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Microfluidic based isolation of circulating tumor cells from whole blood for cancer diagnostics
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. (NANOBIOTECHNOLOGY-CLINICAL MICROFLUIDICS)ORCID iD: 0000-0001-5199-0663
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Detection of circulating tumor cells (CTC) in peripheral blood is indicative of early recognition of tumor progression and such an important biomarker for early diagnosis, staging, monitoring and prognosis of cancer. However, CTC are found in very low concentrations and reliable isolation of these rare cells is challenging. Microfluidics enables precise manipulation of fluids and cells and is ideal for cell sorting methods for clinical diagnostics. The thesis contributes towards the development of microfluidic based CTC isolation methods from peripheral blood. The methods are based on size and immunoaffinity. The first part of the thesis describes the phenomenon of inertial focusing for size based cell separation at high throughputs. In paper 1, we demonstrate continuous filtration of leukocytes from diluted blood, with an efficiency of 78% at a flow rate of 2.2ml/min. In the paper 2, separation of total and subpopulation of leukocytes with a purity of 86% for granulocytes and 91% for lymphocytes is demonstrated. Furthermore, cancer cells spiked into whole blood could be separated at a yield of 88%. Finally, in paper 3 and 4 we unravel parts of the unexplored elasto-inertial microfluidics and was utilized to precisely focus the cells, as part of an integrated optofluidic micro flow cytometer device, capable to simultaneously measure fluorescence and scattering of cells and particles at a rate of 2500 particles/sec (paper 4). Second part of the thesis focuses on acoustophoresis. In (paper 5), a multifunctional acoustic microdevice was developed for isolation of cancer cells from red blood cells with a separation efficiency of 92.4% and trapping efficiency of 93%. In (paper 6), microbubbles activated acoustic cell sorter was developed for affinity based cell separation. As a proof of principle, cancer cells in a suspension were separated at an efficiency of 75%. In the third part, using cellulose nano fibrils (paper 7), we demonstrate efficiently capture and release of cancer cells at a release efficiency of 95%. Finally, a novel, single step self-assembly of spider silk proteins is introduced inside microfluidic channels for effective capture of cancer cells with 85% capture efficiency and subsequent release of captured cells with 95% release efficiency (paper 8). The novel recombinant silk modified microfluidic device was validated using pancreatic cancer patients. In summary, we have developed different microfluidic based isolation technologies for the capture and characterization of CTC.

Place, publisher, year, edition, pages
stockholm: KTH Royal Institute of Technology, 2017. , 109 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:7
National Category
Medical Engineering Medical Biotechnology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-203889ISBN: 978-91-7729-311-8 (electronic)OAI: oai:DiVA.org:kth-203889DiVA: diva2:1083033
Public defence
2017-04-13, Gardaulan, Folkhälsomyndigheten, Nobels väg 18, Solna, Stockholm, 10:00 (English)
Opponent
Supervisors
Funder
EU, FP7, Seventh Framework ProgrammeSwedish Childhood Cancer FoundationSwedish Research Council
Note

QC 20170321

Available from: 2017-03-20 Created: 2017-03-20 Last updated: 2017-03-23Bibliographically approved
List of papers
1. Dean flow-coupled inertial focusing in curved channels
Open this publication in new window or tab >>Dean flow-coupled inertial focusing in curved channels
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2014 (English)In: Biomicrofluidics, ISSN 1932-1058, Vol. 8, no 3, 034117- p.Article in journal (Refereed) Published
Abstract [en]

Passive particle focusing based on inertial microfluidics was recently introduced as a high-throughput alternative to active focusing methods that require an external force field to manipulate particles. In inertial microfluidics, dominant inertial forces cause particles to move across streamlines and occupy equilibrium positions along the faces of walls in flows through straight micro channels. In this study, we systematically analyzed the addition of secondary Dean forces by introducing curvature and show how randomly distributed particles entering a simple u-shaped curved channel are focused to a fixed lateral position exiting the curvature. We found the lateral particle focusing position to be fixed and largely independent of radius of curvature and whether particles entering the curvature are pre-focused (at equilibrium) or randomly distributed. Unlike focusing in straight channels, where focusing typically is limited to channel cross-sections in the range of particle size to create single focusing point, we report here particle focusing in a large cross-section area (channel aspect ratio 1: 10). Furthermore, we describe a simple u-shaped curved channel, with single inlet and four outlets, for filtration applications. We demonstrate continuous focusing and filtration of 10 mu m particles (with > 90% filtration efficiency) from a suspension mixture at throughputs several orders of magnitude higher than flow through straight channels (volume flow rate of 4.25ml/min). Finally, as an example of high throughput cell processing application, white blood cells were continuously processed with a filtration efficiency of 78% with maintained high viability. We expect the study will aid in the fundamental understanding of flow through curved channels and open the door for the development of a whole set of bio-analytical applications.

