Carl Hansen

Professor

Relevant Degree Programs

 

Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - Mar 2019)
Development and application of microscale technology for single-cell sequencing (2018)

No abstract available.

Small volume cell culture technology for the analysis of clonal heterogeneity in mammalian cell populations (2017)

The ability to culture individual cells provides a unique method to assess the heterogeneity of mammalian cell populations. However, there are many challenges when scaling down culture systems due to the complexity of re-creating a stimulating environment at the clonal level. Small volume culture systems such as integrated microfluidic platforms offer the potential to radically alter the throughput of clonal screening through the use of time-lapse imaging, dynamic stimulus control and economy of scale. In particular, the use of automated fluidic control allows for the characterization of single cells in a dynamic microenvironment similar to large-scale culture. This thesis describes how small volume cell culture practices such as the use of conditioned medium and microfluidic technology can be implemented to isolate large numbers of cells in small volumes and evaluate clonal populations under precise medium conditions. For a Chinese Hamster Ovary (CHO) cell system normal growth kinetics and specific productivity were sustained in small volumes. When exposed to conditioned medium from a parental CHO line, clones cultured at sub-mL scales matched the performance of large-scale cultures. A microfluidic bead assay was developed to detect Immunoglobulin G titers secreted from clones in nL volumes. The combination of microfluidic conditioned medium perfusion with the magnetic bead assay allowed for clonal productivity to be evaluated under simulated fed-batch conditions. Lastly, microfluidic cell culture was demonstrated on a human embryonic stem cell (hESC) system through the robust generation of colonies derived from single cells. hESCs propagated in the microfluidic system were observed to match the growth kinetics, marker expression and colony morphologies of larger cultures, while resolving response heterogeneity during differentiation induction. This thesis demonstrates how high-throughput, small volume culture systems can be used to screen clonal populations for therapeutic applications under complex culture conditions.

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Development and application of microfluidic single-cell polymerase chain reaction (2016)

Methods for single-cell analysis are critical to revealing cell-to-cell variability in biological systems, such as during development or onset of disease, where the characteristics of heterogeneity and minority cell populations are obscured by population-averaged measurements. Analysis of individual cells has been limited due to challenges associated with small amounts of starting material, combined with the cost and throughput required to examine large numbers of cells. Microfluidic approaches are well suited to single-cell analysis, providing increased sensitivity, economy of scale, and automation. This thesis presents the development and application of microfluidic technology for single cell gene expression analysis. The foundational contribution of this work is an integrated microfluidic device capable of performing high-precision RT-qPCR measurements of gene expression from hundreds of single cells per run. This device executes all steps of single cell processing including cell capture, cell lysis, reverse transcription, and quantitative PCR. This device is further expanded upon by integrating the single cell and nucleic acid processing capabilities with final measurement of cDNA by high-density digital PCR. The direct quantification of single molecules by digital PCR has advantages over RT-qPCR in the measurement of low abundance transcripts, as well as obviating the need for relative abundance measurements or calibration standards. This technology is demonstrated in over 5,000 individual cell measurements of mRNA, microRNA, and single nucleotide variant detection in a variety of cell types. Finally, this technology is applied to study the performance of lipid nanoparticles in delivery of RNA, and manipulation of gene expression in cells. The microfluidic integration of cell and nucleic acid processing established in this thesis permits analysis of hundreds of single cells in parallel, while improving work flow and reducing technical variation compared to samples prepared in microliter volumes. Ultimately, this advances the tools available for precisely measuring transcripts in single cells, and has application in research and clinical settings.

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A programmable droplet-based microfluidic device for multiparameter single-cell analysis (2013)

The ability of microfluidic systems to perform biological analysis with greater sensitivity, lowercost, and higher throughput relative to conventional methods has now been widely demonstrated.Despite this transformative potential, application innovation and user adoption in biologicalresearch have lagged due to limited access to specialized fabrication facilities and expertise. Inanalogy to how the development of programmable integrated circuits has resulted in the ubiquityand utility of this technology among a broad community of developers and non-expert users, theadvancement of programmable microfluidic devices stands to dramatically enhance thepervasiveness and impact of microfluidic systems.This thesis describes the development and application of a microfluidic device that combines thereconfigurable flow-routing capabilities of integrated microvalve technology with the samplecompartmentalization inherent to mass transport in droplets to achieve programmable fluidhandlingfunctionality. The device allows for the execution of user-defined multistep reactionprotocols in an array of individually addressable nanolitre-volume storage chambers byconsecutively merging programmable sequences of picolitre-volume droplets containingreagents or phenotypically sorted single cells. This functionality is enabled by “flow-controlledwetting,” a novel droplet docking and merging mechanism that exploits the physics of dropletflow through a channel to control the precise location of droplet wetting. The device also allowsfor automated cross-contamination-free recovery of reaction products from individual chambersfor downstream analysis. The combined features of programmability, addressability, andselective recovery provide a general hardware platform that can be reprogrammed for multipleapplications.This versatility is demonstrated by implementing multiple analyses on phenotypically sortedsingle cells including monoclonal culture, genomic PCR, whole genome amplification and wholetranscriptome amplification. These capabilities have been applied to a diverse range ofbiological samples for applications ranging from the identification of microbial communitymembers in environmental samples to the determination of mutation frequencies in humancancer at the single-cell level.

