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PCR amplification and genetic analysis in a microwell cell culturing chip
KTH, Skolan för bioteknologi (BIO), Nanobioteknologi.
KTH, Skolan för bioteknologi (BIO), Nanobioteknologi.
KTH, Skolan för bioteknologi (BIO), Nanobioteknologi.
KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Cellens fysik.ORCID-id: 0000-0003-0578-4003
Visa övriga samt affilieringar
2009 (Engelska)Ingår i: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, s. 3465-3471Artikel i tidskrift (Refereegranskat) 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

Ort, förlag, år, upplaga, sidor
2009. s. 3465-3471
Nyckelord [en]
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
Nationell ämneskategori
Industriell bioteknik
Identifikatorer
URN: urn:nbn:se:kth:diva-11663DOI: 10.1039/b912596eISI: 000272142200001PubMedID: 20024024Scopus ID: 2-s2.0-72849112298OAI: oai:DiVA.org:kth-11663DiVA, id: diva2:279102
Anmärkning
QC 20100728Tillgänglig från: 2009-12-01 Skapad: 2009-12-01 Senast uppdaterad: 2017-12-12Bibliografiskt granskad
Ingår i avhandling
1. Microwell devices for single-cell analyses
Öppna denna publikation i ny flik eller fönster >>Microwell devices for single-cell analyses
2009 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH, 2009. s. xii, 80
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2009:23
Nyckelord
microwell, miniaturization, microfluidics, cell culture, single-cell, clone, imaging, stem cell, cancer, low volume, high-throughput
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:kth:diva-11665 (URN)978-91-7415-477-1 (ISBN)
Disputation
2009-12-11, FR4 Oscar Klein, AlbaNova, Roslagstullsbacken, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Anmärkning
QC 20100728Tillgänglig från: 2009-12-01 Skapad: 2009-12-01 Senast uppdaterad: 2011-11-23Bibliografiskt granskad

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Förlagets fulltextPubMedScopushttp://www.rsc.org/publishing/journals/LC/article.asp?doi=b912596e

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Brismar, Hjalmar

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Lindström, SaraHammond, MariaAndersson-Svahn, HeleneBrismar, HjalmarAhmadian, Afshin
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