Nanowell-Based Immunoassays for Measuring Single-Cell Secretion: Characterization of Transport and Surface Binding

Arrays of subnanoliter wells (nanowells) provide a useful system to isolate single cells and analyze their secreted proteins. Two general approaches have emerged: one that uses open arrays and local capture of secreted proteins, and a second (called microengraving) that relies on closed arrays to capture secreted proteins on a solid substrate, which is subsequently removed from the array. However, the design and operating parameters for efficient capture from these two approaches to analyze single-cell secretion have not been extensively considered. Using numerical simulations, we analyzed the operational envelope for both open and closed formats, as a function of the spatial distribution of capture ligands, their affinities for the protein, and the rates of single-cell secretion. Based on these analyses, we present a modified approach to capture secreted proteins in-well for highly active secreting cells. This simple method for in-well detection should facilitate rapid identification of cell lines with high specific productivities.National Institutes of Health (U.S.)/National Institute of Allergy and Infectious Diseases (U.S.) (5P01AI045757)National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051

Image analysis technique as a tool to identify morphological changes in Trametes versicolor pellets according to exopolysaccharide or laccase production

Image analysis technique was applied to identify morphological changes of pellets from white-rot fungus Trametes versicolor on agitated submerged cultures during the production of exopolysaccharide (EPS) or ligninolytic enzymes. Batch tests with four different experimental conditions were carried out. Two different culture media were used, namely yeast medium or Trametes defined medium and the addition of lignolytic inducers as xylidine or pulp and paper industrial effluent were evaluated. Laccase activity, EPS production, and final biomass contents were determined for batch assays and the pellets morphology was assessed by image analysis techniques. The obtained data allowed establishing the choice of the metabolic pathways according to the experimental conditions, either for laccase enzymatic production in the Trametes defined medium, or for EPS production in the rich Yeast Medium experiments. Furthermore, the image processing and analysis methodology allowed for a better comprehension of the physiological phenomena with respect to the corresponding pellets morphological stages.The authors acknowledge Portucel-Empresa de Celulose e Papel, Cacia, Portugal, SA for the pulp and paper Kraft effluent used in this work. This work was funded by FEDER Funds through the Programa Operacional Factores de Competitividade-COMPETE, and national funds through FCT-Fundacao para a Ciencia e a Tecnologia under the projects PEst-C/CTM/LA/0011/2013 and PEst-C/EQB/LA0020/2013. A. P. M. Tavares acknowledge the financial support of (Programme Ciencia 2008) FCT, Portugal

From nanoliter to large-scale bioreactors: How integrated technologies bring antibody treatments to patients faster

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A high-throughput platform for stem cell niche co-cultures and downstream gene expression analysis

Stem cells reside in 'niches', where support cells provide critical signalling for tissue renewal. Culture methods mimic niche conditions and support the growth of stem cells in vitro. However, current functional assays preclude statistically meaningful studies of clonal stem cells, stem cell-niche interactions, and genetic analysis of single cells and their organoid progeny. Here, we describe a 'microraft array' (MRA) that facilitates high-throughput clonogenic culture and computational identification of single intestinal stem cells (ISCs) and niche cells. We use MRAs to demonstrate that Paneth cells, a known ISC niche component, enhance organoid formation in a contact-dependent manner. MRAs facilitate retrieval of early enteroids for quantitative PCR to correlate functional properties, such as enteroid morphology, with differences in gene expression. MRAs have broad applicability to assaying stem cell-niche interactions and organoid development, and serve as a high-throughput culture platform to interrogate gene expression at early stages of stem cell fate choices

Microfluidic cell culture arrays for clonal expansion and characterization of mammalian cells

Single-cell culture provides a unique means to reveal the heterogeneity within mammalian cell populations. Advances in multilayer soft lithography have enabled the development of high-throughput nanoliter-volume cell culture platforms with integrated and programmable fluidic control to precisely modulate the microenvironment. Coupled with time-lapse imaging, these microfluidic systems allow hundreds of single cells to be monitored simultaneously while providing analytical advantages to characterize each clone. However, there are many challenges associated with the miniaturization of mammalian cell cultures and even greater difficulties for non-adherent cell types. This work shows how microfluidic devices and their control system can be designed to gently trap suspension cells and enable robust clonal expansion. Mouse hematopoietic stem cell (HSC) populations were chosen for their sensitivity and stringent cell culture requirements to demonstrate that normal cell growth and function could be sustained in the microfluidic system. Using microfluidic clonal analysis and image processing it was observed that cells from HSC-enriched populations had highly heterogeneous growth profiles. Automated medium exchange and temporal stimulation were then exploited to show that a high Steel factor (SF) concentration was needed for survival of primary HSCs specifically at the time of exit from quiescence. The ability to perform live immunostaining was combined with genealogical tracing to identify distinct characteristics, such as long cell cycle times and frequent asynchrony of daughter cells, associated with HSC clones exhibiting persistent endothelial protein C receptor expression (EPCR) after in vitro culture. Finally, the flexibility of this microfluidic system was demonstrated with the culture of Chinese hamster ovary (CHO) cells, the most widely used suspension-adapted mammalian cell type for the production of therapeutic recombinant proteins. In this system, the high cell density and the rapid concentration of cell-secreted products in nanoliter-volume chambers were exploited to measure the amount of secreted monoclonal antibodies from single cells and to increase their cloning efficiency. The ability to recover clones from the microfluidic system has allowed the selection and expansion of high-producing cell lines. This thesis demonstrates the potential and adaptability of high-throughput microfluidic single-cell culture systems for both research and therapeutic applications.Applied Science, Faculty ofChemical and Biological Engineering, Department ofGraduat