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Biomedical subjects

Helene Andersson

Publications and source records attributed to Helene Andersson.

16 recordsLinked to original sources
PubMed2006

Rapid melting curve analysis on monolayered beads for high-throughput genotyping of single-nucleotide polymorphisms.

This report describes a rapid solid-phase melting curve analysis method for single-nucleotide polymorphism (SNP) genotyping. The melting curve analysis is based on dynamic allele-specific hybridization (DASH). The DNA duplexes are conjugated on beads that are immobilized on the surface of a microheater chip with integrated heaters and temperature sensors. SNP on PCR products were scored, illustrating the sensitivity and robustness of the system. The method is based on random bead immobilization by microcontact printing. Single-bead detection and multiplexing were performed with a heating rate more than 20 times faster than conventional DASH. Analyses that took more than 15 min could be performed in less that 1 min, enabling ultrarapid SNP analysis. In addition, an array version of the chip was implemented enabling the preparation of an array of bead arrays for high-throughput and rapid SNP genotyping.

Alleles↗
PubMed2006

Viability study of HL60 cells in contact with commonly used microchip materials.

This paper presents a study in which different commonly used microchip materials (silicon oxide, borosilicate glass, and PDMS) were analyzed for their effect on human promyelocytic leukemic (HL60) cells. Copper-coated silicon was analyzed for its toxicity and therefore served as a positive control. With quantitative PCR, the expression of the proliferation marker Cyclin D1 and the apoptosis marker tissue transglutaminase were measured. Flow cytometry was used to analyze the distribution through the different phases of the cell cycle (propidium iodide, PI) and the apoptotic cascade (Annexin V in combination with PI). All microchip materials, with the exception of Cu, appeared to be suitable for HL60 cells, showing a ratio apoptosis/proliferation (R(ap)) comparable to materials used in conventional cell culture (polystyrene). These results were confirmed with cell cycle analysis and apoptosis studies. Precoating the microchip material surfaces with serum favor the proliferation, as demonstrated by a lower R(ap) as compared to uncoated surfaces. The Cu-coated surface appeared to be toxic for HL60 cells, showing over 90% decreased viability within 24 h. From these results, it can be concluded that the chosen protocol is suitable for selection of the cell culture material, and that the most commonly used microchip materials are compatible with HL60 culturing.

Apoptosis↗
PubMed2005

Pyrosequencing in a microfluidic flow-through device.

To explore genome variation meaningfully, there is a critical need for a high-throughput and inexpensive platform for DNA analysis. Pyrosequencing is a nonelectrophoretic bioluminometric DNA sequencing method that uses a four-enzyme mixture reaction to monitor nucleotide incorporation in real time. Currently, the commercialized pyrosequencing technique is limited to a 96-microtiter plate format. However, high throughput and inexpensive pyrosequencing is required to meet the need of screening large numbers of samples. We present here DNA pyrosequencing on a nanoliter-volume microfluidic platform. The microfluidic approach involves the trapping of the DNA on microbeads in an on-chip filter chamber and flow-through of the pyrosequencing reagents to monitor the reaction in real time. Two single-nucleotide polymorphisms were successfully scored to evaluate the microfluidic platform. In addition to significantly reducing reagent costs, microfluidic systems promise to improve the read length by eliminating intermediate product accumulation by constant removal of unincorporated nucleotides and elimination of dilution effects at each reaction cycle in the current plate format. Although only one filter chamber was used in this study, the platform should be readily adaptable to parallel analyses of nanoliter samples using filter chamber arrays to obtain high-throughput DNA analysis.

Base Sequence↗
PubMed2005

A concept for miniaturized 3-D cell culture using an extracellular matrix gel.

This paper presents a novel method to embed, anchor, and cultivate cells in a controlled 3-D flow-through microenvironment. This is realized using an etched silicon pillar flow chamber filled with extracellular matrix (ECM) gel mixed with cells. At 4 degrees C, while in liquid form, ECM gel is mixed with cells and injected into the chamber. Raising the temperature to 37 degrees C results in a gel, with cells embedded. The silicon pillars both stabilize and increase the surface to volume ratio of the gel. During polymerization the gel shrinks, thus creating channels, which enables perfusion through the chip. The pillars increase the mechanical stability of the gel permitting high surface flow rates without surface modifications. Within the structure cells were still viable and proliferating after 6 days of cultivation. Our method thus makes it possible to perform medium- to long-term cultivation of cells in a controlled 3-D environment. This concept opens possibilities to perform studies of cells in a more physiological environment compared to traditional 2-D cultures on flat substrates.

Animals↗
PubMed2004

Apoptotic cell death dynamics of HL60 cells studied using a microfluidic cell trap device.

