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A silicon-glass microwell platform for high-resolution imaging and high-content screening with single cell resolution
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.
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
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2011 (English)In: Biomedical microdevices (Print), ISSN 1387-2176, E-ISSN 1572-8781, Vol. 13, no 4, 683-693 p.Article in journal (Refereed) Published
Abstract [en]

We present a novel microwell array platform suited for various cell-imaging assays where single cell resolution is important. The platform consists of an exchangeable silicon-glass microchip for cell biological applications and a custom made holder that fits in conventional microscopes. The microchips presented here contain arrays of miniature wells, where the well sizes and layout have been designed for different applications, including single cell imaging, studies of cell-cell interactions or ultrasonic manipulation of cells. The device has been designed to be easy to use, to allow long-term assays (spanning several days) with read-outs based on high-resolution imaging or high-content screening. This study is focused on screening applications and an automatic cell counting protocol is described and evaluated. Finally, we have tested the device and automatic counting by studying the selective survival and clonal expansion of 721.221 B cells transfected to express HLA Cw6-GFP compared to untransfected 721.221 B cells when grown under antibiotic selection for 3 days. The device and automated analysis protocol make up the foundation for development of several novel cellular imaging assays.

Place, publisher, year, edition, pages
2011. Vol. 13, no 4, 683-693 p.
Keyword [en]
single cell, microwell, automatic image analysis, screening fluorerscence imaging, clonal expansion
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-30472DOI: 10.1007/s10544-011-9538-2ISI: 000292556900009PubMedID: 21465090Scopus ID: 2-s2.0-80053896769OAI: oai:DiVA.org:kth-30472DiVA: diva2:400348
Note

QC 20110802

Available from: 2011-02-25 Created: 2011-02-25 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Development of Microchip-based Assays to Study Immune Cell Interactions at the Single Cell Level
Open this publication in new window or tab >>Development of Microchip-based Assays to Study Immune Cell Interactions at the Single Cell Level
2011 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Immune cell populations are constantly divided into smaller and smaller subsets defined by newly emerging cellular markers. However, there is a growing awareness of the functional heterogeneities in between cells even within small populations, in addition to the heterogeneity over time. One may ask whether a population is correctly defined only by cellular markers or if the functionality should be regarded as well? Many of today’s techniques only measures at the population level, giving an average estimate of the behavior of that pool of cells, but failing to detect rare possibly important events. Thus, high-throughput experimental approaches to analyze single cells over time are required to address cellular heterogeneity.

Progress in the fields of microfabrication, microscopy and computing have paved the way for increasingly efficient tools for studies on the single cell level, and a variety of devices have been described by others. However, few of them are suitable for long-term imaging of dynamic events such as cell-cell interactions or migration. In addition, for efficient recording of many individual events it is desirable to scale down the cells’ interaction volume; not only to shorten the time to interaction, but also to increase the number of individual events in a given area; thereby pushing a screening approach.

To address these questions, a complete microwell array system for imaging of immune cell responses with single-cell resolution was designed. The platform consists of a range of silicon-glass microchips with arrays of miniature wells for incubation of cells and a custom made holder that fits conventional microscopes. The device has been designed to allow cells to be kept viable for several days in the wells, to be easy to use and to allow high-resolution imaging. Five different designs were fabricated; all with a specific type of assay in mind, and were evaluated regarding biocompatibility and functionality. One design is aimed towards screening applications, making an automatic cell counting protocol necessary in order to analyze the massive amount of data generated; this program is also described and evaluated.

We here show that our silicon microwell platform allows long-term studies (up to several days), with the possibility of both time-lapse and high-resolution imaging of a variety of immune cell behavior. Using time-lapse imaging we confirmed immune cell heterogeneity in NK cell populations regarding both cytotoxicity and migrational behavior. The automatic counting program was tested and showed similar results compared to both manual counting and FACS. In addition, the large numbers of wells that can be simultaneously imaged, provide new statistical information that will lead to a better understanding of the function and regulation of the immune system at the single cell level.

