RESUMEN
The industrial production of cells has a large unmet need for greater process monitoring, in addition to the standard temperature, pH and oxygen concentration determination. Monitoring the cell health by a vast range of fluorescence cell-based assays can greatly improve the feedback control and thereby ensure optimal cell production, by prolonging the fermentation cycle and increasing the bioreactor output. In this work, we report on the development of a fully automated microfluidic system capable of extracting samples directly from a bioreactor, diluting the sample, staining the cells, and determining the total cell and dead cells concentrations, within a time frame of 10.3 min. The platform consists of custom made stepper motor actuated peristaltic pumps and valves, fluidic interconnections, sample to waste liquid management and image cytometry-based detection. The total concentration of cells is determined by brightfield microscopy, while fluorescence detection is used to detect propidium iodide stained non-viable cells. This method can be incorporated into facilities with bioreactors to monitor the cell concentration and viability during the cultivation process. Here, we demonstrate the microfluidic system performance by monitoring in real time the cell concentration and viability of yeast extracted directly from an in-house made bioreactor. This is the first demonstration of using the Dean drag force, generated due to the implementation of a curved microchannel geometry in conjunction with high flow rates, to promote passive mixing of cell samples and thus homogenization of the diluted cell plug. The autonomous operation of the fluidics furthermore allows implementation of intelligent protocols for administering air bubbles from the bioreactor in the microfluidic system, so that these will be guided away from the imaging region, thereby significantly improving both the robustness of the system and the quality of the data.
Asunto(s)
Dispositivos Laboratorio en un Chip , Saccharomyces cerevisiae/citología , Automatización , Reactores Biológicos/microbiología , Supervivencia Celular , Diseño de Equipo , Fermentación , Procesamiento de Imagen Asistido por Computador , Imagen Óptica , Saccharomyces cerevisiae/crecimiento & desarrollo , Factores de TiempoRESUMEN
We present the characterisation and validation of multiplexed 4-terminal (4T) impedance measurements as a method for sensing the spatial location of cell aggregates within large three-dimensional (3D) gelatin scaffolds. The measurements were performed using an array of four rectangular chambers, each having eight platinum needle electrodes for parallel analysis. The electrode positions for current injection and voltage measurements were optimised by means of finite element simulations to maximise the sensitivity field distribution and spatial resolution. Eight different 4T combinations were experimentally tested in terms of the spatial sensitivity. The simulated sensitivity fields were validated using objects (phantoms) with different conductivity and size placed in different positions inside the chamber. This provided the detection limit (volume sensitivity) of 16.5%, i.e. the smallest detectable volume with respect to the size of the measurement chamber. Furthermore, the possibility for quick single frequency analysis was demonstrated by finding a common frequency of 250 kHz for all the presented electrode combinations. As final proof of concept, a high density of human hepatoblastoma (HepG2) cells were encapsulated in gelatin to form artificial 3D cell constructs and detected when placed in different positions inside large gelatin scaffolds. Taken together, these results open new perspectives for impedance-based sensing technologies for non-invasive monitoring in tissue engineering applications providing spatial information of constructs within biologically relevant 3D environments.
Asunto(s)
Impedancia Eléctrica , Ingeniería de Tejidos , Tomografía/métodos , Técnicas de Cultivo de Célula , Electrodos , Gelatina/química , Células Hep G2 , Humanos , Tomografía/instrumentaciónRESUMEN
BACKGROUND: In pulmonary arterial hypertension (PAH), right ventricular (RV) failure is the main cause of mortality. Non-invasive estimation of ventricular-vascular coupling ratio (VVCR), describing contractile response to afterload, could be a valuable tool for monitoring clinical course in children with PAH. This study aimed to test two hypotheses: VVCR by cardiac magnetic resonance (VVCRCMR) correlates with conventional VVCR by right heart catheterization (VVCRRHC) and both correlate with disease severity. METHODS AND RESULTS: Twenty-seven patients diagnosed with idiopathic and associated PAH without post-tricuspid shunt, who underwent RHC and CMR within 17â¯days at two specialized centers for pediatric PAH were retrospectively studied. Clinical functional status and hemodynamic data were collected. Median age at time of MRI was 14.3â¯years (IQR: 11.1-16.8), median PVRi 7.6 WUâ¯×â¯m2 (IQR: 4.1-12.2), median mPAP 40â¯mmâ¯Hg (IQR: 28-55) and median WHO-FC 2 (IQR: 2-3). VVCRCMR, defined as stroke volume/end-systolic volume ratio was compared to VVCRRHC by single-beat pressure method using correlation and Bland-Altman plots. VVCRCMR and VVCRRHC showed a strong correlation (râ¯=â¯0.83, pâ¯<â¯0.001). VVCRCMR and VVCRRHC both correlated with clinical measures of disease severity (pulmonary vascular resistance index [PVRi], mean pulmonary artery pressure [mPAP], mean right atrial pressure [mRAP], and World Health Organization functional class [WHO-FC]; all pâ¯≤â¯0.02). CONCLUSIONS: Non-invasively measured VVCRCMR is feasible in pediatric PAH and comparable to invasively assessed VVCRRHC. Both correlate with functional and hemodynamic measures of disease severity. The role of VVCR assessed by CMR and RHC in clinical decision-making and follow-up in pediatric PAH warrants further clinical investigation.
