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Single Cell Investigations of the Functional Heterogeneity Within Immune Cell Populations: a Microchip-based Study
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics. KTH, Centres, Science for Life Laboratory, SciLifeLab. (Björn Önfelt)
2014 (English)Doctoral 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 measure 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. Here, the design aimed for screening applications is the main focus. In this approach a large amount, tens of thousands, of small wells are imaged two to three times: first directly post-seeding of effector and target cells to register the well’s content, and second after some time has passed to allow for cell-cell interactions. The final read-out is the number of killed target cells in each well, making an automatic cell counting protocol necessary in order to analyze the massive amount of data generated.

We here show that our silicon microwell platform allows long-term studies with the possibility of both time-lapse and high-resolution imaging of a variety of immune cell behavior. Using both time-lapse imaging and the screening approach we confirmed and investigated immune cell heterogeneity within NK cell populations in regards to both cytotoxicity and migrational behavior. In addition, two different types of cytolytic behavior in NK cells, termed fast and slow killing, were described and evaluated in regards to dynamic parameters; like conjugation and attachment time. We could also quantify the type of cytolytic response in relation to serial killing NK cells, and saw that serial killing NK cells more often induced fast target cell death. Further investigations using the screening approach have shown that serial killing NK cells also differ from other NK cells in their morphology, being both larger and with a more elongated shape. So far the platform has been used to gain better understanding of some aspects of NK cell biology, but there is still much left to explore. With the addition of an automatic counting program, the large numbers of wells that can be simultaneously imaged will provide new statistical information and enable higher throughput.

Altogether, our family of techniques 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 such as adoptive cell therapy and stem cell transplantation.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. , viii, 61 p.
Series
TRITA-FYS, ISSN 0280-316X ; 2014:08
National Category
Immunology in the medical area Immunology
Research subject
Biological Physics
Identifiers
URN: urn:nbn:se:kth:diva-142472ISBN: 978-91-7595-028-0 (print)OAI: oai:DiVA.org:kth-142472DiVA: diva2:703081
Public defence
2014-03-25, Air and Fire, Science for Life Laboratories, Tomtebodavägen 23A, 17165, Solna, 09:30 (English)
Opponent
Supervisors
Funder
Swedish Research CouncilSwedish Cancer SocietySwedish Foundation for Strategic Research
Note

QC 20140306

Available from: 2014-03-06 Created: 2014-03-05 Last updated: 2014-03-06Bibliographically 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, e15453- p.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.

Keyword
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, 683-693 p.Article 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.

Keyword
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: 2014-03-06Bibliographically approved
3. Novel microchip-based tools facilitating live cell imaging and assessment of functional heterogeneity within NK cell populations
Open this publication in new window or tab >>Novel microchip-based tools facilitating live cell imaging and assessment of functional heterogeneity within NK cell populations
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2012 (English)In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 3, no OCT, 300- p.Article in journal (Refereed) Published
Abstract [en]

Each individual has a heterogeneous pool of NK cells consisting of cells that may be specialized towards specific functional responses such as secretion of cytokines or killing of tumor cells. Many conventional methods are not fit to characterize heterogeneous populations as they measure the average response of all cells. Thus, there is a need for experimental platforms that provide single cell resolution. In addition, there are transient and stochastic variations in functional responses at the single cell level, calling for methods that allow studies of many events over extended periods of time. This paper presents a versatile microchip platform enabling long-term microscopic studies of individual NK cells interacting with target cells. Each microchip contains an array of microwells, optimized for medium or high-resolution time-lapse imaging of single or multiple NK and target cells, or for screening of thousands of isolated NK-target cell interactions. Individual NK cells confined with target cells in small microwells is a suitable setup for high-content screening and rapid assessment of heterogeneity within populations, while microwells of larger dimensions are appropriate for studies of NK cell migration and sequential interactions with multiple target cells. By combining the chip technology with ultrasonic manipulation, NK and target cells can be forced to interact and positioned with high spatial accuracy within individual microwells.This setup effectively and synchronously creates NK-target conjugates at hundreds of parallel positions in the microchip. Thus, this facilitates assessment of temporal aspects of NK-target cell interactions, e.g., conjugation, immune synapse formation, and cytotoxic events.The microchip platform presented here can be used to effectively address questions related to fundamental functions of NK cells that can lead to better understanding of how the behavior of individual cells add up to give a functional response at the population level.

Keyword
Cell migration, Cytotoxicity, Live cell imaging, Microchip, NK cell, Single cell, Ultrasound
National Category
Other Physics Topics
Identifiers
urn:nbn:se:kth:diva-119183 (URN)10.3389/fimmu.2012.00300 (DOI)2-s2.0-84874217873 (Scopus ID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20130311

Available from: 2013-03-11 Created: 2013-03-08 Last updated: 2017-12-06Bibliographically approved
4. Classification of human natural killer cells based on migration behavior and cytotoxic response
Open this publication in new window or tab >>Classification of human natural killer cells based on migration behavior and cytotoxic response
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2013 (English)In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 121, no 8, 1326-1334 p.Article in journal (Refereed) Published
Abstract [en]

Despite intense scrutiny of the molecular interactions between natural killer (NK) and target cells, few studies have been devoted to dissection of the basic functional heterogeneity in individual NK cell behavior. Using a microchip-based, time-lapse imaging approach allowing the entire contact history of each NK cell to be recorded, in the present study, we were able to quantify how the cytotoxic response varied between individual NK cells. Strikingly, approximately half of the NK cells did not kill any target cells at all, whereas a minority of NK cells was responsible for a majority of the target cell deaths. These dynamic cytotoxicity data allowed categorization of NK cells into 5 distinct classes. A small but particularly active subclass of NK cells killed several target cells in a consecutive fashion. These "serial killers" delivered their lytic hits faster and induced faster target cell death than other NK cells. Fast, necrotic target cell death was correlated with the amount of perforin released by the NK cells. Our data are consistent with a model in which a small fraction of NK cells drives tumor elimination and inflammation.

Keyword
Apoptosis, Cell Communication, Cell Degranulation, Cell Movement, HEK293 Cells, Humans, Immunophenotyping, Killer Cells, Natural, Lymphocyte Activation, Microchip Analytical Procedures, Models, Biological, Necrosis, T-Lymphocytes, Cytotoxic
National Category
Hematology
Identifiers
urn:nbn:se:kth:diva-125773 (URN)10.1182/blood-2012-06-439851 (DOI)000321750000017 ()2-s2.0-84874447340 (Scopus ID)
Funder
Swedish Foundation for Strategic Research Swedish Research CouncilScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20130814

Available from: 2013-08-14 Created: 2013-08-13 Last updated: 2017-12-06Bibliographically approved
5. Microchip screening platform for assessment of cytotoxic effector cells
Open this publication in new window or tab >>Microchip screening platform for assessment of cytotoxic effector cells
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Here we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) or T cells within larger populations. Human primary NK cells or human Epstein-Barr virus (EBV)- specific T cells were distributed across a silicon-glass microchip containing 32 400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis the numbers of effector and live or dead target cells in each well could be assessed at different time-points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75%) were non-cytotoxic to the leukemia cell line K562, some NK cells were able to kill several (≥3) target cells within the 12 hours long experiment. We demonstrate that this assay can be used to enumerate and characterize cytotoxic cells, something that could find clinical applications, e.g. in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy.

National Category
Immunology in the medical area
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-142469 (URN)
Funder
Swedish Foundation for Strategic Research Swedish Cancer SocietySwedish Research Council
Note

QS 2014

Available from: 2014-03-05 Created: 2014-03-05 Last updated: 2014-03-06Bibliographically approved

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