Altogether, our technique enables novel types of cellular imaging assays allowing data collection at a level of resolution not previously obtained – this was shown to be important for performing basic cell biological studies, but may also prove valuable in the proposed future medical applications.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2011. iv, 40 p.
Series
Trita-FYS, ISSN 0280-316X ; 2011:04
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-30443 (URN)978-91-7415-872-4 (ISBN)
Presentation
2011-02-23, FA31, KTH, Roslagstullsbacken 21, Stockholm, 10:30 (English)
Opponent
Supervisors
Note
QC 20110225Available from: 2011-02-25 Created: 2011-02-24 Last updated: 2011-02-25Bibliographically approved
2. Live Single Cell Imaging and Analysis Using Microfluidic Devices
Open this publication in new window or tab >>Live Single Cell Imaging and Analysis Using Microfluidic Devices
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Today many cell biological techniques study large cell populations where an average estimate of individual cells’ behavior is observed. On the other hand, single cell analysis is required for studying functional heterogeneities between cells within populations. This thesis presents work that combines the use of microfluidic devices, optical microscopy and automated image analysis to design various cell biological assays with single cell resolution including cell proliferation, clonal expansion, cell migration, cell-cell interaction and cell viability tracking. In fact, automated high throughput single cell techniques enable new studies in cell biology which are not possible with conventional techniques.

In order to automatically track dynamic behavior of single cells, we developed a microwell based device as well as a droplet microfluidic platform. These high throughput microfluidic assays allow automated time-lapse imaging of encapsulated single cells in micro droplets or confined cells inside microwells. Algorithms for automatic quantification of cells in individual microwells and micro droplets are developed and used for the analysis of cell viability and clonal expansion. The automatic counting protocols include several image analysis steps, e.g. segmentation, feature extraction and classification. The automatic quantification results were evaluated by comparing with manual counting and revealed a high success rate. In combination these automatic cell counting protocols and our microfluidic platforms can provide statistical information to better understand behavior of cells at the individual level under various conditions or treatments in vitro exemplified by the analysis of function and regulation of immune cells. Thus, together these tools can be used for developing new cellular imaging assays with resolution at the single cell level.

To automatically characterize transient migration behavior of natural killer (NK) cells compartmentalized in microwells, we developed a method for single cell tracking. Time-lapse imaging showed that the NK cells often exhibited periods of high motility, interrupted with periods of slow migration or complete arrest. These transient migration arrest periods (TMAPs) often overlapped with periods of conjugations between NK cells and target cells. Such conjugation periods sometimes led to cell-mediated killing of target cells. Analysis of cytotoxic response of NK cells revealed that a small sub-class of NK cells called serial killers was able to kill several target cells. In order to determine a starting time point for cell-cell interaction, a novel technique based on ultrasound was developed to aggregate NK and target cells into the center of the microwells. Therefore, these assays can be used to automatically and rapidly assess functional and migration behavior of cells to detect differences between health and disease or the influence of drugs.

The work presented in this thesis gives good examples of how microfluidic devices combined with automated imaging and image analysis can be helpful to address cell biological questions where single cell resolution is necessary. 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2013. vi, 53 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2013:14
Keyword
Single cell analysis, time-lapse fluorescence imaging, automated image analysis, microwell, droplet microfluidics, NK cells, single cell tracking, migration behavior analysis, cell-cell interaction, optical microscopy, image analysis, image processing, microfluidics, immune cells, tracking, counting, morphology analysis
National Category
Other Medical Biotechnology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-129278 (URN)978-91-7501-846-1 (ISBN)
Public defence
2013-10-18, Gardaulan, Smittskyddsinstitutet, Nobels väg 18, Karolinska Institutet, Solna, 13:00 (English)
Opponent
Supervisors
Funder
Swedish Research Council, 70784
Note