Asunto(s)
Cateterismo Cardíaco/métodos , Imagen por Resonancia Cinemagnética/métodos , Hipertensión Arterial Pulmonar , Disfunción Ventricular Derecha , Cuidados Posteriores/métodos , Niño , Toma de Decisiones Clínicas , Investigación sobre la Eficacia Comparativa , Precisión de la Medición Dimensional , Femenino , Ventrículos Cardíacos/diagnóstico por imagen , Ventrículos Cardíacos/fisiopatología , Humanos , Masculino , Hipertensión Arterial Pulmonar/complicaciones , Hipertensión Arterial Pulmonar/diagnóstico , Hipertensión Arterial Pulmonar/fisiopatología , Índice de Severidad de la Enfermedad , Disfunción Ventricular Derecha/diagnóstico , Disfunción Ventricular Derecha/etiología , Disfunción Ventricular Derecha/fisiopatologíaRESUMEN
We present the use of magnetoresistive sensors integrated in a microfluidic system for real-time studies of the hybridization kinetics of DNA labeled with magnetic nanoparticles to an array of surface-tethered probes. The nanoparticles were magnetized by the magnetic field from the sensor current. A local negative reference ensured that only the specific binding signal was measured. Analysis of the real-time hybridization using a two-compartment model yielded both the association and dissociation constants kon, and koff. The effect of probe modifications with ortho-Twisted Intercalating Nucleic Acid (TINA) was studied. Such modifications have been demonstrated to increase the melting temperature of DNA hybrids in solution and are also relevant for surface-based DNA sensing. Kinetic data for DNA probes with no TINA modification or with TINA modifications at the 5' end (1 × TINA) or at both the 5' and 3' ends (2 × TINA) were compared. TINA modifications were found to provide a relative decrease of koff by a factor of 6-20 at temperatures from 57.5 °C to 60 °C. The values of kon were generally in the range between 0.5-2 × 105 M-1s-1 and showed lower values for the unmodified probe than for the TINA modified probes. The observations correlated well with measured melting temperatures of the DNA hybrids.
Asunto(s)
Técnicas Biosensibles/instrumentación , ADN/análisis , ADN/química , Sustancias Intercalantes/análisis , Sustancias Intercalantes/química , Magnetismo/instrumentación , Humanos , Cinética , Hibridación de Ácido Nucleico , Temperatura de TransiciónRESUMEN
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.
Asunto(s)
Técnicas de Cultivo de Célula , Técnicas Analíticas Microfluídicas , Células CACO-2 , Supervivencia Celular/fisiología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/fisiología , Células HeLa , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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.
Asunto(s)
Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Genoma Humano/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Polímeros/farmacología , División Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Etanolamina , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Polímeros/química , Propiedades de Superficie , Agua/químicaRESUMEN
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.
Asunto(s)
Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Polimetil Metacrilato/química , Secuencia de Bases , Sondas de ADN/genética , Sondas de ADN/metabolismo , Vidrio , Calor , Cinética , Hibridación de Ácido Nucleico , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Plásticos , Reacción en Cadena de la Polimerasa , SilanosRESUMEN
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.
Asunto(s)
Magnetismo/instrumentación , Análisis por Micromatrices/instrumentación , Análisis por Micromatrices/métodos , MicroesferasRESUMEN
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.
Asunto(s)
ADN/química , Análisis de Secuencia por Matrices de Oligonucleótidos/instrumentación , Polimetil Metacrilato/química , Aminas/química , Sondas de ADN/química , Plásticos/química , Compuestos de Sulfhidrilo/químicaRESUMEN
A modular microfluidic airways model system that can simulate the changes in oxygen tension in different compartments of the cystic fibrosis (CF) airways was designed, developed, and tested. The fully reconfigurable system composed of modules with different functionalities: multichannel peristaltic pumps, bubble traps, gas exchange chip, and cell culture chambers. We have successfully applied this system for studying the antibiotic therapy of Pseudomonas aeruginosa, the bacteria mainly responsible for morbidity and mortality in cystic fibrosis, in different oxygen environments. Furthermore, we have mimicked the bacterial reinoculation of the aerobic compartments (lower respiratory tract) from the anaerobic compartments (cystic fibrosis sinuses) following an antibiotic treatment. This effect is hypothesised as the one on the main reasons for recurrent lung infections in cystic fibrosis patients.
RESUMEN
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.