Development of microfluidic devices for chemotaxis of primary stem cells

Zuzana Tatárová
EPFL, 2016
Thèse EPFL

Résumé

Functional analysis of primary tissue-specific stem cells is hampered by their rarity. Here I developed greatly miniaturized microfluidic devices for the multiplexed, quantitative analysis of the chemotactic properties of primary, bone marrow-derived mesenchymal stem cells (MSC). The devices were integrated within a fully customized platform that both increased the viability of stem cells ex vivo and simplified manipulation during multidimensional image acquisition. Since primary stem cells can be isolated only in limited number, I optimized the design for efficient cell trapping from low volume and low concentration cell suspensions. Using nanoliter cell culture volumes and automated microfluidic controls for pulsed medium supply, my platform is able to create stable gradients of chemoattractant secreted from mammalian producer cells within the device, as was visualized by a secreted NeonGreen fluorescent reporter. The design was functionally validated by preferential movement of MSC in serum gradients and by using a CXCL/CXCR ligand/receptor combination in a co-culture design. Stable gradient formation prolonged assay duration and resulted in enhanced response rates for slowly migrating stem cells. Time-lapse video microscopy facilitated determining a number of migratory properties based on single cell analysis. Jackknife-resampling revealed that our assay requires only 120 cells to obtain statistically significant results, enabling new approaches in the research on rare primary stem cells. Compartmentalization of the device not only facilitated such quantitative measurements but will also permit future, high-throughput functional screens.

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https://infoscience.epfl.ch/record/219122?ln=fr

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Zuzana Tatárová
EPFL, 2016
Thèse EPFL

Résumé

Official source

https://infoscience.epfl.ch/record/219122?ln=fr

À propos de ce résultat

Concepts associés (10)

Concepts associés

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Publications associées (3)

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Publications associées

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Publications associées (3)

Microfluidic co-culture platform to quantify chemotaxis of primary stem cells

Zuzana Tatárová

Functional analysis of primary tissue-specific stem cells is hampered by their rarity. Here we describe a greatly miniaturized microfluidic device for the multiplexed, quantitative analysis of the chemotactic properties of primary, bone marrow-derived mesenchymal stem cells ( MSC). The device was integrated within a fully customized platform that both increased the viability of stem cells ex vivo and simplified manipulation during multidimensional acquisition. Since primary stem cells can be isolated only in limited number, we optimized the design for efficient cell trapping from low volume and low concentration cell suspensions. Using nanoliter volumes and automated microfluidic controls for pulsed medium supply, our platform is able to create stable gradients of chemoattractant secreted from mammalian producer cells within the device, as was visualized by a secreted NeonGreen fluorescent reporter. The design was functionally validated by a CXCL/CXCR ligand/receptor combination resulting in preferential migration of primary, non-passaged MSC. Stable gradient formation prolonged assay duration and resulted in enhanced response rates for slowly migrating stem cells. Time-lapse video microscopy facilitated determining a number of migratory properties based on single cell analysis. Jackknife-resampling revealed that our assay requires only 120 cells to obtain statistically significant results, enabling new approaches in the research on rare primary stem cells. Compartmentalization of the device not only facilitated such quantitative measurements but will also permit future, high-throughput functional screens.

