Diffusion-based extraction of DMSO from a cell suspension in a three stream, vertical microchannel

Cells are routinely cryopreserved for investigative and therapeutic applications. The most common cryoprotective agent (CPA), dimethyl sulfoxide (DMSO), is toxic, and must be removed before cells can be used. This study uses a microfluidic device in which three streams flow vertically in parallel through a rectangular channel 500 μm in depth. Two wash streams flow on either side of a DMSO-laden cell stream, allowing DMSO to diffuse into the wash and be removed, and the washed sample to be collected. The ability of the device to extract DMSO from a cell stream was investigated for sample flow rates from 0.5 to 4.0 mL/min (Pe = 1,263-10,100). Recovery of cells from the device was investigated using Jurkat cells (lymphoblasts) in suspensions ranging from 0.5% to 15% cells by volume. Cell recovery was >95% for all conditions investigated, while DMSO removal comparable to a previously developed two-stream device was achieved in either one-quarter the device length, or at four times the flow rate. The high cell recovery is a ~25% improvement over standard cell washing techniques, and high flow rates achieved are uncommon among microfluidic devices, allowing for processing of clinically relevant cell populations.     

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions¹. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions^2,3.The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.     

Towards the realization of label-free biosensors through impedance spectroscopy integrated with IDES technology

Impedance spectroscopy (IS) is a powerful technique for analysis of the complex electrical impedance of a large variety of biological systems, because it is sensitive both to surface phenomena and to changes of bulk properties. A simple and convenient method of analysis of cell properties by IS is described. An interdigitated electrodes configuration was used for the measurements; human epithelial cells were grown on the device to investigate the complex dielectric response as a function of frequency, in order to test the suitability of the device for use as a label-free biosensor. To test the ability of the device to detect channels in the cell membrane, the effect of drugs known to affect membrane integrity was also investigated. The frequency response of the admittance (i.e. the reciprocal of the impedance) can be well fitted by a model based on very simple assumptions about the cells coating the device surface and the current flow; from the calculations, membrane-specific capacitance and information about cell adhesion can be inferred. These preliminary efforts have shown that our configuration could lead to a label-free non-invasive technique for biosensing and cellular behavior monitoring which might prove useful in investigation of the basic properties of cells and the effect of drugs by estimation of some fundamental properties and modification of the electrical characteristics of the device.     

A microfluidic array device for single cell capture and intracellular Ca2+ response analysis induced by dynamic biochemical stimulus

A microfluidic array was constructed for trapping single cell and loading identical dynamic biochemical stimulation for gain a better understanding of Ca²⁺ signaling at single cell resolution in the present study. This microfluidic array consists of multiple radially aligned flow channels with equal intersection angles, which was designed by a combination of stagnation point flow and physical barrier. Numerical simulation results and trajectory analysis have shown the effectiveness of this single cell trapping device. Fluorescent experiment results demonstrated the effects of flow rate and frequency of dynamic stimulus on the profiles of biochemical concentration which exposed on captured cells. In this microarray, the captured single cells in each trapping channels were able to receive identical extracellular dynamic biochemical stimuli which being transmitted from the entrance in the middle of the microfluidic array. Besides, after loading dynamic Adenosine Triphosphate (ATP) stimulation on captured cells by this device, consistent average intracellular Ca²⁺ dynamics phase and cellular heterogeneity were observed in captured single K562 cells. Furthermore, this device is able to be used for investigating cellular respond on single cell resolution to temporally varying environments by modulating the stimulation signal in terms of concentration, pattern, and duration of exposure.     

Spatial and Temporal Control of Cavitation Allows High In Vitro Transfection Efficiency in the Absence of Transfection Reagents or Contrast Agents

Sonoporation using low-frequency high-pressure ultrasound (US) is a non-viral approach for in vitro and in vivo gene delivery. In this study, we developed a new sonoporation device designed for spatial and temporal control of ultrasound cavitation. The regulation system incorporated in the device allowed a real-time control of the cavitation level during sonoporation. This device was evaluated for the in vitro transfection efficiency of a plasmid coding for Green Fluorescent Protein (pEGFP-C1) in adherent and non-adherent cell lines. The transfection efficiency of the device was compared to those observed with lipofection and nucleofection methods. In both adherent and non-adherent cell lines, the sonoporation device allowed high rate of transfection of pEGFP-C1 (40–80%), as determined by flow cytometry analysis of GFP expression, along with a low rate of mortality assessed by propidium iodide staining. The transfection efficiency and toxicity of sonoporation on the non-adherent cell lines Jurkat and K562 were similar to those of nucleofection, while these two cell lines were resistant to transfection by lipofection. Moreover, sonoporation was used to produce three stably transfected human lymphoma and leukemia lines. Significant transfection efficiency was also observed in two fresh samples of human acute myeloid leukemia cells. In conclusion, we developed a user-friendly and cost-effective ultrasound device, well adapted for routine in vitro high-yield transfection experiments and which does not require the use of any transfection reagent or gas micro-bubbles.     