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Microfluidic technologies for rapid, high-throughput screening and selection of monoclonal antibodies from single cells (2013)

Thisthesisdescribesthedevelopmentofnovelmicrofluidictechnologiesforrapid,high-­‐throughputscreeningandselectionofmonoclonalantibodies(mAbs)fromsinglecells.Microfluidicdeviceswereusedtocompartmentalizesingleantibody-­‐secretingcells(ASCs)insmall-­‐volumechambers(i.e.hundredsofpicoliterstonanoliters)inordertoconcentratesecretedmAbsformeasurementofantigenbindingkineticsandaffinitiesusinganovelmicrofluidicfluorescencebeadassay.Microfluidicsingle-­‐cellantibodyscreeningwasperformedonASCsharvestedfromantigen-­‐immunizedmiceandpurifiedbyfluorescence-­‐activatedcellsorting(FACS).FollowingmicrofluidicselectionofASCsproducingantigen-­‐specificmAbs,ASCsweresequentiallyrecoveredfromthemicrofluidicdeviceandsubjectedtosingle-­‐cellRT-­‐PCRtoamplifytheantibody-­‐encodingheavyandlightchaingenes.Antibodygenesforselectedhigh-­‐affinitymAbsaresequencedandclonedintoexpressionvectorsforrecombinantproductioninmammaliancelllines.Nearly200high-­‐affinitymousemAbstothemodelantigenhenegglysozyme(HEL)wereselectedasavalidationofthistechnology,representingaten-­‐foldincreaseinthenumberofhighaffinityanti-­‐HELmAbspreviouslyselectedusingsingle-­‐cellmicro-­‐technologiesandthetraditionalhybridomaapproach.Microfluidicsingle-­‐cellmAbscreeningalsoyieldedimportantinsightsintoaffinitymaturation,immuno-­‐dominance,andantibodystereotypyintheadaptiveimmunesystem.Bycircumventingtime-­‐consuminglimitingdilutionandclonalexpansioninthehybridomaapproach,microfluidicsingle-­‐cellscreeningwillenableselectionofmAbsfromotheranimalspecies(e.g.rabbits,humans)forboththerapeuticandresearchapplications.

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Microfluidic technologies for the integration of liquid chromatography (2013)

The microfabrication technique of Multilayer Soft Lithography (MSL) has emerged as the dominant platform for the development of microfluidic devices in biomedical research. However, Multilayer Soft Lithography, as originally developed, has technological limitations that restrict its effectiveness and versatility in implementing laboratory operations on chip. This thesis solves two of these limitations, namely that channel routing is restricted to planar geometries and that no established methods exist for creating solid-phase columns needed to perform on-chip analytical and preparative separations.The first technological advancement is the development of a new fabrication method using laser ablation that enables the automated fabrication of interlayer connections in MSL-based microfluidic devices. Real-time image recognition and computer control allow for robust wafer-scale registration of laser ablation features with moulded channel structures. This new functionality removes the constraint that all connected device features lie in a single plane, significantly enhancing achievable feature density and fluid handling complexity. To further extend the range of accessible bioanalytical applications using MSL-based devices, this thesis also presents the development of a novel microfluidic column geometry that allows rapid packing of multiple microcolumns in parallel with near-perfect yield. These microcolumns are shown to be of high quality, with plate heights comparable to conventional high-performance capillary columns and superior to what has previously been reported for packed microfluidic columns.Finally, this thesis shows how these new capabilities allow for the implementation of the first fully integrated microfluidic liquid chromatography system that exploits advantages of automation, small sample volume and parallel processing. All elements required for automated sample loading, programmable gradient generation, separation, fluorescent detection, and sample recovery are integrated on a single device. The ability to reliably fabricate three-dimensional microfluidic devices and to integrate highly optimized solid-phase columns will open many new opportunities for on-chip integration, bringing the ultimate goal of complete lab-on-a-chip integration closer to reality.

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Master's Student Supervision (2010-2017)
High-throughput pairing of antigen receptor chains (2015)

The specificity of antigen recognition by a T cell or B cell is determined by its unique T cell receptor (TCR) or B cell receptor (BCR), each consisting of two, paired polypeptide chains (alpha and beta, or heavy and light, respectively). An immense diversity of receptors is created during T cell and B cell development through a process of gene recombination. Previously, this diversity has been studied by extracting RNA from large numbers of cells, amplifying the alpha and beta chain (or heavy and light chain) transcripts, and then deep sequencing. However, through this process, information on correct chain pairing is lost. In this thesis, I present a high-throughput approach for maintaining paired-chain information in next-generation sequencing libraries. Briefly, a bulk cell population is divided into a number of sub-populations, and TCR or BCR transcripts are independently amplified; chains are considered paired if they co-occur in more sub-populations than expected by random chance. Fundamental to this approach is a reliable, sensitive library preparation chemistry in which a sub-population specific index can be incorporated. Such a chemistry was validated on primary human CD8⁺ T cells. This approach for antigen receptor chain pairing will enable in-depth studies of immune dynamics, tracking of disease progression, and personalized immunotherapeutics.

