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Influence of acoustic streaming on ultrasonic particle manipulation in a 100-well ring-transducer microplate
KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.ORCID iD: 0000-0002-7023-4772
KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.ORCID iD: 0000-0002-3996-9279
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.ORCID iD: 0000-0001-5178-7593
Show others and affiliations
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.

Place, publisher, year, edition, pages
2013. Vol. 23, no 3, 035008- p.
Keyword [en]
Radiation Force, Acoustophoresis, Chip, Cell, Devices
National Category
Engineering and Technology Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-119451DOI: 10.1088/0960-1317/23/3/035008ISI: 000314816800009Scopus ID: 2-s2.0-84878090655OAI: oai:DiVA.org:kth-119451DiVA: diva2:611257
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
In thesis
1. Ultrasound-assisted Interactions of Natural Killer Cells with Cancer Cells and Solid Tumors
Open this publication in new window or tab >>Ultrasound-assisted Interactions of Natural Killer Cells with Cancer Cells and Solid Tumors
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
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:nbn:se:kth:diva-158522 (URN)978-91-7595-419-6 (ISBN)
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
2. Ultrasonic Fluid and Cell Manipulation
Open this publication in new window or tab >>Ultrasonic Fluid and Cell Manipulation
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
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:nbn:se:kth:diva-166779 (URN)978-91-7595-559-9 (ISBN)
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

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