Influence of serum level,cell line,flow type and viscosity on flow-induced lysis of suspended mammalian cells

An investigation was conducted of the parametric dependence of cell lysis observed when mammalian cells growing in suspension are subjected intermittently to intense hydrodynamic forces. Two flow devices were tested: one consisting of a sudden contraction into a short length of capillary tubing, in which turbulent flow is obtained, and another consisting of a smoothly converging and diverging tube, in which laminar flow is obtained. Changes in the cell line and the serum level in which the cells were grown and subjected to flow trauma both affected the specific lysis rate (fraction of cells lysed per pass through the flow device) in the turbulent flow device. The threshold value of the average wall shear stress level was approximately the same in the turbulent and laminar flow devices (1500–1800 dyn/cm²). Increasing the viscosity of the medium with 70,000 MW dextran had no effect on the specific lysis rate in either flow device.     

A disposable and cost efficient microfluidic device for the rapid chip-based electrical detection of DNA

Requirements for a point-of-care device are an easy and robust read-out and – above all – a simple handling. We integrated an established robust electrical read-out for DNA-chips into a microfluidic device, thereby creating an automated analysis system that combines the necessary steps for a chip-based analysis. It is based on the electrical detection of biotin-labeled DNA in a gap between two microstructured electrodes on the surface of a DNA-chip. The biotin serves as binding molecule for streptavidin-conjugated horseradish peroxidase. A following enzyme-induced silver deposition bridges the gap by a conductive layer. The miniaturized chip gives the possibility to realize a durable system suitable for point-of-care applications.To enable an initial automation, all corresponding process steps were executed in a miniaturized silicone flow cell. The required defined temperatures for the hybridization and the washing steps can be adjusted by a heating foil.This paper characterizes the performance of the flow cell based system in terms of reaction speed and analysis time, sensitivity as well as specificity, and the comparison to a conventional system, without flow cell. These first steps of automation and integration will help to realize a laboratory-independent bioanalytical tool, for the use outside of specialized laboratories for fast analysis of different chemical and biological applications.     

Miniaturized device for agitating a high-density yeast suspension that is suitable for in vivo nuclear magnetic resonance applications

In vivo nuclear magnetic resonance (NMR) monitoring requires a high-density cell suspension, where cell precipitation should be avoided. We have designed a miniaturized cell agitator that fits entirely into an 8-mm NMR probe but that, being mounted into the instrument, is situated outside of the sensitive area. The device consists of two glass tubes connected in a way that, when gas flow is blown through them, creates influx of cell suspension into the device that returns through apertures. This flow creates continuous circular vortex of the cell suspension in the whole sample volume, whereas there are no moving mechanical parts or gas bubbles crossing the instrument’s sensitive area. The gas flow controls conditions of the cell suspension and removes volatile waste metabolites.     

An ultra scale-down method to investigate monoclonal antibody processing during tangential flow filtration using ultrafiltration membranes

The availability of material for experimental studies is a key constraint in the development of full-scale bioprocesses. This is especially true for the later stages in a bioprocess sequence such as purification and formulation, where the product is at a relatively high concentration and traditional scale-down models can require significant volumes. Using a combination of critical flow regime analysis, bioprocess modelling, and experimentation, ultra scale-down (USD) methods can yield bioprocess information using only millilitre quantities before embarking on highly demanding full-scale studies. In this study the performance of a pilot-scale tangential flow filtration (TFF) system based on a membrane flat-sheet cassette using pumped flow was predicted by devising an USD device comprising a stirred cell using a rotating disc. The USD device operates with just 2.1 cm² of membrane area and, for example, just 1.7 mL of feed for diafiltration studies. The novel features of the design involve optimisation of the disc location and the membrane configuration to yield an approximately uniform shear rate. This is characterised using computational fluid dynamics for a defined layer above the membrane surface. A pilot-scale TFF device operating at ~500-fold larger feed volume and membrane area was characterised in terms of the shear rate derived from flow rate-pressure drop relationships for the cassette. Good agreement was achieved between the USD and TFF devices for the flux and resistance values at equivalent average shear rates for a monoclonal antibody diafiltration stage.     

