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Microfluidic device for generating a stepwise concentration gradient on a microwell slide for cell analysis
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
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2013 (English)In: Biomicrofluidics, ISSN 1932-1058, E-ISSN 1932-1058, Vol. 7, no 6, p. 064115-Article in journal (Refereed) Published
Abstract [en]

Understanding biomolecular gradients and their role in biological processes is essential for fully comprehending the underlying mechanisms of cells in living tissue. Conventional in vitro gradient-generating methods are unpredictable and difficult to characterize, owing to temporal and spatial fluctuations. The field of microfluidics enables complex user-defined gradients to be generated based on a detailed understanding of fluidic behavior at the μm-scale. By using microfluidic gradients created by flow, it is possible to develop rapid and dynamic stepwise concentration gradients. However, cells exposed to stepwise gradients can be perturbed by signals from neighboring cells exposed to another concentration. Hence, there is a need for a device that generates a stepwise gradient at discrete and isolated locations. Here, we present a microfluidic device for generating a stepwise concentration gradient, which utilizes a microwell slide’s pre-defined compartmentalized structure to physically separate different reagent concentrations. The gradient was generated due to flow resistance in the microchannel configuration of the device, which was designed using hydraulic analogy and theoretically verified by computational fluidic dynamics simulations. The device had two reagent channels and two dilutant channels, leading to eight chambers, each containing 4 microwells. A dose-dependency assay was performed using bovine aortic endothelial cells treated with saponin. High reproducibility between experiments was confirmed by evaluating the number of living cells in a live-dead assay. Our device generates a fully mixed fluid profile using a simple microchannel configuration and could be used in various gradient studies, e.g., screening for cytostatics or antibiotics.

Place, publisher, year, edition, pages
2013. Vol. 7, no 6, p. 064115-
Keywords [en]
Chemotaxis, Culture, Systems
National Category
Biomedical Laboratory Science/Technology
Identifiers
URN: urn:nbn:se:kth:diva-140190DOI: 10.1063/1.4846435ISI: 000329292200018Scopus ID: 2-s2.0-84891443639OAI: oai:DiVA.org:kth-140190DiVA, id: diva2:688635
Funder
VinnovaSwedish Foundation for Strategic Research Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20140117

Available from: 2014-01-17 Created: 2014-01-17 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Miniaturised Microwell-based Cell Assays
Open this publication in new window or tab >>Miniaturised Microwell-based Cell Assays
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cell heterogeneity in genetically identical cell populations is becoming a well-known and important phenomenon in cell biology. Current methods commonly utilise population-based analysis founded on averaged result. Hence there is a need for high-throughput cell assays on the single-cell level. By using miniaturised devices it is possible to enhance spatial and temporal control of the individual cells, increase the potential throughput and minimise the needed sample and reagent volume while enabling a wide range of biological applications.

This thesis is based on the results generated with a miniaturised microwell slide for cell assays. The microwell slide’s high-throughput compartmentalised configuration enables several hundred isolated experiments to be run simultaneously. The bottom of the wells is made out of a thin glass slide, which supports high-resolution imaging. The slide has a standardised format and its’ compatibility with conventional instruments is used extensively throughout the thesis. The presented papers all contribute to the development of the microwell slide by adding technical value or increasing the number of potential applications. For example, the slide was success-fully implemented as a chip-to-world output format for single microfluidic droplets in Paper I, by interfacing two miniaturised systems with fluorescence-activated cell sorting. In Paper II and III, microfluidic channels were integrated to increase spatial and temporal control of the added samples and reagents. In Paper II an automated stepwise concentration gradient generator was developed delivering a drug gradient to adherent mammalian cells in designated wells. In Paper III fluidic-imposed shear stress on endothelial cells was studied. In Paper IV, the slide was functionalised by coating the surfaces of the wells with several antibiotics at a defined concentration range. The coated slide was used for multiplex antibiotic susceptibility testing of bacterial pathogens, using an algorithm-based identification of the point defining lag to exponential phase transition. It successfully determined the pathogens susceptibility profile in 3-5 hours. Finally, in Paper V, a method to retrieve bacteria colonies with a desired phenotype from the wells for downstream genetic analysis was developed. In summary, the presented work has furthered the development of miniaturised high-throughput tools for various cell heterogeneity assays.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. p. iv, 70
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:2
Keywords
microwell, miniaturisation, high-resolution imaging, high-throughput, cell culture, single-cell, clone, heterogeneity, antibiotic susceptibility testing, concentration gradient, interfacing, microchannels, cell retrieval
National Category
Biomedical Laboratory Science/Technology
Identifiers
urn:nbn:se:kth:diva-140113 (URN)978-91-7501-982-6 (ISBN)
Public defence
2014-02-07, Air & Fire - Gamma building, Tomtebodavägen 23A, Solna, 10:00 (English)
Opponent
Supervisors
Note

QC 20140117. The abstract published on January 24th, 2014.

Available from: 2014-01-17 Created: 2014-01-17 Last updated: 2014-01-24Bibliographically approved

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