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A microwell array device with integrated microfluidic components for enhanced single-cell analysis
KTH, School of Biotechnology (BIO), Nano Biotechnology.
KTH, School of Biotechnology (BIO), Nano Biotechnology.
2009 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 30, no 24, 4166-4171 p.Article in journal (Refereed) Published
Abstract [en]

Increasing awareness of the importance of cell heterogeneity in many biological and medical contexts is prompting increasing interest in systems that allow single-cell analysis rather than conventional bulk analysis (which provides average values for variables of interest from large numbers of cells). Recently, we presented a microwell chip for long-term, high-throughput single-cell analysis. The chip has proved to be useful for purposes such as screening individual cancer and stem cells for protein/gene markers. However, liquids in the wells can only be added or changed by manually rinsing the chip, or parts of it. This procedure has several well-known drawbacks - including risks of cross-contamination, large dead volumes and laboriousness - but there have been few previous attempts to integrate liquid rinsing/switching channels in "ready-to-use" systems for single-cell analysis. Here we present a microwell system designed (using flow simulations) for single-cell analysis with integrated microfluidic components (microchannels, magnetically driven micropumps and reservoirs) for supplying the cell culture wells with reagents, or rinsing, thus facilitating controlled, directed liquid handling. It can be used totally independently, since tubing is not essential. The practical utility of the integrated system has been demonstrated by culturing endothelial cells in the microwells, and successfully applying live-cell Calcein AM staining. Systems such as this combining high-density microwell chips with microfluidic components have great potential in numerous screening applications, such as exploring the important, but frequently undetected, heterogeneity in drug responses among individual cells.

Place, publisher, year, edition, pages
2009. Vol. 30, no 24, 4166-4171 p.
Keyword [en]
Cell culture; Microfluidics; Micropumps; Microwells; Single-cell analysis
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-11664DOI: 10.1002/elps.200900572ISI: 000273187800002PubMedID: 19938185Scopus ID: 2-s2.0-72749110928OAI: oai:DiVA.org:kth-11664DiVA: diva2:279106
Note
QC 20100728Available from: 2009-12-01 Created: 2009-12-01 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Microwell devices for single-cell analyses
Open this publication in new window or tab >>Microwell devices for single-cell analyses
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Powerful tools for detailed cellular studies are emerging, increasing the knowledge ofthe ultimate target of all drugs: the living cell. Today, cells are commonly analyzed inensembles, i.e. thousands of cells per sample, yielding results on the average responseof the cells. However, cellular heterogeneity implies the importance of studying howindividual cells respond, one by one, in order to learn more about drug targeting andcellular behavior. In vitro assays offering low volume sampling and rapid analysis in ahigh-throughput manner are of great interest in a wide range of single-cellapplications.

This work presents a microwell device in silicon and glass, developed using standardmicrofabrication techniques. The chip was designed to allow flow-cytometric cellsorting, a controlled way of analyzing and sorting individual cells for dynamic cultureand clone formation, previously shown in larger multiwell plates only. Dependent onthe application, minor modifications to the original device were made resulting in agroup of microwell devices suitable for various applications. Leukemic cancer cellswere analyzed with regard to their clonogenic properties and a method forinvestigation of drug response of critical importance to predict long-term clinicaloutcome, is presented. Stem cells from human and mouse were maintainedpluripotent in a screening assay, also shown useful in studies on neural differentiation.For integrated liquid handling, a fluidic system was integrated onto the chip fordirected and controlled addition of reagents in various cell-based assays. The chip wasproduced in a slide format and used as an imaging tool for low-volume sampling withthe ability to run many samples in parallel, demonstrated in a protein-binding assay fora novel bispecific affinity protein. Moving from cells and proteins into geneticanalysis, a method for screening genes from clones in a rapid manner was shown bygene amplification and mutation analysis in individual wells. In summary, a microwelldevice with associated methods were developed and applied in a range of biologicalinvestigations, particularly interesting from a cell-heterogeneity perspective.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. xii, 80 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:23
Keyword
microwell, miniaturization, microfluidics, cell culture, single-cell, clone, imaging, stem cell, cancer, low volume, high-throughput
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-11665 (URN)978-91-7415-477-1 (ISBN)
Public defence
2009-12-11, FR4 Oscar Klein, AlbaNova, Roslagstullsbacken, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20100728Available from: 2009-12-01 Created: 2009-12-01 Last updated: 2011-11-23Bibliographically approved

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Publisher's full textPubMedScopushttp://www3.interscience.wiley.com/journal/123191070/abstract

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