Selective Retrieval of Individual Cells from Microfluidic Arrays Combining Dielectrophoretic Force and Directed Hydrodynamic Flow

Hydrodynamic-based microfluidic platforms enable single-cell arraying and analysis over time. Despite the advantages of established microfluidic systems, long-term analysis and proliferation of cells selected in such devices require off-chip recovery of cells as well as an investigation of on-chip analysis on cell phenotype, requirements still largely unmet. Here, we introduce a device for single-cell isolation, selective retrieval and off-chip recovery. To this end, singularly addressable three-dimensional electrodes are embedded within a microfluidic channel, allowing the selective release of single cells from their trapping site through application of a negative dielectrophoretic (DEP) force. Selective capture and release are carried out in standard culture medium and cells can be subsequently mitigated towards a recovery well using micro-engineered hybrid SU-8/PDMS pneumatic valves. Importantly, transcriptional analysis of recovered cells revealed only marginal alteration of their molecular profile upon DEP application, underscored by minor transcriptional changes induced upon injection into the microfluidic device. Therefore, the established microfluidic system combining targeted DEP manipulation with downstream hydrodynamic coordination of single cells provides a powerful means to handle and manipulate individual cells within one device.

Microtechnologies to Map the Fate of Single Stem Cells and their Progeny

Stefan Kobel

Since stem cells have the unique ability to produce more of themselves (i.e. to "self-renew") and to generate specialized tissue cells, they are an ideal source of cells for regenerative medicine and in vitro tissue models. In order to fully exploit this potential, we have to better understand the mechanisms that regulate stem cell behavior at a single cell level. However, current in vitro methodologies to study single cells are limited by their throughput, do not afford to distinguish the fate of daughter cells or track the progeny of a single cell over long time periods. This thesis addresses these shortcomings by developing miniaturized devices to handle and analyze single live stem cells. To this end, a micro-structured platform was first established that consisted of miniaturized wells with diameters of 100 µm that were arranged in a regular grid. Single hematopoietic stem cells (HSCs) were then seeded into these "microwell arrays" and, since the HSCs could not leave the microwells, they could be tracked over many days, and their dynamics and proliferation rates could be studied. However, in order to analyze the daughter cells of these single HSCs individually, the daughter cells have to be physically separated by micromanipulation. For this purpose, a microdevice was developed to reliably manipulate single cells using hydrodynamic single cell trapping. This platform was thoroughly characterized using computational simulations combined with particle flow velocimetry and live-cell time-lapse microscopy. The optimization of design parameters towards increasing trapping efficiency ensured a nearly perfect efficiency of single cell trapping (97%) and a successful re-capture of daughter cells generated by dividing mother cells. Furthermore, it was demonstrated that cell trapping under very low flow is sufficiently gentle to enable long-term cell trapping in the microfluidic environment. To identify and track captured cells in these microfluidic single cell traps, automated image analysis tools were developed. A reliable and fast algorithm was generated to identify the borders of microchannels that were indicative for the position of single cell traps. These detected edges were used to individualize the single cell traps with a sub-pixel resolution such that it was possible to segment single HSCs on the chip by thresholding. Accordingly, single HSCs were discovered with efficiencies as high as >95%, allowing the automated tracking of microfluidic single cell trapping, as well as the detection of cell cycle phases of trapped single HSCs engineered with dual fluorescence reporter system marking G1 and S/G2-M. To actively micromanipulate single cells for downstream cell-fate analyses, on-chip valves were integrated on the microfluidic platform to precisely control the direction of medium flow in the microfluidic channels. This approach enabled a proof-of-concept study to demonstrate a more complex single cell manipulation: Cells were first captured in single cell traps and transferred into a culture chamber for clonal expansion. Subsequently, the progeny of these single cells was removed from the culture chamber and trapped in a series of single cells traps. Importantly, since these traps could be addressed individually, a reliable physical separation of the daughter cells was achieved. To enable individual analyses of these separated daughter cells, the last aim of this thesis was to interface a microfluidic chip with standard multititer plates in order to efficiently recover the cells or the cDNA of the single cell generated on-chip from extracted mRNA. For this purpose, the bottom of the chip was equipped with an array of access holes that matched the layout of a 1536-well plate. These access holes were linked to a single cell trap via an analysis chamber, where the HSCs were lysed and their mRNA was reverse transcribed using bead-coupled primers. In this vain, the obtained cDNA was coupled to these beads that could then removed from the chip into a 1536-well by centrifugation. These beads could then be analyzed using standard bench-top PCR machines. In conclusion, this thesis presented several microfluidic modules to handle and analyze single stem cells in high-throughput. In a proof-of-concept study, these modules were applied to automatically capture single cells, culture them, and subsequently separate the daughter cells for downstream cell-fate analyses, thereby greatly enhancing the throughput compared to manual micromanipulation. During the development of this system, it became apparent how crucial reliable and gentle cell handling is to perform complex experimental tasks with live cells on microfluidic chips. The further integration of such microfluidic modules into multi-functional lab-on-a-chip devices will open the door to experimental paradigms previously not imaginable and promises novel insights in stem cell biology.

