In conclusion, we found that the maximum fluorescence enhancement of the fluorophores (Cy7) is about 2.3 times at a fluorophores-nanoparticles separation of approximately 9-12 nm. This work contributes some research information towards the design of optical biochip platforms based on plasmon-enhanced fluorescence.

Powerful tools for detailed cellular studies are emerging, increasing the knowledge ofthe ultimate target of all drugs: the living cell. Today, cells are commonly analyzed inensembles, i.e. thousands of cells per sample, yielding results on the average responseof the cells. However, cellular heterogeneity implies the importance of studying howindividual cells respond, one by one, in order to learn more about drug targeting andcellular behavior. In vitro assays offering low volume sampling and rapid analysis in ahigh-throughput manner are of great interest in a wide range of single-cellapplications. This work presents a microwell device in silicon and glass, developed using standardmicrofabrication techniques. The chip was designed to allow flow-cytometric cellsorting, a controlled way of analyzing and sorting individual cells for dynamic cultureand clone formation, previously shown in larger multiwell plates only. Dependent onthe application, minor modifications to the original device were made resulting in agroup of microwell devices suitable for various applications. Leukemic cancer cellswere analyzed with regard to their clonogenic properties and a method forinvestigation of drug response of critical importance to predict long-term clinicaloutcome, is presented. Stem cells from human and mouse were maintainedpluripotent in a screening assay, also shown useful in studies on neural differentiation.For integrated liquid handling, a fluidic system was integrated onto the chip fordirected and controlled addition of reagents in various cell-based assays. The chip wasproduced in a slide format and used as an imaging tool for low-volume sampling withthe ability to run many samples in parallel, demonstrated in a protein-binding assay fora novel bispecific affinity protein. Moving from cells and proteins into geneticanalysis, a method for screening genes from clones in a rapid manner was shown bygene amplification and mutation analysis in individual wells. In summary, a microwelldevice with associated methods were developed and applied in a range of biologicalinvestigations, particularly interesting from a cell-heterogeneity perspective. / QC 20100728

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.

Today many cell biological techniques study large cell populations where an average estimate of individual cells’ behavior is observed. On the other hand, single cell analysis is required for studying functional heterogeneities between cells within populations. This thesis presents work that combines the use of microfluidic devices, optical microscopy and automated image analysis to design various cell biological assays with single cell resolution including cell proliferation, clonal expansion, cell migration, cell-cell interaction and cell viability tracking. In fact, automated high throughput single cell techniques enable new studies in cell biology which are not possible with conventional techniques. In order to automatically track dynamic behavior of single cells, we developed a microwell based device as well as a droplet microfluidic platform. These high throughput microfluidic assays allow automated time-lapse imaging of encapsulated single cells in micro droplets or confined cells inside microwells. Algorithms for automatic quantification of cells in individual microwells and micro droplets are developed and used for the analysis of cell viability and clonal expansion. The automatic counting protocols include several image analysis steps, e.g. segmentation, feature extraction and classification. The automatic quantification results were evaluated by comparing with manual counting and revealed a high success rate. In combination these automatic cell counting protocols and our microfluidic platforms can provide statistical information to better understand behavior of cells at the individual level under various conditions or treatments in vitro exemplified by the analysis of function and regulation of immune cells. Thus, together these tools can be used for developing new cellular imaging assays with resolution at the single cell level. To automatically characterize transient migration behavior of natural killer (NK) cells compartmentalized in microwells, we developed a method for single cell tracking. Time-lapse imaging showed that the NK cells often exhibited periods of high motility, interrupted with periods of slow migration or complete arrest. These transient migration arrest periods (TMAPs) often overlapped with periods of conjugations between NK cells and target cells. Such conjugation periods sometimes led to cell-mediated killing of target cells. Analysis of cytotoxic response of NK cells revealed that a small sub-class of NK cells called serial killers was able to kill several target cells. In order to determine a starting time point for cell-cell interaction, a novel technique based on ultrasound was developed to aggregate NK and target cells into the center of the microwells. Therefore, these assays can be used to automatically and rapidly assess functional and migration behavior of cells to detect differences between health and disease or the influence of drugs. The work presented in this thesis gives good examples of how microfluidic devices combined with automated imaging and image analysis can be helpful to address cell biological questions where single cell resolution is necessary. / <p>QC 20130927</p>

No description available.