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Ultrasound-assisted Interactions of Natural Killer Cells with Cancer Cells and Solid Tumors
KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.ORCID iD: 0000-0002-3139-1724
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this Thesis, we have developed a microtechnology-based method for culturing and visualizing high numbers of individual cells and cell-cell interactions over extended periods of time. The foundation of the device is a silicon-glass multiwell microplate (also referred as microchip) directly compatible with fluorescence microscopy. The initial microchip design involved thousands of square wells of sizes up to 80 µm, for screening large numbers of cell-cell interactions at the single cell level. Biocompatibility and confinement tests proved the feasibility of the idea, and further investigation showed the conservation of immune cellular processes within the wells. Although the system is very reliable for screening, limitations related to synchronization of the interaction events, and the inability to maintain conjugations for long time periods, led to the development of a novel ultrasonic manipulation multiwell microdevice.

The main components of the ultrasonic device is a 100-well silicon-glass microchip and an ultrasonic transducer. The transducer is used for ultrasonic actuation on the chip with a frequency causing half-wave resonances in each of the wells (2.0-2.5 MHz for wells with sizes 300-350 µm). Therefore, cells in suspension are directed by acoustic radiation forces towards a pressure node formed in the center of each well. This method allows simultaneous aggregation of cells in all wells and sustains cells confined within a small area for long time periods (even up to several days).

The biological target of investigation in this Thesis is the natural killer (NK) cells and their functional properties. NK cells belong to the lymphatic group and they are important factors for host defense and immune regulation. They are characterized by the ability to interact with virus infected cells and cancer cells upon contact, and under suitable conditions they can induce target cell death. We have utilized the ultrasonic microdevice to induce NK-target cell interactions at the single cell level. Our results confirm a heterogeneity within IL-2 activated NK cell populations, with some cells being inactive, while others are capable to kill quickly and in a consecutive manner.

Furthermore, we have integrated the ultrasonic microdevice in a temperature regulation system that allows to actuate with high-voltage ultrasound, but still sustain the cell physiological temperature. Using this system we have been able to induce formation of up to 100 solid tumors (HepG2 cells) in parallel without using surface modification or hydrogels. Finally, we used the tumors as targets for investigating NK cells ability to infiltrate and kill solid tumors. 

To summarize, a method is presented for investigating individual NK cell behavior against target cells and solid tumors. Although we have utilized our technique to investigate NK cells, there is no limitation of the target of investigation. In the future, the device could be used for any type of cells where interactions at the single cell level can reveal critical information, but also to form solid tumors of primary cancer cells for toxicology studies.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. , vi, 72 p.
Series
TRITA-FYS, ISSN 0280-316X ; 2014:79
Keyword [en]
Natural killer cell, cytotoxicity, heterogeneity, multiwell microchip, biocompatibility, ultrasonic cell manipulation, 3D cell culture, solid tumor, spheroid, high-resolution imaging
National Category
Engineering and Technology
Research subject
Biological Physics
Identifiers
URN: urn:nbn:se:kth:diva-158522ISBN: 978-91-7595-419-6 (print)OAI: oai:DiVA.org:kth-158522DiVA: diva2:777994
Public defence
2015-01-30, Sal FD5, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

QC 20150113

Available from: 2015-01-13 Created: 2015-01-09 Last updated: 2015-01-13Bibliographically approved
List of papers
1. Ultrasound-controlled cell aggregation in a multi-well chip
Open this publication in new window or tab >>Ultrasound-controlled cell aggregation in a multi-well chip
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2010 (English)In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 10, no 20, 2727-2732 p.Article in journal (Refereed) Published
Abstract [en]

We demonstrate a microplate platform for parallelized manipulation of particles or cells by frequency-modulated ultrasound. The device, consisting of a silicon-glass microchip and a single ultrasonic transducer, enables aggregation, positioning and high-resolution microscopy of cells distributed in an array of 100 microwells centered on the microchip. We characterize the system in terms of temperature control, aggregation and positioning efficiency, and cell viability. We use time-lapse imaging to show that cells continuously exposed to ultrasound are able to divide and remain viable for at least 12 hours inside the device. Thus, the device can be used to induce and maintain aggregation in a parallelized fashion, facilitating long-term microscopy studies of, e.g., cell-cell interactions.

Keyword
article, cell interaction, cell viability, device, human, human cell, microchip analysis, microscopy, priority journal, thermoregulation, ultrasound, ultrasound transducer
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-26656 (URN)10.1039/c004707d (DOI)000282314200012 ()2-s2.0-77957655329 (Scopus ID)
Note
QC 20101203Available from: 2010-12-03 Created: 2010-11-26 Last updated: 2017-12-12Bibliographically approved
2. 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
3. Influence of acoustic streaming on ultrasonic particle manipulation in a 100-well ring-transducer microplate
Open this publication in new window or tab >>Influence of acoustic streaming on ultrasonic particle manipulation in a 100-well ring-transducer microplate
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2013 (English)In: Journal of Micromechanics and Microengineering, ISSN 0960-1317, E-ISSN 1361-6439, Vol. 23, no 3, 035008- p.Article in journal (Refereed) Published
Abstract [en]