QC 20130927

Available from: 2013-09-26 Created: 2013-09-25 Last updated: 2014-02-28Bibliographically approved
3. Single Cell Investigations of the Functional Heterogeneity Within Immune Cell Populations: a Microchip-based Study
Open this publication in new window or tab >>Single Cell Investigations of the Functional Heterogeneity Within Immune Cell Populations: a Microchip-based Study
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Immune cell populations are constantly divided into smaller and smaller subsets defined by newly emerging cellular markers. However, there is a growing awareness of the functional heterogeneities in between cells even within small populations, in addition to the heterogeneity over time. One may ask whether a population is correctly defined only by cellular markers or if the functionality should be regarded as well? Many of today’s techniques only measure at the population level, giving an average estimate of the behavior of that pool of cells, but failing to detect rare possibly important events. Thus, high-throughput experimental approaches to analyze single cells over time are required to address cellular heterogeneity.

Progress in the fields of microfabrication, microscopy and computing have paved the way for increasingly efficient tools for studies on the single cell level, and a variety of devices have been described by others. However, few of them are suitable for long-term imaging of dynamic events such as cell-cell interactions or migration. In addition, for efficient recording of many individual events it is desirable to scale down the cells’ interaction volume; not only to shorten the time to interaction, but also to increase the number of individual events in a given area; thereby pushing a screening approach.

To address these questions, a complete microwell array system for imaging of immune cell responses with single-cell resolution was designed. The platform consists of a range of silicon-glass microchips with arrays of miniature wells for incubation of cells and a custom made holder that fits conventional microscopes. The device has been designed to allow cells to be kept viable for several days in the wells, to be easy to use and to allow high-resolution imaging. Five different designs were fabricated; all with a specific type of assay in mind, and were evaluated regarding biocompatibility and functionality. Here, the design aimed for screening applications is the main focus. In this approach a large amount, tens of thousands, of small wells are imaged two to three times: first directly post-seeding of effector and target cells to register the well’s content, and second after some time has passed to allow for cell-cell interactions. The final read-out is the number of killed target cells in each well, making an automatic cell counting protocol necessary in order to analyze the massive amount of data generated.

We here show that our silicon microwell platform allows long-term studies with the possibility of both time-lapse and high-resolution imaging of a variety of immune cell behavior. Using both time-lapse imaging and the screening approach we confirmed and investigated immune cell heterogeneity within NK cell populations in regards to both cytotoxicity and migrational behavior. In addition, two different types of cytolytic behavior in NK cells, termed fast and slow killing, were described and evaluated in regards to dynamic parameters; like conjugation and attachment time. We could also quantify the type of cytolytic response in relation to serial killing NK cells, and saw that serial killing NK cells more often induced fast target cell death. Further investigations using the screening approach have shown that serial killing NK cells also differ from other NK cells in their morphology, being both larger and with a more elongated shape. So far the platform has been used to gain better understanding of some aspects of NK cell biology, but there is still much left to explore. With the addition of an automatic counting program, the large numbers of wells that can be simultaneously imaged will provide new statistical information and enable higher throughput.

Altogether, our family of techniques enables novel types of cellular imaging assays allowing data collection at a level of resolution not previously obtained – this was shown to be important for performing basic cell biological studies, but may also prove valuable in the proposed future medical applications such as adoptive cell therapy and stem cell transplantation.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. viii, 61 p.
Series
TRITA-FYS, ISSN 0280-316X ; 2014:08
National Category
Immunology in the medical area Immunology
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-142472 (URN)978-91-7595-028-0 (ISBN)
Public defence
2014-03-25, Air and Fire, Science for Life Laboratories, Tomtebodavägen 23A, 17165, Solna, 09:30 (English)
Opponent
Supervisors
Funder
Swedish Research CouncilSwedish Cancer SocietySwedish Foundation for Strategic Research
Note

QC 20140306

Available from: 2014-03-06 Created: 2014-03-05 Last updated: 2014-03-06Bibliographically approved

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