Keyword
Continuous Particle Separation, Microfluidic Device, Poiseuille Flow, Tumor-Cells, Microchannels, Filtration, Filter
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-149227 (URN)10.1063/1.4884306 (DOI)000339004500017 ()
Funder
EU, FP7, Seventh Framework ProgrammeSwedish Research CouncilScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20140819

Available from: 2014-08-19 Created: 2014-08-18 Last updated: 2017-03-21Bibliographically approved
2. Inertial microfluidcs combined with selective cell lysis for high throughput separation of nucleated cells from whole blood
Open this publication in new window or tab >>Inertial microfluidcs combined with selective cell lysis for high throughput separation of nucleated cells from whole blood
2017 (English)Article in journal (Other academic) Submitted
Abstract [en]

The ability to rapidly analyze and extract information from peripheral blood cells has the potential of providing a wealth of new information about immune function and general health of the patient. In spite of the tremendous progress achieved in the field of leukocyte analysis, one of the major impediments for routine analysis is the enrichment of cell populations from heterogeneous sources such as blood, as the currently used techniques tend to be laborious. Moreover, the isolation of small and transient cell populations in blood, like circulating tumor cells during cancer metastasis, is even more challenging. Here, we report an integrated device for label-free continuous flow separation of nucleated cells from unprocessed whole blood at high throughput. The method utilizes osmometric behavior of cells to completely remove red blood cells and flow characteristics in spiral microchannels for inertial focusing and separation of nucleated cells. Using an integrated device with two outlets, we isolated total leukocytes at a high yield of 99%. Furthermore cancer cells spiked into whole blood could be separated at a yield of 88% while 80 % of leukocyte could be depleted into separate outlet by simply changing the resistance between the two outlets. Finally, using a three-outlet integrated device, we demonstrate fractionation of leukocyte into subpopulation. The device continuously separates granulocytes at a purity of 86%, monocyte at a purity of 43% and lymphocytes at a purity of 91% simultaneously. Finally, a cell activation study of the immune system using blood from healthy subjects, stimulated ex vivo with Lipopolysaccharides (LPS), confirmed that the high operational flow rate of the device does not alter the activation levels of leukocytes or introduce artifacts. Hence, the simple, high-throughput and low-cost integrated device requiring neither external force fields nor mechanical parts to operate should readily be applicable to sort nucleated cells as stand-alone and/or as integrated lab-on-a-chip devices with high-throughput requirements 

Place, publisher, year, edition, pages
RSC Publishing, 2017
Keyword
inertial microfluidics, nucleated cells, selective cell lysis
National Category
Medical Engineering
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-203241 (URN)
Funder
Swedish Childhood Cancer FoundationEU, FP7, Seventh Framework ProgrammeSwedish Research Council
Note

QC 20170320

Available from: 2017-03-14 Created: 2017-03-14 Last updated: 2017-03-21Bibliographically approved
3. Extended elasto-inertial microfluidics for high throughput separation in low aspect ratio spiral microchannels
Open this publication in new window or tab >>Extended elasto-inertial microfluidics for high throughput separation in low aspect ratio spiral microchannels
(English)Manuscript (preprint) (Other (popular science, discussion, etc.))
Abstract [en]

Particle manipulation in viscoelastic fluid has received substantial interest because this phenomenon provides high-quality focusing. In this work, we report elasto-inertial particle focusing in spiral channels at one to two orders of magnitude higher Reynolds numbers than previously reported. We systematically investigate the interaction between inertial forces, viscoelastic forces using PEO as an elasticity enhancer and the Dean drag forces in microchannels, and report single stream particle focusing at Re > 100. Using a novel integrated 2-spiral microdevice, we applied the method to continuous focus and separate particles and report differential migration and separation 15μm from 10 μm particles at flow rate of 1 ml/min. A separation efficiency of 99% for the 15 μm and 91% for the 10μm particles was achieved. 