A single-cell surgery microfluidic device for transplanting tumor cytoplasm into dendritic cells without nuclei mixing

This study aimed to demonstrate the feasibility of generating tumor cell vaccine models by single-cell surgery in a microfluidic device that integrates one-to-one electrofusion, shear flow reseparation, and on-device culture. The device was microfabricated from polydimethylsiloxane (PDMS) and consisted of microorifices (aperture size: ∼3 μm) for one-to-one fusion, and microcages for on-device culture. Using the device, we could achieve one-to-one electrofusion of leukemic plasmacytoid dendritic cells (DC-like cells) and Jurkat cells with a fusion efficiency of ∼ 80%. Fusion via the narrow microorifices allowed DC-like cells to acquire cytoplasmic contents of the Jurkat cells while preventing nuclei mixing. After fusion, the DC-like cells were selectively reseparated from the Jurkat cells by shear flow application to generate tumor nuclei-free antigen-recipient DC-like (tarDC-like) cells. When cultured as single cells on the device, these cells could survive under gentle medium perfusion with a median survival time of 11.5 h, although a few cells could survive longer than 36 h. Overall, this study demonstrates single-cell surgery in a microfluidic device for potential generation of dendritic cell vaccines which are uncontaminated with tumor nucleic materials. We believe that this study will inspire the generation of safer tumor cell vaccines for cancer immunotherapy.     

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO₂ tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.     

On-line determination of biomass in a microalga bioreactor using a novel computerized flow injection analysis system

A flow injection analysis (FIA) device has been developed, which is able to assay successfully for biomass in a microalga bioreactor. The device is fully computerized and is operated via diluting small aliquots of the culture followed by measuring optical density (OD); this figure is then accurately correlated with biomass, in terms of both cell number and ash-free dry weight, during the entire culture time. Furthermore, the device is not expensive, is highly versatile, and is easy to operate owing to specifically developed, user-friendly software. The growth rate and biomass productivity of Pavlova lutheri, cultivated under batch and semicontinuous modes, were monitored as experimental testing model.     

Fast imaging in flow: a means of combining flow-cytometry and image analysis.

The morphological identification of cells by flow cytometry is difficult. Usually cell sorting and microscopical analysis have to be used in addition. Morphological analysis is simplified by taking cell pictures from a range of particular interest immediately during flow cytometric analysis. Instruments using the video scanning technique for fluorescence imaging are slow and expensive (8, 10). Morphological information can also be obtained by transmission imaging of cells in flow, which requires shorter exposure times. Therefore a cell volume activated flow imaging device has been developed which operates at flow speeds up to 5 m/sec and which depicts transmission images of selected cells on a 16-mm film by a nsec flashlamp illumination. An electronic unit detects the particles in the optically accessible orifice, performs the pulse height analysis, triggers the flashlamp if particles are in the preselcted range of interest and feeds the film. The instrument is capable of delivering up to 150 pictures per second and works either as a flow microscope in which the cells in the preselected volume range are directly observed, or as a picture system in which the cell pictures are stored on the 16-mm film for documentation or for image analysis.     

A new set up for multi-analyte sensing: at-line bio-process monitoring

A multi-analyte sensing device is described, for simultaneous at-line monitoring of glucose, ethanol, pO?-value and cell density. It consists of a dual biosensor, a modified microscope and a fiber optical pO?-sensor that are integrated into a flow analysis (FA) system. The biosensor is based on a conventional thin layer flow-through cell equipped with a gold (Au) dual electrode (serial configuration). The biosensors with no cross-talking were produced by modifying the electrochemical transducers. Each Au surface was initially modified by self-assembled monolayer (SAM) of cysteamine. Alcohol oxidase (AOx) and pyranose oxidase (PyOx) were immobilized each onto a gold surface by means of PAMAM (polyamidoamine) dendrimer via glutaraldehyde cross-linking. The responses for glucose and ethanol were linear up to 0.5 mM. The operational stability of the biosensors was very promising, after 11 h continuous operation, only 6.0% of the initial activity was lost. The potential of the described biosensor was demonstrated by parallel determination of ethanol and glucose in yeast fermentation process. Simultaneously the cell density of the culture was monitored with an in situ microscope (ISM), which was integrated into the FA system. Both the used in situ microscope and the image processing algorithm used for the analysis of the acquired image data are described. Furthermore the pO?-value was monitored using a fiber optical sensor, which was embedded in a flow cell. The multi-sensor device allows the at-line monitoring of several process values without the need for further sampling or time consuming offline measurements.     

