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Microwell devices for single-cell analyses
KTH, School of Biotechnology (BIO), Nano Biotechnology. (Proteomics)
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 [en]
microwell, miniaturization, microfluidics, cell culture, single-cell, clone, imaging, stem cell, cancer, low volume, high-throughput
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-11665ISBN: 978-91-7415-477-1 (print)OAI: oai:DiVA.org:kth-11665DiVA: diva2:279114
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
List of papers
1. Towards high-throughput single cell/clone cultivation and analysis
Open this publication in new window or tab >>Towards high-throughput single cell/clone cultivation and analysis
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.

Keyword
clonal analysis; high-throughput microplate; leukaemia; single cell clone analysis; single cell cultivation array
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11660 (URN)10.1002/elps.200700536 (DOI)000254882800003 ()2-s2.0-41549162810 (Scopus ID)
Note
QC 20100728Available from: 2009-12-01 Created: 2009-12-01 Last updated: 2017-12-12Bibliographically approved
2. High-Density Microwell Chip for Culture and Analysis of Stem Cells
Open this publication in new window or tab >>High-Density Microwell Chip for Culture and Analysis of Stem Cells
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2009 (English)In: PLos ONE, ISSN 1932-6203, Vol. 4, no 9, e6997- p.Article in journal (Refereed) Published
Abstract [en]

With recent findings on the role of reprogramming factors on stem cells, in vitro screening assays for studying (de)differentiation is of great interest. We developed a miniaturized stem cell screening chip that is easily accessible and provides means of rapidly studying thousands of individual stem/progenitor cell samples, using low reagent volumes. For example, screening of 700,000 substances would take less than two days, using this platform combined with a conventional bio-imaging system. The microwell chip has standard slide format and consists of 672 wells in total. Each well holds 500 nl, a volume small enough to drastically decrease reagent costs but large enough to allow utilization of standard laboratory equipment. Results presented here include weeklong culturing and differentiation assays of mouse embryonic stem cells, mouse adult neural stem cells, and human embryonic stem cells. The possibility to either maintain the cells as stem/progenitor cells or to study cell differentiation of stem/progenitor cells over time is demonstrated. Clonality is critical for stem cell research, and was accomplished in the microwell chips by isolation and clonal analysis of single mouse embryonic stem cells using flow cytometric cell-sorting. Protocols for practical handling of the microwell chips are presented, describing a rapid and user-friendly method for the simultaneous study of thousands of stem cell cultures in small microwells. This microwell chip has high potential for a wide range of applications, for example directed differentiation assays and screening of reprogramming factors, opening up considerable opportunities in the stem cell field.

Keyword
animal cell; article; biochip; cell cloning; cell culture; cell differentiation; cell screening; cell selection; controlled study; embryo; embryonic stem cell; female; flow cytometry; human; human cell; microwell chip; molecular imaging; mouse; neural stem cell; nonhuman; stem cell; animal; C57BL mouse; cell separation; culture technique; cytology; DNA microarray; equipment design; metabolism; methodology; nerve cell
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11661 (URN)10.1371/journal.pone.0006997 (DOI)000269796300013 ()2-s2.0-70349206057 (Scopus ID)
Note
QC 20100728Available from: 2009-12-01 Created: 2009-12-01 Last updated: 2011-10-31Bibliographically approved
3. Engineering and characterization of a bispecific HER2 × EGFR-binding affibody molecule
Open this publication in new window or tab >>Engineering and characterization of a bispecific HER2 × EGFR-binding affibody molecule
Show others...
2009 (English)In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 54, 121-131 p.Article in journal (Refereed) Published
Abstract [en]

