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Miniaturised Microwell-based Cell Assays
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
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. , iv, 70 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:2
Keyword [en]
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: urn:nbn:se:kth:diva-140113ISBN: 978-91-7501-982-6 (print)OAI: oai:DiVA.org:kth-140113DiVA: diva2:688603
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
List of papers
1. Interfacing picoliter droplet microfluidics with addressable microliter compartments using fluorescence activated cell sorting
Open this publication in new window or tab >>Interfacing picoliter droplet microfluidics with addressable microliter compartments using fluorescence activated cell sorting
2014 (English)In: Sensors and actuators. B, Chemical, ISSN 0925-4005, E-ISSN 1873-3077, Vol. 194, 249-254 p.Article in journal (Refereed) Published
Abstract [en]

Droplet microfluidic platforms have, while enabling high-throughput manipulations and the assaying of single cell scale compartments, been lacking interfacing to allow macro scale access to the output from droplet microfluidic operations. Here, we present a simple and high-throughput method for individually directing cell containing droplets to an addressable and macro scale accessible microwell slide for downstream analysis. Picoliter aqueous droplets containing low gelling point agarose and eGFP expressing Escherichia coli (E. coli) are created in a microfluidic device, solidified to agarose beads and transferred into an aqueous buffer. A Fluorescence activated cell sorter (FACS) is used to sort agarose beads containing cells into microwells in which the growth and expansion of cell colonies is monitored. We demonstrate fast sorting and high accuracy positioning of sorted 15 μm gelled droplet agarose beads into microwells (14 × 48) on a 25 mm × 75 mm microscope slide format using a FACS with a 100 μm nozzle and an xy-stage. The interfacing method presented here enables the products of high-throughput or single cell scale droplet microfluidics assays to be output to a wide range of microtiter plate formats familiar to biological researchers lowering the barriers for utilization of these microfluidic platforms.

Keyword
Droplet microfluidics, Fluorescence activated cell sorting, Agarose beads, Microwell slide, Escherichia coli
National Category
Biomedical Laboratory Science/Technology
Identifiers
urn:nbn:se:kth:diva-140109 (URN)10.1016/j.snb.2013.12.089 (DOI)000331575400033 ()2-s2.0-84892492768 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceSwedish Research Council
Note

QC 20140117

Available from: 2014-01-17 Created: 2014-01-17 Last updated: 2017-12-06Bibliographically approved
2. Microfluidic device for generating a stepwise concentration gradient on a microwell slide for cell analysis
Open this publication in new window or tab >>Microfluidic device for generating a stepwise concentration gradient on a microwell slide for cell analysis
Show others...
2013 (English)In: Biomicrofluidics, ISSN 1932-1058, E-ISSN 1932-1058, Vol. 7, no 6, 064115- p.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.

Keyword
Chemotaxis, Culture, Systems
National Category
Biomedical Laboratory Science/Technology
Identifiers
urn:nbn:se:kth:diva-140190 (URN)10.1063/1.4846435 (DOI)000329292200018 ()2-s2.0-84891443639 (Scopus ID)
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
3. A microfluidic device towards shear stress analysis on clonal expanded endothelial cells
Open this publication in new window or tab >>A microfluidic device towards shear stress analysis on clonal expanded endothelial cells
2013 (English)Manuscript (preprint) (Other academic)
National Category
Biomedical Laboratory Science/Technology
Identifiers
urn:nbn:se:kth:diva-140110 (URN)
Note

QS 2014

Available from: 2014-01-17 Created: 2014-01-17 Last updated: 2014-01-17Bibliographically approved
4. Bacterial nano-scale cultures for rapid multiplexed antibiotic susceptibility testing
Open this publication in new window or tab >>Bacterial nano-scale cultures for rapid multiplexed antibiotic susceptibility testing
Show others...
2013 (English)Manuscript (preprint) (Other academic)
National Category
Biomedical Laboratory Science/Technology
Identifiers
urn:nbn:se:kth:diva-140111 (URN)
Note

QS 2014

Available from: 2014-01-17 Created: 2014-01-17 Last updated: 2014-01-17Bibliographically approved
5. Dynamic optical screening of single-bacterium and retrieval of the subsequent liquid colony for genetic analysis
Open this publication in new window or tab >>Dynamic optical screening of single-bacterium and retrieval of the subsequent liquid colony for genetic analysis
2014 (English)Manuscript (preprint) (Other academic)
National Category
Biomedical Laboratory Science/Technology
Identifiers
urn:nbn:se:kth:diva-140112 (URN)
Note

QS 2014

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

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Output format
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