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Ultrasonic Handling of Living Cells in Microfluidic Systems
KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics. (Biomedicinsk och röntgenfysik)
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Microfluidic chips have become a powerful tool in research where biological cells are processed and/or analyzed. One method for contactless cell manipulation in microfluidic chips that has gained an increasing amount of attention the last decade is ultrasonic standing wave (USW) technology. This Thesis explores the biocompatibility of USW technology applied to microfluidic chips, and presents a novel USW-based method for serial processing and accurate characterization of living cells.

The biocompatibility has been investigated by measuring the proliferation rate of cells after they had been trapped and aggregated inside a chip by ultrasound. No negative influence was observed after continuous exposure to 0.85 MPa pressure amplitudes for up to 75 min. Furthermore, the heat generation in the fluid channel caused by the ultrasound has been measured and used in a regulation scheme where the temperature can be controlled around any relevant temperature (e.g. 37‰) with ±0.1‰ accuracy for more than 12 hours. The proliferation rate and temperature investigations suggest that USW technology applied to microfluidic chips is a biocompatiblemethod useful for long-term handling of living cells.

We have introduced a new concept of contactless ultrasonic ”caging” of single cells or small aggregates of cells. These cages are channel segments in the microfluidic chips that are geometrically designed to resonate at one or several actuation frequencies. The actuation is performed remotely by up to five external frequency specific wedge transducers, where each transducer produces a localized and spatially confined standing wave with a specific orientation of its corresponding radiation force field. By multi-frequency actuation, sophisticated and flexible force fields are realized by both overlapping and separated single fields. The Thesis describes two different cages: A sub-mm ”micro-cage” for tree-dimensional manipulationof single cells, and a 5-mm ”mini-cage” for selective retention of small cell aggregates (up to approx. 10^3 cells) from a continuously feeding sample flow. Finally,our microfluidic chips were also designed to be compatible with high-resolution optical microscopy. We have demonstrated sub-μm-resolution confocal fluorescence and trans-illumination microscopy imaging of ultrasonically caged living cells.

Place, publisher, year, edition, pages
Stockholm: KTH , 2009. , xi, 53 p.
Series
Trita-FYS, ISSN 0280-316X ; 2009:56
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-11500ISBN: 978-91-7415-466-5 (print)OAI: oai:DiVA.org:kth-11500DiVA: diva2:277312
Public defence
2009-11-27, FB42, Roslagstullsbacken 21, AlbaNova Universitetscentrum, Kungliga Tekniska Högskolan., Stockholm., 10:00 (English)
Opponent
Supervisors
Note
QC 20100811Available from: 2009-11-17 Created: 2009-11-17 Last updated: 2011-10-04Bibliographically approved
List of papers
1. Proliferation and viability of adherent cells manipulated by standing-wave ultrasound in a microfluidic chip
Open this publication in new window or tab >>Proliferation and viability of adherent cells manipulated by standing-wave ultrasound in a microfluidic chip
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2007 (English)In: Ultrasound in Medicine and Biology, ISSN 0301-5629, E-ISSN 1879-291X, Vol. 33, 145-151 p.Article in journal (Refereed) Published
Abstract [en]

Ultrasonic-standing-wave (USW) technology has potential to become a standard method for gentle and contactless cell handling in microfluidic chips. We investigate the viability of adherent cells exposed to USWs by studying the proliferation rate of recultured cells following ultrasonic trapping and aggregation of low cell numbers in a microfluidic chip. The cells form 2-D aggregates inside the chip and the aggregates are held against a continuous flow of cell culture medium perpendicular to the propagation direction of the standing wave. No deviations in the doubling time from expected values (24 to 48 h) were observed for COS-7 cells held in the trap at acoustic pressure amplitudes up to 0.85 MPa and for times ranging between 30 and 75 min. Thus, the results demonstrate the potential of ultrasonic standing waves as a tool for gentle manipulation of low cell numbers in microfluidic systems.