This paper presents the design, fabrication and first results of a microfluidic cell trap device for analysis of apoptosis. The microfluidic silicon-glass chip enables the immobilization of cells and real-time monitoring of the apoptotic process. Induction of apoptosis, either electric field mediated or chemically induced with tumour necrosis factor (TNF-alpha), in combination with cycloheximide (CHX), was addressed. Exposure of cells to the appropriate fluorescent dyes, FLICA and PI, allows one to discriminate between viable, apoptotic and necrotic cells. The results showed that the onset of apoptosis and the transitions during the course of the cell death cascade were followed in chemically induced apoptotic HL60 cells. For the case of electric field mediated cell death, the distinction between apoptotic and necrotic stage was not clear. This paper presents the first results to analyse programmed cell death dynamics using this apoptosis chip and a first step towards an integrated apoptosis chip for high-throughput drug screening on a single cellular level.

Apoptosis↗
PubMed2004

Microfabrication and microfluidics for tissue engineering: state of the art and future opportunities.

An introductory overview of the use of microfluidic devices for tissue engineering is presented. After a brief description of the background of tissue engineering, different application areas of microfluidic devices are examined. Among these are methods for patterning cells, topographical control over cells and tissues, and bioreactors. Examples where microfluidic devices have been employed are presented such as basal lamina, vascular tissue, liver, bone, cartilage and neurons. It is concluded that until today, microfluidic devices have not been used extensively in tissue engineering. Major contributions are expected in two areas. The first is growth of complex tissue, where microfluidic structures ensure a steady blood supply, thereby circumventing the well-known problem of providing larger tissue structures with a continuous flow of oxygen and nutrition, and withdrawal of waste products. The second, and probably more important function of microfluidics, combined with micro/nanotechnology, lies in the development of in vitro physiological systems for studying fundamental biological phenomena.

Animals↗
PubMed2004

Genotyping by dynamic heating of monolayered beads on a microheated surface.

A miniaturized bead-based dynamic allele-specific hybridization (DASH) approach for single-nucleotide polymorphism analysis is presented. Chips with integrated heater and temperature sensors for open-surface DNA analysis were microfabricated. Microcontact printing using a poly(dimethylsiloxane) (PDMS) stamp was employed to create monolayers of immobilized beads on the surface of the chip. This chip allows fast, well-controllable temperature ramping. The temperature distribution was homogeneous over the entire heater area. All three possible variants of an SNP site of a synthesized oligonucleotide were accurately scored using the bead-based DASH approach. Our assay has a nonoptimized temperature ramping rate of 4 degrees C-6 degrees C/min compared to earlier reported values of 2 degrees C-3 degrees C/min, thereby reducing the total analysis time by a factor of 2. Reliable DASH measurement data from areas as small as 12 x 13 microm was achieved. Our bead-based DASH approach has enabled a dramatic volume reduction and is a step towards developing a cost-effective high-throughput DASH method on arrays of single beads.

Alleles↗
PubMed2004

The potential of autofluorescence for the detection of single living cells for label-free cell sorting in microfluidic systems.

A novel method for studying unlabeled living mammalian cells based on their autofluorescence (AF) signal in a prototype microfluidic device is presented. When combined, cellular AF detection and microfluidic devices have the potential to facilitate high-throughput analysis of different cell populations. To demonstrate this, unlabeled cultured cells in microfluidic devices were excited with a 488 nm excitation light and the AF emission (> 505 nm) was detected using a confocal fluorescence microscope (CFM). For example, a simple microfluidic three-port glass microstructure was used together with conventional electroosmotic flow (EOF) to switch the direction of the fluid flow. As a means to test the potential of AF-based cell sorting in this microfluidic device, granulocytes were successfully differentiated from human red blood cells (RBCs) based on differences in AF. This study demonstrated the use of a simple microfabricated device to perform high-throughput live cell detection and differentiation without the need for cell-specific fluorescent labeling dyes and thereby reducing the sample preparation time. Hence, the combined use of microfluidic devices and cell AF may have many applications in single-cell analysis.

Equipment Design↗
PubMed2004

Microtechnologies and nanotechnologies for single-cell analysis.

Many efforts are currently underway to try and mimic the properties of single cells with the aim of designing chips that are as efficient as cells. However, cells are nature's nanotechnology engineering at the scale of atoms and molecules, and it might be better to envision a microchip that utilizes a single cell as an experimentation platform. A novel, so-called laboratory-in-a-cell concept has been described, where advantage is taken of micro- and nanotechnological tools to enable precise control of the biochemical cellular environment; these tools also offer the possibility to analyse the composition of single cells. Methods for single-cell handling and analysis are being developed and will be required for this concept to progress further.

Animals↗
PubMed2003

Single nucleotide polymorphism analysis by allele-specific primer extension with real-time bioluminescence detection in a microfluidic device.

