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Towards high-throughput single cell/clone cultivation and analysis
KTH, School of Biotechnology (BIO), Nano Biotechnology. (Proteomics)
KTH, School of Biotechnology (BIO), Nano Biotechnology.
2008 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 29, 1219-1227 p.Article in journal (Refereed) Published
Abstract [en]

In order to better understand cellular processes and behavior, a controlled way of studying high numbers of single cells and their clone formation is greatly needed. Numerous ways of ordering single cells into arrays have previously been described, but platforms in which each cell/clone can be addressed to an exact position in the microplate, cultivated for weeks and treated separately in a high-throughput manner have until now been missing. Here, a novel microplate developed for high-throughput single cell/clone cultivation and analysis is presented. Rapid single cell seeding into microwells, using conventional flow cytometry, allows several thousands of single cells to be cultivated, short-term (72 h) or long-term (10-14 days), and analyzed individually. By controlled sorting of individual cells to predefined locations in the microplate, analysis of single cell heterogeneity and clonogenic properties related to drug sensitivity can be accomplished. Additionally, the platform requires remarkably low number of cells, a major advantage when screening limited amounts of patient cell samples. By seeding single cells into the microplate it is possible to analyze the cells for over 14 generations, ending up with more than 10 000 cells in each well. Described here is a proof-of-concept on compartmentalization and cultivation of thousands of individual cells enabling heterogeneity analysis of various cells/clones and their response to different drugs.

Place, publisher, year, edition, pages
2008. Vol. 29, 1219-1227 p.
Keyword [en]
clonal analysis; high-throughput microplate; leukaemia; single cell clone analysis; single cell cultivation array
National Category
Industrial Biotechnology
URN: urn:nbn:se:kth:diva-11660DOI: 10.1002/elps.200700536ISI: 000254882800003ScopusID: 2-s2.0-41549162810OAI: diva2:279086
QC 20100728Available from: 2009-12-01 Created: 2009-12-01 Last updated: 2010-12-06Bibliographically 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.
Trita-BIO-Report, ISSN 1654-2312 ; 2009:23
microwell, miniaturization, microfluidics, cell culture, single-cell, clone, imaging, stem cell, cancer, low volume, high-throughput
National Category
Biochemistry and Molecular Biology
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)
QC 20100728Available from: 2009-12-01 Created: 2009-12-01 Last updated: 2011-11-23Bibliographically approved

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