Keyword
mammalian-cells; retention; sedimentation; aggregation; perfusion; channels; driven; matrix; filter; trap
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-10913 (URN)10.1016/j.ultrasmedbio.2006.07.024 (DOI)000243243700017 ()2-s2.0-33845626925 (Scopus ID)
Note
QC 20100730Available from: 2009-08-18 Created: 2009-08-18 Last updated: 2017-12-13Bibliographically approved
2. Temperature regulation during ultrasonic manipulation for long-term cell handling in a microfluidic chip
Open this publication in new window or tab >>Temperature regulation during ultrasonic manipulation for long-term cell handling in a microfluidic chip
2007 (English)In: Journal of Micromechanics and Microengineering, ISSN 0960-1317, E-ISSN 1361-6439, Vol. 17, 2469-2474 p.Article in journal (Refereed) Published
Abstract [en]

Regulation by the use of ultrasonic standing wave technology in a microfluidic chip. The system is based on a microfabricated silicon structure sandwiched between two glass layers, and an external ultrasonic transducer using a refractive wedge placed on top of the chip for efficient coupling of ultrasound into the microchannel. The chip is fully transparent and compatible with any kind of high-resolution optical microscopy. The temperature regulation method uses calibration data of the temperature increase due to the ultrasonic actuation for determining the temperature of the surrounding air and microscope table, controlled by a warm-air heating unit and a heatable mounting frame. The heating methods are independent of each other, resulting in a flexible choice of ultrasonic actuation voltage and flow rate for different cell and particle manipulation purposes. Our results indicate that it is possible to perform stable temperature regulation with an accuracy of the order of +/- 0.1 degrees C around any physiologically relevant temperature (e.g., 37 degrees C) with high temporal stability and repeatability. The purpose is to use ultrasound for long-term cell and/or particle handling in a microfluidic chip while controlling and maintaining the biocompatibility of the system.

Keyword
technology; retention
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-10914 (URN)10.1088/0960-1317/17/12/012 (DOI)000251767100012 ()2-s2.0-36949032927 (Scopus ID)
Note
QC 20100730Available from: 2009-08-18 Created: 2009-08-18 Last updated: 2017-12-13Bibliographically approved
3. Selective bioparticle retention and characterization in a chip-integrated confocal ultrasonic cavity
Open this publication in new window or tab >>Selective bioparticle retention and characterization in a chip-integrated confocal ultrasonic cavity
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2009 (English)In: Biotechnology and Bioengineering, ISSN 0006-3592, E-ISSN 1097-0290, Vol. 103, 323-328 p.Article in journal (Refereed) Published
Abstract [en]

We demonstrate selective retention and positioning of cells or other bioparticles by ultrasonic manipulation in a microfluidic expansion chamber during microfluidic perfusion. The chamber is designed as a confocal ultrasonic resonator for maximum confinement of the ultrasonic force field at the chamber center, where the cells are trapped. We investigate the resonant modes in the expansion chamber and its connecting inlet channel by theoretical modeling and experimental verification during no-flow conditions. Furthermore, by triple-frequency ultrasonic actuation during continuous microfluidic sample feeding, a set of several manipulation functions performed in series is demonstrated: sample bypass-injection-aggregation and retention-positioning. Finally, we demonstrate transillumination microscopy imaging Of Ultrasonically trapped COS-7 cell aggregates.

Keyword
ultrasonic manipulation; cell characterization; microfluidic chip
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-10917 (URN)10.1002/bit.22255 (DOI)000266078200009 ()19170245 (PubMedID)2-s2.0-65549156499 (Scopus ID)
Note
QC 20100730Available from: 2009-08-18 Created: 2009-08-18 Last updated: 2017-12-13Bibliographically approved
4. Wedge transducer design for two-dimensional ultrasonic manipulation in a microfluidic chip
Open this publication in new window or tab >>Wedge transducer design for two-dimensional ultrasonic manipulation in a microfluidic chip
2008 (English)In: Journal of Micromechanics and Microengineering, ISSN 0960-1317, E-ISSN 1361-6439, Vol. 18, 095025- p.Article in journal (Refereed) Published
Abstract [en]

We analyze and optimize the design of wedge transducers used for the excitation of resonances in the channel of a microfluidic chip in order to efficiently manipulate particles or cells in more than one dimension. The design procedure is based on (1) theoretical modeling of acoustic resonances in the transducer-chip system and calculation of the force fields in the fluid channel, (2) full-system resonance characterization by impedance spectroscopy and (3) image analysis of the particle distribution after ultrasonic manipulation. We optimize the transducer design in terms of actuation frequency, wedge angle and placement on top of the chip, and we characterize and compare the coupling effects in orthogonal directions between single- and dual-frequency ultrasonic actuation. The design results are verified by demonstrating arraying and alignment of particles in two dimensions. Since the device is compatible with high-resolution optical microscopy, the target application is dynamic cell characterization combined with improved microfluidic sample transport.