A microfluidic approach for rapid bioluminescent real-time detection of single nucleotide polymorphism (SNP) is presented. The method is based on single-step primer extension using pyrosequencing chemistry to monitor nucleotide incorporations in real-time. The method takes advantage of the fact that the reaction kinetics differ between matched and mismatched primer-template configurations. We show here that monitoring the initial reaction in real time accurately scores SNPs by comparing the initial reaction kinetics between matched and mismatched configurations. Thus, no additional treatment is required to improve the sequence specificity of the extension, which has been the case for many allele-specific extension assays. The microfluidic approach was evaluated using four SNPs. Three of the SNPs included primer-template configurations that have been previously reported to be difficult to resolve by allele-specific primer extension. All SNPs investigated were successfully scored. Using the microfluidic device, the volume for the bioluminescent assay was reduced dramatically, thus offering a cost-effective and fast SNP analysis method.

Alleles↗
PubMed2003

Single-nucleotide polymorphism analysis by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device.

We describe a microfluidic approach for allele-specific extension of fluorescently labeled nucleotides for scoring of single-nucleotide polymorphism (SNP). The method takes advantage of the fact that the reaction kinetics differs between matched and mismatched configurations of allele-specific primers hybridized to DNA template. A microfluidic flow-through device for biochemical reactions on beads was used to take advantage of the reaction kinetics to increase the sequence specificity of the DNA polymerase, discriminating mismatched configurations from matched. The volume of the reaction chamber was 12.5 nL. All three possible variants of an SNP site at codon 72 of the p53 gene were scored using our approach. This work demonstrates the possibility of scoring SNP by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device. The sensitive detection system and easy microfabrication of the microfluidic device enable further miniaturization and production of an array format of microfluidic devices for high-throughput SNP analysis.

Alleles↗
PubMed2003

Prevalence of dyslipidemic risk factors in hemodialysis and CAPD patients.

BACKGROUND: Dyslipidemic factors obviously contribute to the high cardiovascular risk in dialysis patients but are often an underestimated problem. Therefore, we determined the prevalence of dyslipidemic factors in a large group of unselected hemodialysis (N = 564) and CAPD (N = 168) patients. METHODS: We used the recently published recommendations of the Medical Experts Group concerning cardiovascular risk factors for the categorization of dyslipidemic factors. These were total cholesterol>200 mg/dL, low-density lipoprotein (LDL) cholesterol>100 mg/dL, high-density lipoprotein (HDL) cholesterol <40 mg/dL, triglycerides>180 mg/dL, and Lp(a)>30 mg/dL. RESULTS: CAPD patients had, in sum, a markedly worse lipid profile when compared with HD patients. They had higher frequencies of elevated total cholesterol (67% vs. 34%), triglycerides (47% vs. 28%), and Lp(a) concentrations (37% vs. 30%) when compared with HD patients. In both patient groups, about two thirds of the patients had LDL cholesterol above 100 mg/dL and HDL cholesterol below 40 mg/dL. When we analyzed the total frequency of dyslipidemic factors, we observed that the CAPD group included a markedly higher number of patients with three or four concurrent dyslipidemic factors than HD patients (P < 0.001). Furthermore, we analyzed apolipoprotein A-IV (apoA-IV), which was recently shown to be associated with cardiovascular disease, and which was about twice as high in both patient groups when compared with controls (P < 0.001). CONCLUSIONS: Dyslipidemic risk factors are highly prevalent in dialysis patients, and the concomitant occurrence of several risk factors in a given patient is more often observed in CAPD than HD patients.

Adult↗
PubMed2003

Molecular fluorescence excitation-emission matrices relevant to tissue spectroscopy.

In vivo and ex vivo studies of fluorescence from endogenous and exogenous molecules in tissues and cells are common for applications such as detection or characterization of early disease. A systematic determination of the excitation-emission matrices (EEM) of known and putative endogenous fluorophores and a number of exogenous fluorescent photodynamic therapy drugs has been performed in solution. The excitation wavelength range was 250-520 nm, with fluorescence emission spectra collected in the range 260-750 nm. In addition, EEM of intact normal and adenomatous human colon tissues are presented as an example of the relationship to the EEM of constituent fluorophores and illustrating the effects of tissue chromophore absorption. As a means to make this large quantity of spectral data generally available, an interactive database has been developed. This currently includes EEM and also absorption spectra of 35 different endogenous and exogenous fluorophores and chromophores and six photosensitizing agents. It is intended to maintain and extend this database in the public domain, accessible through the Photochemistry and Photobiology website (http://www.aspjournal. com/).

Colon↗
PubMed2002

Expandable microspheres for the handling of liquids.

Two novel concepts for controlled handling of liquids in microfluidic systems are presented: a one-shot micropump and a normally open one-shot valve based on thermo-expanding Expancel microspheres. Expancel microspheres are small spherical plastic particles that, when heated, increase their volume considerably. We show that liquid volumes in the nanoliter range can be actuated against a counter pressure of at least 100 kPa and fluid flow can be inhibited in a microchannel against pressures of at least 100 kPa.

Journal Article↗