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Development of Microchip-based Assays to Study Immune Cell Interactions at the Single Cell Level
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
2011 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Immune cell populations are constantly divided into smaller and smaller subsets defined by newly emerging cellular markers. However, there is a growing awareness of the functional heterogeneities in between cells even within small populations, in addition to the heterogeneity over time. One may ask whether a population is correctly defined only by cellular markers or if the functionality should be regarded as well? Many of today’s techniques only measures at the population level, giving an average estimate of the behavior of that pool of cells, but failing to detect rare possibly important events. Thus, high-throughput experimental approaches to analyze single cells over time are required to address cellular heterogeneity.

Progress in the fields of microfabrication, microscopy and computing have paved the way for increasingly efficient tools for studies on the single cell level, and a variety of devices have been described by others. However, few of them are suitable for long-term imaging of dynamic events such as cell-cell interactions or migration. In addition, for efficient recording of many individual events it is desirable to scale down the cells’ interaction volume; not only to shorten the time to interaction, but also to increase the number of individual events in a given area; thereby pushing a screening approach.

To address these questions, a complete microwell array system for imaging of immune cell responses with single-cell resolution was designed. The platform consists of a range of silicon-glass microchips with arrays of miniature wells for incubation of cells and a custom made holder that fits conventional microscopes. The device has been designed to allow cells to be kept viable for several days in the wells, to be easy to use and to allow high-resolution imaging. Five different designs were fabricated; all with a specific type of assay in mind, and were evaluated regarding biocompatibility and functionality. One design is aimed towards screening applications, making an automatic cell counting protocol necessary in order to analyze the massive amount of data generated; this program is also described and evaluated.

We here show that our silicon microwell platform allows long-term studies (up to several days), with the possibility of both time-lapse and high-resolution imaging of a variety of immune cell behavior. Using time-lapse imaging we confirmed immune cell heterogeneity in NK cell populations regarding both cytotoxicity and migrational behavior. The automatic counting program was tested and showed similar results compared to both manual counting and FACS. In addition, the large numbers of wells that can be simultaneously imaged, provide new statistical information that will lead to a better understanding of the function and regulation of the immune system at the single cell level.

Altogether, our technique enables novel types of cellular imaging assays allowing data collection at a level of resolution not previously obtained – this was shown to be important for performing basic cell biological studies, but may also prove valuable in the proposed future medical applications.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology , 2011. , p. iv, 40
Series
Trita-FYS, ISSN 0280-316X ; 2011:04
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-30443ISBN: 978-91-7415-872-4 (print)OAI: oai:DiVA.org:kth-30443DiVA, id: diva2:399969
Presentation
2011-02-23, FA31, KTH, Roslagstullsbacken 21, Stockholm, 10:30 (English)
Opponent
Supervisors
Note
QC 20110225Available from: 2011-02-25 Created: 2011-02-24 Last updated: 2011-02-25Bibliographically approved
List of papers
1. Imaging Immune Surveillance of Individual Natural Killer Cells Confined in Microwell Arrays
Open this publication in new window or tab >>Imaging Immune Surveillance of Individual Natural Killer Cells Confined in Microwell Arrays
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2010 (English)In: PLOS ONE, ISSN 1932-6203, Vol. 5, no 11, p. e15453-Article in journal (Refereed) Published
Abstract [en]

New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level.

Keywords
t-cells, single cells, imunological synapse, microfluidic device, limph-node, on-chip, activation, platform, segregation, cytometry
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-27053 (URN)10.1371/journal.pone.0015453 (DOI)000284147700028 ()2-s2.0-78649726238 (Scopus ID)
Note

QC 20101213

Available from: 2010-12-13 Created: 2010-12-06 Last updated: 2015-01-13Bibliographically approved
2. A silicon-glass microwell platform for high-resolution imaging and high-content screening with single cell resolution
Open this publication in new window or tab >>A silicon-glass microwell platform for high-resolution imaging and high-content screening with single cell resolution
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2011 (English)In: Biomedical microdevices (Print), ISSN 1387-2176, E-ISSN 1572-8781, Vol. 13, no 4, p. 683-693Article in journal (Refereed) Published
Abstract [en]

We present a novel microwell array platform suited for various cell-imaging assays where single cell resolution is important. The platform consists of an exchangeable silicon-glass microchip for cell biological applications and a custom made holder that fits in conventional microscopes. The microchips presented here contain arrays of miniature wells, where the well sizes and layout have been designed for different applications, including single cell imaging, studies of cell-cell interactions or ultrasonic manipulation of cells. The device has been designed to be easy to use, to allow long-term assays (spanning several days) with read-outs based on high-resolution imaging or high-content screening. This study is focused on screening applications and an automatic cell counting protocol is described and evaluated. Finally, we have tested the device and automatic counting by studying the selective survival and clonal expansion of 721.221 B cells transfected to express HLA Cw6-GFP compared to untransfected 721.221 B cells when grown under antibiotic selection for 3 days. The device and automated analysis protocol make up the foundation for development of several novel cellular imaging assays.

Keywords
single cell, microwell, automatic image analysis, screening fluorerscence imaging, clonal expansion
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-30472 (URN)10.1007/s10544-011-9538-2 (DOI)000292556900009 ()21465090 (PubMedID)2-s2.0-80053896769 (Scopus ID)
Note

QC 20110802

Available from: 2011-02-25 Created: 2011-02-25 Last updated: 2017-12-11Bibliographically approved

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