We characterize and quantify the performance of ultrasonic particle aggregation and positioning in a 100-well microplate. We analyze the result when operating a planar ultrasonic ring transducer at different single actuation frequencies in the range 2.20-2.40 MHz, and compare with the result obtained from different schemes of frequency-modulated actuation. Compared to our previously used wedge transducer design, the ring transducer has a larger contact area facing the microplate, resulting in lower temperature increase for a given actuation voltage. Furthermore, we analyze the dynamics of acoustic streaming occurring simultaneously with the particle trapping in the wells of the microplate, and we define an adaptive ultrasonic actuation scheme for optimizing both efficiency and robustness of the method. The device is designed as a tool for ultrasound-mediated cell aggregation and positioning. This is a method for high-resolution optical characterization of time-dependent cellular processes at the level of single cells. In this paper, we demonstrate how to operate our device in order to optimize the scanning time of 3D confocal microscopy with the aim to perform high-resolution time-lapse imaging of cells or cell-cell interactions in a highly parallel manner.

Keyword
Radiation Force, Acoustophoresis, Chip, Cell, Devices
National Category
Engineering and Technology Biological Sciences
Identifiers
urn:nbn:se:kth:diva-119451 (URN)10.1088/0960-1317/23/3/035008 (DOI)000314816800009 ()2-s2.0-84878090655 (Scopus ID)
Funder
Swedish Research Council, 2011-5230EU, FP7, Seventh Framework Programme
Note

QC 20130315

Available from: 2013-03-15 Created: 2013-03-14 Last updated: 2017-12-06Bibliographically approved
4. Live cell imaging in a micro-array of acoustic traps facilitates quantification of natural killer cell heterogeneity
Open this publication in new window or tab >>Live cell imaging in a micro-array of acoustic traps facilitates quantification of natural killer cell heterogeneity
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2013 (English)In: Integrative Biology, ISSN 1757-9694, E-ISSN 1757-9708, Vol. 5, no 4, 712-719 p.Article in journal (Refereed) Published
Abstract [en]

Natural killer (NK) cells kill virus-infected or cancer cells through the release of cytotoxic granules into a tight intercellular contact. NK cell populations comprise individual cells with varying sensitivity to distinct input signals, leading to disparate responses. To resolve this NK cell heterogeneity, we have designed a novel assay based on ultrasound-assisted cell-cell aggregation in a multiwell chip allowing high-resolution time-lapse imaging of one hundred NK-target cell interactions in parallel. Studying human NK cells' ability to kill MHC class I deficient tumor cells, we show that approximately two thirds of the NK cells display cytotoxicity, with some NK cells being particularly active, killing up to six target cells during the assay. We also report that simultaneous interaction with several susceptible target cells increases the cytotoxic responsiveness of NK cells, which could be coupled to a previously unknown regulatory mechanism with implications for NK-mediated tumor elimination.

Keyword
Nk Cells, Education, Cytotoxicity, Lymphocytes, Secretion
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-121500 (URN)10.1039/c3ib20253d (DOI)000316692700008 ()2-s2.0-84878096273 (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 20130506

Available from: 2013-05-06 Created: 2013-04-29 Last updated: 2017-12-06Bibliographically approved
5. Ultrasound-Induced Cell-Cell Interaction Studies in a Multi-Well Microplate
Open this publication in new window or tab >>Ultrasound-Induced Cell-Cell Interaction Studies in a Multi-Well Microplate
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2014 (English)In: Micromachines, ISSN 2072-666X, E-ISSN 2072-666X, Vol. 5, no 1, 27-49 p.Article, review/survey (Refereed) Published
Abstract [en]

This review describes the use of ultrasound for inducing and retaining cell-cell contact in multi-well microplates combined with live-cell fluorescence microscopy. This platform has been used for studying the interaction between natural killer (NK) cells and cancer cells at the level of individual cells. The review includes basic principles of ultrasonic particle manipulation, design criteria when building a multi-well microplate device for this purpose, biocompatibility aspects, and finally, two examples of biological applications: Dynamic imaging of the inhibitory immune synapse, and studies of the heterogeneity in killing dynamics of NK cells interacting with cancer cells.

Keyword
ultrasound, lab-on-a-chip, acoustofluidics, acoustic trapping, natural killer cells
National Category
Nano Technology Biological Sciences
Identifiers
urn:nbn:se:kth:diva-144959 (URN)10.3390/mi5010027 (DOI)000333674700003 ()2-s2.0-84902585350 (Scopus ID)
Funder
Swedish Foundation for Strategic Research EU, FP7, Seventh Framework ProgrammeSwedish Research Council
Note

QC 20140505

Available from: 2014-05-05 Created: 2014-05-05 Last updated: 2017-12-05Bibliographically approved
6. Ultrasound-assisted three-dimensional tumor formation in a multi well microplate for monitoring natural killer cell functional behavior
Open this publication in new window or tab >>Ultrasound-assisted three-dimensional tumor formation in a multi well microplate for monitoring natural killer cell functional behavior
(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:kth:diva-158882 (URN)
Note

QS 2015

Available from: 2015-01-13 Created: 2015-01-13 Last updated: 2015-01-13Bibliographically approved

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Christakou, Athanasia

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