Keyword
Inertial microfluidics, extended-elasto inertial microfluidics, circulating tumor cells, particle separation
National Category
Medical Engineering
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-203887 (URN)
Funder
Swedish Childhood Cancer Foundation
Note

QC 20170320

Available from: 2017-03-20 Created: 2017-03-20 Last updated: 2017-03-21Bibliographically approved
4. All silica fibre microflow cytometer
Open this publication in new window or tab >>All silica fibre microflow cytometer
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Flow cytometry is currently the gold standard for analysis of cells in the medical laboratory and biomedical research. Fuelled by the need of point-of-care diagnosis, a significant effort has been made to miniaturize and reduce cost of flow cytometers. However, despite recent advances, current microsystems remain less versatile and much slower than their large-scale counterparts. In this work, an all-silica fibre microflow cytometer is presented that measures fluorescence and scattering from particles and cells. It integrates cell transport in circular capillaries and light delivery by optical fibres   Single-stream cell focusing is performed by Elasto-inertial microfluidics to guarantee optical accuracy and sensitivity.  The capability of this technique is extended to high flow rates (up to 800 µl/min), enabling throughput of 2500 particles/s. The robust, portable and low-cost system described here could be the basis for a point-of-care flow cytometer with a performance comparable to commercial systems. 

Keyword
Flow cytometry, fiber optics
National Category
Clinical Medicine
Identifiers
urn:nbn:se:kth:diva-203953 (URN)
Note

QC 20170321

Available from: 2017-03-20 Created: 2017-03-20 Last updated: 2017-03-21Bibliographically approved
5. On-chip ultrasonic sample preparation for cell based assays
Open this publication in new window or tab >>On-chip ultrasonic sample preparation for cell based assays
2015 (English)In: RSC Advances, ISSN 2046-2069, E-ISSN 2046-2069, Vol. 5, no 91, 74304-74311 p.Article in journal (Refereed) Published
Abstract [en]

We demonstrate an acoustophoresis method for size-based separation, isolation, up-concentration and trapping of cells that can be used for on-chip sample preparation combined with high resolution imaging for cell-based assays. The method combines three frequency-specific acoustophoresis functions in a sequence by actuating three separate channel zones simultaneously: zones for pre-alignment, size-based separation, and trapping. We characterize the mutual interference between the acoustic radiation forces between the different zones by measuring the spatial distribution of the acoustic energy density during different schemes of ultrasonic actuation, and use this information for optimizing the driving frequencies and voltages of the three utilized ultrasonic transducers attached to the chip, and the flow rates of the pumps. By the use of hydrodynamic defocusing of the pre-aligned cells in the separation zone, a cell population from a complex sample can be isolated and trapped with very high purity, followed by dynamic fluorescence analysis. We exemplify the method's potential by isolating A549 lung cancer cells from red blood cells with 100% purity, 92% separation efficiency, and 93% trapping efficiency resulting in a 130× up-concentration factor during 15 minutes of continuous sample processing through the chip. Furthermore, we demonstrate an on-chip fluorescence assay of the isolated cancer cells by monitoring the dynamic uptake and release of a fluorescence probe in individual trapped cells. The ability to combine isolation of individual cells from a complex sample with high-resolution image analysis holds great promise for applications in cellular and molecular diagnostics.

Place, publisher, year, edition, pages
RSC Publishing, 2015
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-166823 (URN)10.1039/c5ra16865a (DOI)000361116500020 ()2-s2.0-84941242035 (ScopusID)
Note

Updated from manuscript to article.