Keyword
standing-wave; separation; particles; channels; bioassays; cells
National Category
Condensed Matter Physics
Identifiers
urn:nbn:se:kth:diva-10915 (URN)10.1088/0960-1317/18/9/095025 (DOI)000259590700025 ()2-s2.0-54749116541 (Scopus ID)
Note
QC 20100730Available from: 2009-08-18 Created: 2009-08-18 Last updated: 2017-12-13Bibliographically approved
5. A three-dimensional ultrasonic cage for characterization of individual cells
Open this publication in new window or tab >>A three-dimensional ultrasonic cage for characterization of individual cells
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2008 (English)In: Applied Physics Letters, ISSN 0003-6951, E-ISSN 1077-3118, Vol. 93, 063901- p.Article in journal (Refereed) Published
Abstract [en]

We demonstrate enrichment, controlled aggregation, and manipulation of microparticles and cells by an ultrasonic cage integrated in a microfluidic chip compatible with high-resolution optical microscopy. The cage is designed as a dual-frequency resonant filleted square box integrated in the fluid channel. Individual particles may be trapped three dimensionally, and the dimensionality of one-dimensional to three-dimensional aggregates can be controlled. We investigate the dependence of the shape and position of a microparticle aggregate on the actuation voltages and aggregate size, and demonstrate optical monitoring of individually trapped live cells with submicrometer resolution.

Keyword
optical manipulation; microfluidic chip; particles; traps
National Category
Condensed Matter Physics
Identifiers
urn:nbn:se:kth:diva-10916 (URN)10.1063/1.2971030 (DOI)000258491000076 ()2-s2.0-49749149572 (Scopus ID)
Note
QC 20100730Available from: 2009-08-18 Created: 2009-08-18 Last updated: 2017-12-13Bibliographically approved
6. Spatial confinement of ultrasonic force fields in microfluidic chips
Open this publication in new window or tab >>Spatial confinement of ultrasonic force fields in microfluidic chips
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2009 (English)In: Ultrasonics, ISSN 0041-624X, E-ISSN 1874-9968, Vol. 49, 112-119 p.Article in journal (Refereed) Published
Abstract [en]

We demonstrate and investigate multiple localized ultrasonic manipulation functions in series in microfluidic chips. The manipulation functions are based on spatially separated and confined ultrasonic primary radiation force fields, obtained by local matching of the resonance condition of the microfluidic channel. The channel segments are remotely actuated by the use of frequency-specific external transducers with refracting wedges placed on top of the chips. The force field in each channel segment is characterized by the use of micrometer-resolution particle image velocimetry ( micro-PIV). The confinement of the ultrasonic fields during single-or dual-segment actuation, as well as the cross-talk between two adjacent. fields, is characterized and quantified. Our results show that the field confinement typically scales with the acoustic wavelength, and that the cross-talk is insignificant between adjacent. fields. The goal is to define design strategies for implementing several spatially separated ultrasonic manipulation functions in series for use in advanced particle or cell handling and processing applications. One such proof-of-concept application is demonstrated, where. flow-through-mode operation of a chip with. flow splitting elements is used for two-dimensional pre-alignment and addressable merging of particle tracks.

Keyword
Ultrasonic manipulation; Acoustic radiation force; Microfluidic chip; Particle image velocimetry; Spatial confinement; Cell handling
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-10912 (URN)10.1016/j.ultras.2008.06.012 (DOI)000261834200017 ()2-s2.0-56949083339 (Scopus ID)
Note
QC 20100730Available from: 2009-08-18 Created: 2009-08-18 Last updated: 2017-12-13Bibliographically approved

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