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A microfluidic approach for antibody screening from single cells with numerical methods for modelling solid-substrate capture (2013)

Antibodies are the fastest growing class of therapeutics and are also ubiquitousreagents in diagnostic and research applications. Despite the advanceof many applications of antibodies in basic and applied research, the majorityof novel antibody discovery and screening still typically relies on thecostly, time-consuming and ine cient hybridoma approach; the developmentof improved approaches for the high-throughput screening and selection ofmonoclonal antibodies is thus of high interest.In this thesis a novel approach combining the concentration enhancementand sample manipulation bene ts of polydimethylsiloxane (PDMS)multilayer microuidics devices is validated for the high-throughput prescreeningof large samples of primary antibody secreting cells. Dual functionalizedbeads are co-incubated with antibody secreting cells to captureboth antibodies and their associated messenger RNA (mRNA) transcriptsto couple sequence with a nity information in downstream uorescence activatedcell sorting (FACS). This is done in a parallel way for simultaneouscell processing over a large planar array. The device is validated with quantitativereverse transcription polymerase chain reaction (RT-qPCR) to verifysuccessful complementary DNA (cDNA) synthesis on the bead surface andthat the beads retain functional antibodies throughout the incubation andreverse transcription process.As part of this work, I also present a numerical modelling pipeline todesign and characterize performance of di usion based solid substrate cellsecretion assays in microuidic chambers prior to time-consuming fabricationand experimentation.

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Study of non-genetic variability and heritability of pheromone signaling in Saccharomyces cerevisiae on a microfluidic device (2012)

Clonal populations of cells exhibit variability in gene expression despite genetic identity. Single cell technologies have helped identify various sources of such variability. Intrinsic noise in biochemical reactions as well as variability introduced by cell cycle progression and division have been suggested to play a significant role. However, there is a paucity of experimental platforms that can simultaneously measure gene expression and track cell cycle and division through multiple generations in a fully automated fashion. In this thesis I describe a microfluidic-based approach for performing such studies which integrate high- resolution live cell microscopy and automated image analysis to track lineages of multiple yeast strains for up to 8 generations in temporally and chemically controlled environments. This technology is applied to the quantitative study of non-genetic inheritance of the pheromone mitogen activated protein kinase signaling response. These studies demonstrate that the capacity to respond to pheromone is non-genetically passed on to progeny and that this response correlation is maintained between cells that are multiple generations apart. Deletions in the pheromone pathway were found to affect the strength of these correlations. While Δfus3 cells were the most correlated of all screened strains, Δste50 elicited dramatic asymmetry in response between mothers and their daughters leading to highly heterogeneous phenotype. Comparing expression with cell cycle phase and cell age, we present a previously unrecognized role of FUS3 in cell cycle regulation and reveal the pathway’s sensitivity to asymmetric division in the absence of STE50. Our results contribute to the understanding of the origins of heterogeneity in a monoclonal population and elucidate the role of division processes and the cell cycle in giving rise to this cell-to-cell variability.

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Microfluidic technology for high-throughput single cell gene expression analysis (2010)

Transcription measurements with single cell resolution are critical to understanding variable responses in immunity, measuring stochastic noise in gene expression, and assessing the disease and developmental state of heterogeneous populations. The latter is particularly important in stem cell science, developmental biology, and cancer, where minority cells may be most significant. To see these populations requires the quick and cost-effective measurement of hundreds to thousands of individual cells. Quantitative real-time polymerase chain reaction (RT-qPCR) is a sensitive method for quantitative analysis of transcript levels that provides excellent sensitivity and dynamic range in the detection of transcripts. However, the use of RT-qPCR is generally limited to ensemble measurements of bulk cells or plasma, and is blind to minority cell populations. This aggregation obscures the underlying biological response and variability. To address this limitation, we exploit recent advances in scalable microfluidics to develop robust lab-on-chip technology capable of highly parallel and cost-effective measurements of transcript levels from single cells. The microfluidic device integrates single-cell capture, lysis, reverse transcription of contained RNA, and precise measurement of cDNA using RT-qPCR. We demonstrate this system in the study of microRNA expression in a cell line representing chronic myelogenous leaukemia, pluripotency markers in differentiating human embryonic stem cells, and the detection of somatic mutations in a primary breast cancer sample. The ability to screen isolated cells by simultaneously measuring the fraction of cells expressing a specific gene and quantifying the abundance of expression, may provide a new modality for the early detection of disease such as cancer.

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