QC 20151008

Available from: 2015-05-19 Created: 2015-05-19 Last updated: 2017-03-21Bibliographically approved
6. MicroBubble Activated Acoustic Cell Sorting: BAACS
Open this publication in new window or tab >>MicroBubble Activated Acoustic Cell Sorting: BAACS
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(English)In: Biomedical microdevices (Print), ISSN 1387-2176, E-ISSN 1572-8781Article in journal (Refereed) Submitted
Abstract [en]

Acoustophoresis, the ability to acoustically manipulate particles and cells inside a microfluidic channel, is a critical enabling technology for cell-sorting applications. However, one of the major impediments for routine use of acoustophoresis at clinical laboratory has been the reliance on the inherent physical properties of cells for separation. Here, we present a microfluidic-based microBubble-Activated Acoustic Cell Sorting (BAACS) method that rely on the specific binding of target cells to microbubbles conjugated with specific antibodies on their surface for continuous cell separation using ultrasonic standing wave. In acoustophoresis, cells being positive acoustic contrast particles migrate to pressure nodes. On the contrary we show that air-filled polymer-shelled microbubbles being strong negative acoustic contrast particles migrate to pressure antinodes at acoustic pressure amplitudes as low as 60 kPa. As a proof of principle, using the BAACS strategy, we demonstrate the separation of cancer cell line in a suspension with better than 75% efficiency. Moreover, 100% of the microbubble-cell conjugates migrated to the anti-node. Hence a better upstream affinity-capture has the potential to provide higher sorting efficiency. The BAACS technique may potentially provide a simplistic approach for similar sized selective isolation of cells, and is suited for applications in point of care.

Keyword
Cell sorting, acoustophoresis, microbubble, contrast agent, microfluidic separation
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-200302 (URN)
Funder
EU, FP7, Seventh Framework Programme, 115153
Note

QC 20170124

Available from: 2017-01-24 Created: 2017-01-24 Last updated: 2017-03-21Bibliographically approved
7. Layer-by-layer system based cellulose nanofibrils for capture and release of cells in microfluidic device
Open this publication in new window or tab >>Layer-by-layer system based cellulose nanofibrils for capture and release of cells in microfluidic device
(English)Manuscript (preprint) (Other (popular science, discussion, etc.))
Abstract [en]

Selective isolation of cells, without inducing any phenotypic changes and maintaining cell viability will preserve the information necessary for down stream analysis. Here we present an ultra thin coating on the surface of disposable microfluidic device based on cellulose nanofibrils, that is modified to capture cells and for later release. Layer-by-layer technique facilitates the production of the thin coating of cellulose onto polymeric surfaces and modified to form affinity based cell capture surface. We demonstrate an  efficiently capture and release of cells, the release is done by selectively degrading 

Keyword
layer-by-layer, cellulose nano fibrils, circulating tumor cells, capture and release
National Category
Biomaterials Science
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-203248 (URN)
Note

QC 20170320

Available from: 2017-03-14 Created: 2017-03-14 Last updated: 2017-03-21Bibliographically approved
8. Microfluidic based circulating tumor cell isolation and release from whole blood of pancreatic cancer patients using bio-functionalized recombinant spider silk
Open this publication in new window or tab >>Microfluidic based circulating tumor cell isolation and release from whole blood of pancreatic cancer patients using bio-functionalized recombinant spider silk
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

A bio-functionalized microsystem was developed for the capture and release of cancer cells from whole blood. Effective isolation and purification of circulating tumor cells from whole blood provides important capability for clinical application and biological research. Here, we demonstrate a single step surface modification procedure for a microfluidic device based on self-assembly of recombinant spider silk harbouring an affinity domain for antibody binding. The surfaces of microfluidic devices were conjugated/equipped with anti-EpCAM antibody for selective isolation of pancreatic cancer cells from spiked whole blood and finally circulating tumor cells from pancreatic cancer patients. Moreover, a protease-cleavage site in the recombinant spider silk proteins provides the unique option to release the captured cancer cells on command from the device without compromising the cell’s viability. Our approach offers a simple, easy and robust surface modification process with a 85% cancer cell capture efficiency. Subsequent addition of a site-specific protease results in the release of 95% of captured cells from the bio functionalized microfluidic systems. 

Keyword
CIRCULATING TUMOR CELLS, SPIDER SILK, MICROFLUIDICS, CAPTURE AND RELEASE
National Category
Biomaterials Science
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-203249 (URN)
Funder
EU, FP7, Seventh Framework Programme
Note

QC 20170320

Available from: 2017-03-15 Created: 2017-03-15 Last updated: 2017-03-21Bibliographically approved

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