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Ultrasonic Fluid and Cell Manipulation
KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics. (Ultrasonics - Martin Wiklund)ORCID iD: 0000-0002-7023-4772
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

During the last decade, ultrasonic manipulation has matured into an important tool with a wide range of applications, from fundamental cell biological research to clinical and industrial implementations. The contactless nature of ultrasound makes it possible to manipulate living cells in a gentle way, e.g., for positioning, sorting, and aggregation. However, when manipulating cells using ultrasound, especially using high acoustic amplitudes, a great deal of heat can be generated. This constitutes a challenge, since the viability of cells is dependent on a stable physiological temperature around 37°C.

     In this Thesis we present applications of ultrasonic manipulation of fluids, particles, and cells in temperature-controlled micrometer-sized devices fabricated using well established etching techniques, directly compatible with high-resolution fluorescence microscopy. Furthermore, we present ultrasonic manipulation in larger up to centimeter-sized devices optimized for fluid mixing and cell lysis. In the present work, two new ultrasonic manipulation platforms have been developed implementing temperature control. These platforms are much improved with increased performance and usability compared to previous platforms. Also, two new ultrasonic platforms utilizing low-frequency ultrasound for solubilization and cell lysis of microliter-volumed and milliliter-volumed samples have been designed and implemented.

     We have applied ultrasound to synchronize the interaction between large numbers of immune, natural killer cells, and cancer cells to study the cytotoxic response, on a single cell level. A heterogeneity was found among the natural killer cell population, i.e., some cells displayed high cytotoxic response while others were dormant. Furthermore, we have used temperature-controlled ultrasound to form up to 100, in parallel, solid cancer HepG2 tumors in a glass-silicon multi-well microplate. Next, we investigated the immune cells cytotoxic response against the solid tumors. We found a correlation between the number of immune cells compared to the size of the tumor and the cytotoxic outcome, i.e., if the tumor could be defeated.

            Finally, the effect of high acoustic pressure amplitudes in the MPa-range on cell viability has been studied in a newly developed platform optimized for long-term stable temperature control, independent on the applied ultrasound power. Lastly, we present two applications of ultrasonic fluid mixing and lysis of cells. One platform is optimized for small microliter-sized volumes in plastic disposable chips and another is optimized for large milliliter-sized volumes in plastic test tubes. The latter platform has been implemented for clinical sputum sample solubilization and cell lysis for genomic DNA extraction for subsequent pathogen detection

Abstract [sv]

Ultraljudsmanipulering har under de senaste tio åren mognat och utvecklats till ett verktyg med ett brett användningsområde. Idag kan man finna applikationer inom allt från cellbiologisk grundforskning till industri samt sjukvård. Ultraljudsmanipuleringens kontaktlösa natur gör det till en varsam metod för att manipulera celler, till exempel inom positionering, sortering och aggregering. När ultraljud med hög amplitud används kan värmeutvecklingen, som är oundviklig, bli ett problem. För att kunna säkerställa hög cellviabilitet krävs temperaturkontroll som kan hålla en fysiologisk, stabil temperatur på 37°C.

     I denna avhandling presenterar vi tillämpningar av temperaturkontrollerad ultraljudsmanipulering i mikrometerstora anordningar fabricerade med väletablerade etsningstekniker.  Dessa anordningar är optimerade för att vara fullt kompatibla med högupplöst fluorescensmikroskopi.  Vi demonstrerar även ultraljudsmanipulering i centimeterstora anordningar optimerade för omrörning och blandning av vätskor samt lysering av celler. Två nya plattformar för ultraljudsmanipulering med inbyggd temperaturkontroll har utvecklats. Dessa två plattformar erbjuder ökad prestanda, flexibilitet samt även användarvänlighet. Utöver dessa plattformar har ytterligare två anordningar för lågfrekvent ultraljudssolubilisering och cellysering av mikroliter- och milliliterstora prover konstruerats.

     I denna avhandling har vi tillämpat ultraljud för att synkronisera interaktionen mellan populationer utav immunceller (natural killer-celler) och cancerceller för att på cellnivå studera det cytotoxiska gensvaret. Vi fann en heterogenitet hos immuncellspopulationen. Det manifesterade sig i en fördelning av immuncellerna, från celler med stort cytotoxiskt gensvar till inaktiva immunceller. Vi har dessutom använt temperaturkontrollerad ultrasljudsmanipulering för att skapa solida cancertumörer utav HepG2-cancerceller, upp till 100 stycken parallellt, i en multihåls-mikrotiterplatta bestående av glas och kisel. Med hjälp av dessa tumörer har vi studerat det cytotoxiska gensvaret från immuncellerna. Vi fann att förhållandet mellan antalet immunceller och storleken på tumören bestämde utfallet, det vill säga om tumören kunde bekämpas.

     Vi presenterar dessutom effekten utav högamplitudsultraljudsexponering av cancerceller i en plattform speciellt designad för höga tryckamplituder med implementerad ultraljudseffektsoberoende temperaturkontroll. Slutligen presenterar vi två tillämpningar av ultraljud för vätskeblandning och cellysering. Den första tillämpningen är anpassad för små volymer i plastchip för engångsbruk och den andra är optimerad för större volymer i plastprovrör. Den senare tillämpningen är speciellt framtagen för ultraljudssolubilisering och cellysering utav kliniska sputumprover för att möjliggöra DNA-extrahering för detektion av smittämnen.     