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication,System Setup,and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g. cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.     

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.     

Small-volume rapid-mix device for subsecond kinetic analysis in flow cytometry.

BACKGROUND: Rapid-mix flow cytometry has emerged as a powerful tool for mechanistic analysis of ligand binding, cell response, and molecular assembly. Although progress has come from improving sample delivery capabilities, little attention has been paid to the volumetric requirements associated with precious biological reagents. METHODS: By using programmable syringes, valves, and other fluidic components, we created a modular, precisely regulated rapid-mix device for the delivery of small-volume samples to the flow cytometer. The device was tested using a bead-based assay in which the binding kinetics between native biotin and fluorescein biotin-bearing beads were characterized. RESULTS: Bead suspensions and reagents paired in 35- to 45-microl aliquots were efficiently mixed by the device and delivered to the flow cytometer. Kinetic data associated with the fluorescein biotin beads were analyzed and used to calibrate the performance characteristics of the device in terms of sample delivery and mixing efficiency. CONCLUSION: The rapid-mix device is capable of detecting subsecond kinetics of biological reactions using microliter volume of samples. Dimensions of the device have been minimized, and the quantitative aspects of sample delivery and analysis have been optimized. Further, the modular design has been optimized for adaptation to a variety of experimental protocols.     

Microdevice with integrated dialysis probe and biosensor array for continuous multi-analyte monitoring

The simultaneous on-line determination of glucose and lactate using a microdevice that consisted of a dialysis sampling system incorporated to the flow-through cell of a microfabricated biosensor array is presented. The fluidic connections between the different device's components were realized by subsequent processing of stacked dry resist layers on a plastic support that provided also the means for electric connections. The performance of the device was evaluated in vitro. The cross-talk effect on the downstream sensor was investigated and found to be negligible. Recoveries of over 95% for both analytes were achieved when flow rates of the perfusion fluid 相似文献   

Cell damage of microcarrier cultures as a function of local energy dissipation created by a rapid extensional flow

Microcarrier cultures of Chinese hamster ovary cells were subjected to a range of energy dissipations created by an abrupt contraction. These flow conditions can be characterized as a rapidly transient, extensional, and shear flow. Cell damage was measured using a lactate dehydrogenase assay. The laminar flow in the device was modeled using two commercial, computation fluid-dynamic codes: POLYFLOW and FLUENT. Cell damage was correlated to numerical values of energy dissipation. The magnitude of energy dissipation at which cell damage began to be detected, 10(4) ergs cm(-3) s(-1) (10(4) cm(2) s(-3)), is consistent with values of energy dissipation estimated in bioreactors operated under conditions which result in cell damage. This magnitude of energy dissipation is orders of magnitude lower than those values reported to cause damage to suspended animals cells which is also consistent with generally accepted experimental observations. Finally, an analysis and discussion of the presence and relative importance with re- spect to cell damage of shear vs. extensional flow is included.     

Considerations on the flow configuration of membrane chromatography devices for the purification of human basic fibroblast growth factor from crude lysates

Membrane chromatography possesses numerous advantages such as operation at high flow rates, low back pressure, ease of handling and scale up, which make the membrane adsorber process a viable alternative to conventional packed column chromatography. A purification process for the isolation of human recombinant basic fibroblast growth factor (FGF‐2) based on membrane chromatography was investigated using devices with different flow configurations. In the first process, the FGF‐2 capture step was performed with an axial flow device, while the alternative method achieved direct capture of FGF‐2 from unclarified cell lysate with a tangential flow device. In both processes, FGF‐2 purities exceeded 82% and the purified cytokine displayed high biological activity. Binding capacity (BC) from fermentation broth of the axial flow device was 28 mg/mL. This was 50% higher than the BC obtained with the tangential flow device under particle‐free supernatant conditions (18 mg/mL) and 150% higher compared to the BC achieved with unclarified cell lysate (11 mg/mL). While membrane chromatography in tangential flow mode omits clarification and thus reduces the number of stages in the downstream process, it displays lower peak resolution and leads to a lower overall process yield.