Biomedical subjects

M Dufva

Publications and source records attributed to M Dufva.

6 recordsLinked to original sources

PubMed2006

A biocompatible micro cell culture chamber (microCCC) for the culturing and on-line monitoring of eukaryote cells.

We have previously shown that a polymeric (PMMA) chip with medium perfusion and integrated heat regulation provides sufficiently precise heat regulation, pH-control and medium exchange to support cell growth for weeks. However, it was unclear how closely the cells cultured in the chip resembled cells cultured in the culture flask. In the current study, gene expression profiles of cells cultured in the chip were compared with gene expression profiles of cells cultured in culture flasks. The results showed that there were only two genes that were differently expressed in cells grown in the cell culture chip compared to cell culture flasks. The cell culture chip could without further modification support cell growth of two other cell lines. Light coming from the microscope lamp during optical recordings of the cells was the only external factor identified, that could have a negative effect on cell survival. Low grade light exposure was however compatible with optical recordings as well as cell viability. These results strongly indicate that a cell culture chip could be constructed that allowed for on-line optical recording of cellular events without affecting the cell culturing condition compared to cell cultured in culture flasks incubated in a dark and CO2 conditioned incubator.

Caco-2 Cells↗

PubMed2006

Whole genome expression profiling using DNA microarray for determining biocompatibility of polymeric surfaces.

There is an ever increasing need to find surfaces that are biocompatible for applications like medical implants and microfluidics-based cell culture systems. The biocompatibility of five different surfaces with different hydrophobicity was determined using gene expression profiling as well as more conventional methods to determine biocompatibility such as cellular growth rate, morphology and the hydrophobicity of the surfaces. HeLa cells grown on polymethylmethacrylate (PMMA) or a SU-8 surface treated with HNO3-ceric ammonium nitrate (HNO3-CAN) and ethanolamine showed no differences in growth rate, morphology or gene expression profiles as compared to HeLa cells grown in cell culture flasks. Cells grown on SU-8 treated with only HNO3-CAN showed almost the same growth rate (36 +/- 1 h) and similar morphology as cells grown in cell culture flasks (32 +/- 1 h), indicating good biocompatibility. However, more than 200 genes showed different expression levels in cells grown on SU-8 treated with HNO3-CAN compared to cells grown in cell culture flasks. This shows that gene expression profiling is a simple and precise method for determining differences in cells grown on different surfaces that are otherwise difficult to find using conventional methods. It is particularly noteworthy that no correlation was found between surface hydrophobicity and biocompatibility.

Biocompatible Materials↗

PubMed2005

On-chip magnetic bead microarray using hydrodynamic focusing in a passive magnetic separator.

Implementing DNA and protein microarrays into lab-on-a-chip systems can be problematic since these are sensitive to heat and strong chemicals. Here, we describe the functionalization of a microchannel with two types of magnetic beads using hydrodynamic focusing combined with a passive magnetic separator with arrays of soft magnetic elements. The soft magnetic elements placed on both sides of the channel are magnetized by a relatively weak applied external magnetic field (21 mT) and provide magnetic field gradients attracting magnetic beads. Flows with two differently functionalized magnetic beads and a separating barrier flow are introduced simultaneously at the two channel sides and the centre of the microfluidic channel, respectively. On-chip experiments with fluorescence labeled beads demonstrate that the two types of beads are captured at each of the channel sidewalls. On-chip hybridization experiments show that the microfluidic systems can be functionalized with two sets of beads carrying different probes that selectively recognize a single base pair mismatch in target DNA. By switching the places of the two types of beads it is shown that the microsystem can be cleaned and functionalized repeatedly with different beads with no cross-talk between experiments.

Magnetics↗

PubMed2004

One-step immobilization of aminated and thiolated DNA onto poly(methylmethacrylate) (PMMA) substrates.

Direct immobilisation of modified DNA oligonucleotides (aminated or thiolated) onto a plastic substrate, poly(methylmethacrylate), (PMMA) is described. Using the methyl esters present on non-modified PMMA, it was possible to establish a covalent bond between the electron donor of a DNA probe and the C terminal ester of the PMMA substrate. Since the procedure consists of a single brief wash in isopropanol or ethanol, the procedure is simple and environmentally friendly. The new immobilization strategy was characterized by analysing DNA microarray performance. The new procedure resulted in probe- and hybridization densities that were greater or equivalent to those obtained with commercially available surfaces and other procedures to immobilize DNA onto PMMA. The described chemistry selectively immobilized the DNA via terminal thiol or amine groups indicating that probe orientation could be controlled. Furthermore, the chemical bond between the immobilized DNA and the PMMA could endure repeated heat cycling with only 50% probe loss after 20 cycles, indicating that the chemistry could be used in integrated PCR/microarray devices.

Amines↗

PubMed2004

Functionalization of poly(methyl methacrylate) (PMMA) as a substrate for DNA microarrays.

A chemical procedure was developed to functionalize poly(methyl methacrylate) (PMMA) substrates. PMMA is reacted with hexamethylene diamine to yield an aminated surface for immobilizing DNA in microarrays. The density of primary NH2 groups was 0.29 nmol/cm2. The availability of these primary amines was confirmed by the immobilization of DNA probes and hybridization with a complementary DNA strand. The hybridization signal and the hybridization efficiency of the chemically aminated PMMA slides were comparable to the hybridization signal and the hybridization efficiency obtained from differently chemically modified PMMA slides, silanized glass, commercial silylated glass and commercial plastic Euray trade mark slides. Immobilized and hybridized densities of 10 and 0.75 pmol/cm2, respectively, were observed for microarrays on chemically aminated PMMA. The immobilized probes were heat stable since the hybridization performance of microarrays subjected to 20 PCR heat cycles was only reduced by 4%. In conclusion, this new strategy to modify PMMA provides a robust procedure to immobilize DNA, which is a very useful substrate for fabricating single use diagnostics devices with integrated functions, like sample preparation, treatment and detection using microfabrication and microelectronic techniques.

Base Sequence↗

PubMed1995

Differential regulation of macrophage scavenger receptor isoforms: mRNA quantification using the polymerase chain reaction.

There are two isoforms of the macrophage scavenger receptor (MSR I and II). Both are expressed on macrophages and mediate internalization of oxidized lipoproteins and several other ligands. MSR expression is regulated by cytokines but the individual regulation of each isoform is not well documented. We have therefore developed a PCR method to quantify mRNA levels of MSR isoforms. The analysis is based on relating the amount of reverse transcribed and amplified human macrophage MSR transcripts to a synthetic internal standard, using a 32P-labeled 5'-primer to allow quantitation of the products. Each MSR isoform and its corresponding standard amplified with equal efficiency and the amount of MSR mRNA could be determined from 1 to 100 ng of total RNA. Using this method, we estimated that each monocyte-derived macrophage contains 10-130 molecules of MSR I and 30-640 copies of MSR II mRNA. Both isoforms were down-regulated by bacterial endotoxin (LPS), but the effect was more pronounced for MSR II transcripts. However, cycloheximide induced a selective degradation of MSR I transcripts, leaving MSR II levels unaltered. This suggests that both transcriptional and posttranscriptional control mechanisms are important in the regulation of MSR expression.

Base Sequence↗