EPFL2012

Kinetic Analysis of Transcriptional and Post-Transcriptional Processes During Circadian Cycles

Laura Symul

Every morning the sun rises, flooding the earth with its light and heat and every night it disappears leaving the Earth in the darkness of the night. Organisms living on this planet have adapted to this solar rhythm and have spread their activities throughout the day. Early in the development of life, an internal molecular clock appeared in primitive organisms and the evolutionary advantage of being able to anticipate, rather than simply passively face, environmental changes led virtually all organisms to have an internal timing system called "circadian clock." In most species, this clock is constituted of a genetic network ticking in every cell of the body. This genetic network organized in interlocked feedback loops generates oscillations with a 24-hour period. A variable number of proteins and RNAs is thus accumulating in a rhythmic fashion. For example, in mouse liver, which was the main biological model I used during my thesis, it is estimated that thousands of genes are expressed periodically. The molecular processes leading to rhythmic gene expression and protein accumulation are highly regulated. Dynamical control is needed to sustain the oscillations and to maintain their timing. During my PhD, I was particularly interested in the regulation of rhythmic gene activation via transcription factor binding in their promoter region. I also focused on gene transcription itself and its dynamics as well as on transcription associated changes in epigenetic marks. Another interest was given to the regulation of transcripts (messenger RNA) after transcription, especially to understand whether their degradation in the cytoplasm was regulated rhythmically. Finally, I contributed to a project aiming at the detection of transcripts whose translation into protein was specifically regulated in a circadian manner. The four projects on which I worked were done in collaborations with experimental biology labs. Our partners performed the experiments and provided the biological material while I was in charge of the data processing and their statistical analysis. For some projects, I also developed different mathematical models for a more accurate and complete understanding of the phenomena studied and to take into account the inherent variability of biological data. Notably in the project on the post-transcriptional regulation of transcripts, the model-based approach we developed allowed estimating the role of this regulation in a way that is less prone to arbitrary choice of criteria or thresholds compared to previous studies addressing this question. To summarize, the results I obtained show that, in human cell lines, the major transcription factor of the clock (the heterodimer CLOCK/BMAL1) binds to DNA at specific binding sites and their number is comparable to the number of binding sites observed in the mouse liver. However the fraction of rhythmic transcript among the genes with CLOCK/BMAL1 binding sites is about 10 times lower than the corresponding fraction observed in mouse liver. The results of the second project showed that, in mouse liver, rhythmic transcription is followed by dynamical changes in epigenetic marks with delays specific to the marks (tri-methylation of lysines 4 and 36 of histone 3 of nucleosomes) and we highlighted the role of post-transcriptional regulation for the rhythmic expression of a subset of mRNAs. In the third project, we further investigated the regulation at the post-transcriptional level and combining measurements of pre-mRNA and mRNA with a mathematical model, we showed that the post-transcriptional regulation plays a limited role in the rhythmic accumulation of transcripts and is involved in the regulation of only 20 % of rhythmic transcripts. Laboratories of Michael Brunner (University of Heidelberg, Germany), Frederic Gachon, Nouria Hernandez (University of Lausanne, Switzerland) and Ueli Schibler (University of Geneva, Switzerland) 3 Finally, helped by the analysis I performed on micro array data from an experiment aiming at comparing the fraction of translated mRNA to the total accumulation of these mRNAs, the team of Frederic Gachon showed that several transcripts implied in the composition of ribosomes are translated specifically during the night, when rodents are active. All of these results and their links with other published studies on circadian rhythms, show that biological tissues have largely incorporated the clock for the regulation of many other functional pathways in order to optimally synchronize their physiological actions with environmental changes due to the alternation of days and nights. In mouse liver, links with the metabolism are particularly strong and disruptions of the clock induce phenotypes reflecting a disturbed metabolism.

EPFL2013

Implementation and Characterization of Dynamic Genetic Networks in Vitro

Henrike Marie Niederholtmeyer

Transcription and translation (TX-TL) can be performed in vitro, outside of cells, allowing the assembly and analysis of genetic networks. This approach to engineering biological networks in a less complex and more controllable environment could one day allow rapid prototyping of network designs before implementing them in living cells. Furthermore, the in vitro approach provides insight into how natural biological systems are built and is instructive to define the rules for engineering biological systems from bottom up. Despite progress in engineering TX-TL mixes for higher yields and longer synthesis times it remains challenging to implement complex genetic networks, such as oscillators, in vitro. The reason is that the reactions are usually performed in a batch format, where reaction products accumulate and synthesis rates decline over time. We addressed the problems associated with batch reactions by developing a microfluidic chip with nanoliter-scale reactors that exchange reagents at dilution rates matching those of dividing bacteria. In these nano-reactors we can run TX-TL reactions in continuous mode keeping synthesis rates at constant steady state levels for more than 30h. The setup allows close control over the reaction conditions such as dilution rates and DNA template concentration, and to monitor mRNA and reporter protein levels in real time. We can test any genetic program of our choice just by adding the DNA templates coding for the desired functions. We implemented diverse regulatory mechanisms on the transcriptional, translational, and posttranslational levels, including RNA polymerases, transcriptional repression, translational activation, and proteolysis. As a proof of concept for this reactor-based approach to engineering genetic networks we designed and implemented a novel genetic oscillator. Its network architecture consists of a positive feedback loop coupled to delayed negative feedback. Varying dilution rates and DNA template concentrations we mapped its phase diagram showing that steady state conditions were necessary to produce oscillations. The period of oscillations could be tuned by dilution rate. To demonstrate that in vitro synthetic biology is useful for prototyping of dynamic genetic networks, we compared the behavior of biomolecular ring oscillators in a cell-free framework and Escherichia coli. We implemented and characterized the ârepressilatorâ, a three-node negative feedback oscillator in vitro. We then used our cell-free framework to engineer novel three-node, four-node, and five-node negative feedback architectures going from the characterization of circuit components to the rapid analysis of complete networks. We validated our cell-free approach by transferring the three-node and five-node oscillators to E. coli, resulting in robust and synchronized oscillations reflecting our in vitro observations. Our results demonstrate that comprehensive circuit characterization can be rapidly performed in a cell-free system and that these in vitro results have direct applicability in vivo. The reactor-based approach to in vitro synthetic biology, which was developed in this thesis, will thus enable a quantitative and more complete characterization of synthetic and natural genetic networks.

EPFL2015