Royal Soc Chemistry2016

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Bacterial cells behave as individuals despite being genetically identical and subject to the same environment. Although the underlying mechanisms of cellular individuality are not well understood, temporal variation of gene expression and protein levels in individual cells could act as sources for phenotypic heterogeneity of populations. Bacterial persistence in fluctuating environments relies on such diversity, allowing some individuals, by chance, to survive a challenge that would kill an unadapted and phenotypically homogeneous population. Bacterial persistence is a non-genetic, metastable phenomenon that is readily lost upon subculture of surviving cells in the absence of the environmental stress. A medically relevant example of persistence is the fractional killing of bacteria by antibiotics, whereby a subpopulation of cells survives indefinitely despite prolonged exposure to a bactericidal drug. It is hypothesized in the case of tuberculosis (TB) that drug therapy may require administration of multiple drugs for 6-9 months in order to eliminate a subpopulation of "persister" cells. The focus of this thesis is to understand how persisters tolerate antibiotics that kill their genetically identical siblings. To address this question, automated time-lapse fluorescence microcopy was applied in conjunction with microfluidic and microelectromechanical systems (MEMS) to study bacterial responses to antibiotics in real time at the single-cell level. A genetic approach was employed to identify mutants of Mycobacterium smegmatis with altered rates of persistence against the first-line anti-TB drug isoniazid (INH). A microfluidic platform was designed for high-throughput screening of transposon (Tn) mutant libraries. A pilot screen of 1,100 Tn mutants revealed 11 mutants that showed increased (persister-up, PU) or decreased (persister-down, PD) survival when exposed to isoniazid. These mutants were further characterized in comparison to wild-type cells using the single-cell microfluidics platform. This single-cell analysis revealed distinct modes of persistence within each category, which were otherwise masked at the population level. The results indicate that persistence behavior was the cumulative result of the dynamics of cell division and cell lysis. Mutants with decreased persistence exhibited behavior at single-cell level that challenges the accepted mechanism of isoniazid-mediated killing. Therefore, these results yield new insights into antibiotic persistence and isoniazid's mode of action. Persisters cannot be easily studied in non-fractionated populations of cells where they represent a small percentage of the total. As a complementary approach, a method was developed based on dielectrophoretic (DEP) purification of naturally occurred low frequency persister cells from an antibiotic-treated bacterial culture. Purification of persister cells would allow for downstream analysis using conventional bulk/population level transcriptomic and proteomic approaches. The crossover frequencies for live and dead cells were determined to enable selective trapping or release from the device. This approach enabled a proof-of-concept enrichment of live bacteria in the mixture of live and dead cells after INH treatment, based on their dielectric properties. Quantitative confirmation of the enriched/isolated cell populations was attempted using flow cytometry. The high-throughput microfluidic screening and single-cell approaches developed in this thesis could, in principal, be applied to the genetic analysis of a wide variety of dynamic cellular processes that can be imaged by time- lapse fluorescence microscopy, such as gene expression, cell division, chromosome replication and segregation, and protein localization. The biochemical analysis of live cells isolated by dielectrophoresis-based separation would provide useful information about the underlying persistence mechanism. Dielectrophoresis could also be a valuable analytical tool for mutant characterization based on their intrinsic dielectric properties, either at single-cell or population levels. In conclusion, this thesis presents several new microfabricated tools to identify and understand bacterial persistence. The thesis illustrates how integration of microfluidic tools with automated time-lapse microscopy allows hidden characteristics that are present but invisible in the macro- world of batch cultures to be examined in a quantitative and high-throughput manner in the micro-world of single-cell analysis.

EPFL2012

Chargement

Microfluidic culture platforms for C. elegans bacterial interaction and digestion studies at single-organism resolution

Vittorio Viri

Increasing interest in understanding fundamental biological processes, among which aging and nutrition, has led to the study of simple and powerful model organisms, as the round-worm Caenorhabditis elegans (C. elegans). The nematode, selected by Sydney Brenner , shares slightly over one-third of its genetic base code with human beings, making it an optimal model organism for many human related studies. It has been established that bacteria, besides being the principal nutrition source for these nematodes, play an active role in their aging process. In later life stage, i.e. after the larval development, the bacterial grinder of the worms loses efficiency permitting viable bacteria to reach and colonize the gut of the nematodes . Intestinal persistency of bacteria is highly related both to the strain of the hosted organism and of the host; in the first case the pathogenicity of bacteria determines colonization whereas in the second case the speed of the ingestion and digestion mechanism adopted by the host is one of the causes that can determine the occurrence of a condition of illness. Conventional methods commonly employed by biologists for C. elegans observation, widely applied in studies of interaction between worms and bacteria, food intake and processing analysis, result in some cases to be time consuming and difficult to operate. In order to solve these study related issues, such as observations of digestion related pheno-types on individual worms during the entire developmental time and reproducible food stimulation, microfluidics techniques, based on control of microliters of fluid in a laminar flow regime conditions, revealed to be useful for a consistent increase of the overall accuracy. Technical improvement in research and new analytical tools allowed single worm reversible immobilization and imaging and high throughput analysis, but gathering phenotypic information at single-cell or individual organism level, with higher throughput and better precision than conventional methods, still remains challenging. Combining the multiple technical advantages brought by microfluidics to the study of C. elegans, we focus our attention in developing new devices and protocols aiming to investigate how microbiota interact with the host organism. In particular, we intend to develop a novel microfluidic approach for performing long-term live imaging, bacterial processing and standardized food intake assays in an automated way. Worms sorting and immobilization by passive PDMS structures and accurate food concentration dispens-ing will be accomplished. We intend to obtain quantitative measurement of C. elegans food digestion, intake and development during the adult stage first and then in all stages, showing how digestion process, food intake and bacterial hosting is highly correlated with multiple characterizing phenotypes. To obtain food absorption measurement at single worm resolution, we apply an optical method of quantification using green fluorescent protein (GFP) labeled E. coli bacteria together with fluorescent microscopy techniques. Feeding behaviour and physiological digestion response under different exposure conditions will be tested in order to investigate how these factors impact on the development of the organism.

EPFL2021

Chargement

EPFL2012

Microfluidic culture platforms for C. elegans bacterial interaction and digestion studies at single-organism resolution

Vittorio Viri

EPFL2021