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Novel microchip-based tools facilitating live cell imaging and assessment of functional heterogeneity within NK cell populations
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.ORCID iD: 0000-0002-6019-8157
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.ORCID iD: 0000-0002-3996-9279
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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.

Place, publisher, year, edition, pages
2012. Vol. 3, no OCT, 300- p.
Keyword [en]
Cell migration, Cytotoxicity, Live cell imaging, Microchip, NK cell, Single cell, Ultrasound
National Category
Other Physics Topics
Identifiers
URN: urn:nbn:se:kth:diva-119183DOI: 10.3389/fimmu.2012.00300Scopus ID: 2-s2.0-84874217873OAI: oai:DiVA.org:kth-119183DiVA: diva2:610341
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
In thesis
1. Single Cell Investigations of the Functional Heterogeneity Within Immune Cell Populations: a Microchip-based Study
Open this publication in new window or tab >>Single Cell Investigations of the Functional Heterogeneity Within Immune Cell Populations: a Microchip-based Study
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:nbn:se:kth:diva-142472 (URN)978-91-7595-028-0 (ISBN)
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
2. Quantitative approaches to studying NK cell functional heterogeneity
Open this publication in new window or tab >>Quantitative approaches to studying NK cell functional heterogeneity
2014 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

It is commonly stated that the cell is the smallest functional unit of life. By analogy, then, the immune cell is the smallest functional unit of the immune system. Natural killer (NK) cells are effector cells of the innate immune system that are responsible for mediating cellular cytotoxicity against virally infected or neoplastically transformed cells. Many phenotypically distinct subpopulations of NK cells have been discovered, usually by dividing cells on the basis of cell-surface markers. These subpopulations are typically described as related to activation or developmental status of the cells. However, how these distinct phenotypes correlate with behavior in e.g. NK–target interactions is less widely understood. There is therefore a need to study NK cell behavior down at the single-cell level. The aim of this thesis is to approach methods that quantitatively describe these single-cell-level behavioral differences of NK cells.

Using a newly developed single-cell imaging and screening assay, we trap small populations of NK and target cells inside microwells, where they can be imaged over extended periods of time. We have performed experiments on both resting and IL-2-activated NK cells and quantified their cytotoxic behavior. One major discovery was that a small population of NK cells mediate a majority of the cytotoxicity directed against target cells. A particularly cytotoxic group of cells, which we termed “serial killers”, displayed faster and more effective cytotoxicity.

Also, we identified differences between resting and activated NK cells in regard to their migration and contact dynamics. Activated NK cells were found to more readily adhere to targets cells than did NK cells freshly isolated from peripheral blood. Apart form migration and contact dynamics, we have also quantified killing behavior, where NK cells can be seen to exhibit a behavior we term multiple lytic hits on the basis of analyzing target cell fluorescence profiles.

We have quantified these heterogeneities and developed tools that can be used to further study and elucidate differences in the behavior of single immune cells. These methods, and automated single-cell analysis methods, will likely play a more important role in the study of immune responses in the future.

 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. xiii, 35 p.
Series
TRITA-FYS, ISSN 0280-316X ; 2014:21
National Category
Cell Biology
Identifiers
urn:nbn:se:kth:diva-146281 (URN)978-91-7595-166-9 (ISBN)
Presentation
2014-05-27, sal Air, Gamma, Science for Life Laboratory, Science for Life Laboratory, Solna, 13:00 (English)
Opponent
Supervisors
Note

QC 20140611

Available from: 2014-06-11 Created: 2014-06-11 Last updated: 2014-06-11Bibliographically approved

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