HER2 (human epidermal-growth-factor receptor-2; ErbB2) and EGFR (epidermal-growth-factor receptor) are overexpressed in various forms of cancer, and the co-expression of both HER2 and EGFR has been reported in a number of studies. The simultaneous targeting of HER2 and EGFR has been discussed as a strategy with which to potentially increase efficiency and selectivity in molecular imaging and therapy of certain cancers. In an effort to generate a molecule capable of bispecifically targeting HER2 and EGFR, a gene fragment encoding a bivalent HER2-binding affibody molecule was genetically fused in-frame with a bivalent EGFR-binding affibody molecule via a (G(4)S)(3) [(Gly(4)-Ser)(3)]-encoding gene fragment. The encoded 30 kDa affibody construct (Z(HER2))(2)-(G(4)S)(3)-(Z(EGFR))(2), with potential for bs (bispecific) binding to HER2 and EGFR, was expressed in Escherichia coli and characterized in terms of its binding capabilities. The retained ability to bind HER2 and EGFR separately was demonstrated using both biosensor technology and flow-cytometric analysis, the latter using HER2- and EGFR-overexpressing cells. Furthermore, simultaneous binding to HER2 and EGFR was demonstrated in: (i) a sandwich format employing real-time biospecific interaction analysis where the bs affibody molecule bound immobilized EGFR and soluble HER2; (ii) immunofluorescence microscopy, where the bs affibody molecule bound EGFR-overexpressing cells and soluble HER2; and (iii) a cell-cell interaction analysis where the bs affibody molecule bound HER2-overexpressing SKBR-3 cells and EGFR-overexpressing A-431 cells. This is, to our knowledge, the first reported bs affinity protein with potential ability for the simultaneous targeting of HER2 and EGFR. The potential future use of this and similar constructs, capable of bs targeting of receptors to increase the efficacy and selectivity in imaging and therapy, is discussed.

Keyword
bispecifc (bs) affibody molecule; human epidermal-growth-factor receptor-3 (ErbB); microwell array; protein engineering; tumour targeting
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11662 (URN)10.1042/BA20090096 (DOI)000270769000006 ()2-s2.0-70350561680 (Scopus ID)
Note
QC 20100728Available from: 2009-12-01 Created: 2009-12-01 Last updated: 2017-12-12Bibliographically approved
4. PCR amplification and genetic analysis in a microwell cell culturing chip
Open this publication in new window or tab >>PCR amplification and genetic analysis in a microwell cell culturing chip
Show others...
2009 (English)In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, 3465-3471 p.Article in journal (Refereed) Published
Abstract [en]

We have previously described a microwell chip designed for high throughput, long-term single-cell culturing and clonal analysis in individual wells providing a controlled way of studying high numbers of individual adherent or non-adherent cells. Here we present a method for the genetic analysis of cells cultured on-chip by PCR and minisequencing, demonstrated using two human adherent cell lines: one wild type and one with a single-base mutation in the p53 gene. Five wild type or mutated cells were seeded per well (in a defined set of wells, each holding 500 nL of culture medium) in a 672-microwell chip. The cell chip was incubated overnight, or cultured for up to five days, depending on the desired colony size, after which the cells were lysed and subjected to PCR directly in the wells. PCR products were detected, in the wells, using a biotinylated primer and a fluorescently labelled primer, allowing the products to be captured on streptavidin-coated magnetic beads and detected by a fluorescence microscope. In addition, to enable genetic analysis by minisequencing, the double-stranded PCR products were denatured and the immobilized strands were kept in the wells by applying a magnetic field from the bottom of the wells while the wells were washed, a minisequencing reaction mixture was added, and after incubation in appropriate conditions the expected genotypes were detected in the investigated microwells, simultaneously, by an array scanner. We anticipate that the technique could be used in mutation frequency screening, providing the ability to correlate cells' proliferative heterogeneity to their genetic heterogeneity, in hundreds of samples simultaneously. The presented method of single-cell culture and DNA amplification thus offers a potentially powerful alternative to single-cell PCR, with advantageous robustness and sensitivity

Keyword
adherent cell; article; biotinylation; controlled study; culture medium; fluorescence microscopy; gene mutation; genetic heterogeneity; genetic screening; genotype; human; human cell; lab on a chip; magnetic field; magnetism; mutation rate; polymerase chain reaction; priority journal; sequence analysis; tumor suppressor gene; wild type
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11663 (URN)10.1039/b912596e (DOI)000272142200001 ()20024024 (PubMedID)2-s2.0-72849112298 (Scopus ID)
Note
QC 20100728Available from: 2009-12-01 Created: 2009-12-01 Last updated: 2017-12-12Bibliographically approved
5. A microwell array device with integrated microfluidic components for enhanced single-cell analysis
Open this publication in new window or tab >>A microwell array device with integrated microfluidic components for enhanced single-cell analysis
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.

Keyword
Cell culture; Microfluidics; Micropumps; Microwells; Single-cell analysis
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11664 (URN)10.1002/elps.200900572 (DOI)000273187800002 ()19938185 (PubMedID)2-s2.0-72749110928 (Scopus ID)
Note
QC 20100728Available from: 2009-12-01 Created: 2009-12-01 Last updated: 2017-12-12Bibliographically approved

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