 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2015. , ix, 68 p.
Series
TRITA-FYS, ISSN 0280-316X ; 2015:24
Keyword [en]
3D cell culture, Acoustic streaming, Acoustofluidics, Cell manipulation, High-resolution imaging, Multi-well, Microplate, Natural killer cell, Piezo, Radiation force, Solid tumor, Solubilization, Spheroid, Standing wave, Temperature control, Transducer, Trapping, Ultrasonic
National Category
Other Physics Topics
Research subject
Physics; Biological Physics
Identifiers
URN: urn:nbn:se:kth:diva-166779ISBN: 978-91-7595-559-9 (print)OAI: oai:DiVA.org:kth-166779DiVA: diva2:812243
Public defence
2015-06-12, AlbaNova FD5, Roslagstullsbacken 21, KTH, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

QC 20150522

Available from: 2015-05-22 Created: 2015-05-18 Last updated: 2015-05-26Bibliographically 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. Acoustofluidics 14: Applications of acoustic streaming in microfluidic devices
Open this publication in new window or tab >>Acoustofluidics 14: Applications of acoustic streaming in microfluidic devices
2012 (English)In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 12, no 14, 2438-2451 p.Article in journal, Editorial material (Other academic) Published
Abstract [en]

In part 14 of the tutorial series "Acoustofluidics - exploiting ultrasonic standing wave forces and acoustic streaming in microfluidic systems for cell and particle manipulation", we provide a qualitative description of acoustic streaming and review its applications in lab-on-a-chip devices. The paper covers boundary layer driven streaming, including Schlichting and Rayleigh streaming, Eckart streaming in the bulk fluid, cavitation microstreaming and surface-acoustic-wave-driven streaming.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-99105 (URN)10.1039/c2lc40203c (DOI)000305532600002 ()2-s2.0-84862207850 (Scopus ID)
Note
QC 20120717Available from: 2012-07-17 Created: 2012-07-13 Last updated: 2017-12-07Bibliographically approved
3. 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
4. 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
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. Ultrasonic three-dimensional cell culture on chip for dynamic studies of tumor immune surveillance by natural killer cells
Open this publication in new window or tab >>Ultrasonic three-dimensional cell culture on chip for dynamic studies of tumor immune surveillance by natural killer cells
(English)Manuscript (preprint) (Other academic)
National Category
Nano Technology Biological Sciences
Identifiers
urn:nbn:se:kth:diva-167643 (URN)
Note

QS 2015

Available from: 2015-05-22 Created: 2015-05-22 Last updated: 2015-05-22Bibliographically approved
7. Temperature-controlled MPa-pressure ultrasonic cell manipulation in a microfluidic chip
Open this publication in new window or tab >>Temperature-controlled MPa-pressure ultrasonic cell manipulation in a microfluidic chip
2015 (English)In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 15, no 16, 3341-3349 p.Article in journal (Refereed) Published
Abstract [en]

We study the temperature-independent impact on cell viability of relevant physical parameters during long-term, high-acoustic-pressure ultrasonic exposure in a microfluidic chip designed for ultrasonic-standing-wave trapping and aggregation of cells. We use a light-intensity method and 5 mum polymer beads for accurate acoustic pressure calibration before injecting cells into the device, and we monitor the viability of A549 lung cancer cells trapped during one hour in an ultrasonic standing wave with 1 MPa pressure amplitude. The microfluidic chip is actuated by a novel temperature-controlled ultrasonic transducer capable of keeping the temperature stable around 37 °C with an accuracy better than ±0.2 °C, independently on the ultrasonic power and heat produced by the system, thereby decoupling any temperature effect from other relevant effects on cells caused by the high-pressure acoustic field. We demonstrate that frequency-modulated ultrasonic actuation can produce acoustic pressures of equally high magnitudes as with single-frequency actuation, and we show that A549 lung cancer cells can be exposed to 1 MPa standing-wave acoustic pressure amplitudes for one hour without compromising cell viability. At this pressure level, we also measure the acoustic streaming induced around the trapped cell aggregate, and conclude that cell viability is not affected by streaming velocities of the order of 100 mum s(-1). Our results are important when implementing acoustophoresis methods in various clinical and biomedical applications.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-166828 (URN)10.1039/c5lc00490j (DOI)000358609500011 ()26156858 (PubMedID)2-s2.0-84938342105 (Scopus ID)
Note

Updated from "Manuscript" to "Article". QC 20150814

Available from: 2015-05-19 Created: 2015-05-19 Last updated: 2017-12-04Bibliographically approved
8. Acoustic micro-vortexing of fluids, beads and cells in disposable microfluidic chips
Open this publication in new window or tab >>Acoustic micro-vortexing of fluids, beads and cells in disposable microfluidic chips
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(English)Manuscript (preprint) (Other academic)
National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-166825 (URN)
Note

QS 2015

Available from: 2015-05-19 Created: 2015-05-19 Last updated: 2015-05-22Bibliographically approved

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