Continuous Cell Separator Research Articles

Improving an intensified and integrated continuous bioprocess platform for biologics manufacturing.

The WuXi Biologics' Ultra-high Productivity platform (WuXiUP) technology is an innovative and integrated platform of continuous biomanufacturing. Through process intensification, the platform enables continuous manufacturing of almost any type of biologics and delivers processes with ultra-high productivity. In this paper, a new case study producing a monoclonal antibody (mAb) via the WuXiUP process was further optimized. Key process parameters like culture temperature, basal media, and perfusion rate were evaluated to ensure an enhanced and robust process. To improve process efficiency for downstream processing, a continuous dual-pore size hollow fiber cell separation and product harvest system were also designed to complement the increased harvest volume from upstream production. In this case study, a significant protein concentration increase and harvest volume reduction were achieved by the application of the new WuXiUP platform.

A portable sperm cell purification instrument based on continuous flow acoustophoretic separation of sperm cells for on-site forensic sample pretreatment.

Obtaining a purified male sperm specimen from the original forensic sample mixtures is a critical procedure in identifying the criminal suspect in the forensic analysis of sexual assault crimes. Differential extraction (DE) has been used as the gold standard for extracting DNA preferentially from sexual assault samples, however it is laborious, time-consuming and inefficient. In this report a continuous flow two-step acoustic cell separation scheme is proposed for the removal of cell debris and cell-free genomic contaminants, followed by separation of sperm cells from epithelial cells. Based on the above method, a portable forensic sperm cell purification instrument has been constructed for on-site pretreatment of forensic samples in sexual assault crimes, where removal of contaminants and isolation of sperm cells could be automatically performed without the use of cell lysis buffer.

A Continuous Cell Separation and Collection Approach on a Microfilter and Negative Dielectrophoresis Combined Chip.

Cell separation plays an important role in the fields of analytical chemistry and biomedicine. To solve the blockage problem and improve the separation throughput in the traditional microstructure filtration-based separation approach, a continuous cell separation and collection approach via micropost array railing on a microfilter and negative dielectrophoresis combined chip is proposed. By tilting the micropost array at a certain angle, microparticles or cells enter the collection area under micropost array railing. The effects of the inclination angle of the micropost array and the electrode distance on the microparticle collection efficiency were investigated. Based on the optimized microfluidic chip structure, 37- and 16.3-μm particles were collected with 85% and 89% efficiencies, respectively. Additionally, algal cells were separated and collected by using the optimized microchip. The chip also had good separation and collection effects on biological samples, which effectively solved the blockage problem and improved the separation throughput, laying a foundation for subsequent microstructure filtration separation-based research and application.

Applied electric field analysis and numerical investigations of the continuous cell separation in a dielectrophoresis-based microfluidic channel

Dielectrophoresis (DEP) is known as an attractive and frugal technique to manipulate biological particles in microfluidics. This study presents the advanced solution strategy of a DEP-based microfluidic channel for focusing and separating cancerous cells in continuous flow. Theoretical calculations were carried out to define the favorable parameters in the electric field operation of the microchip. A simulation model was also used to explore the performance of the design in the isolation of circulating tumor cells (CTCs). It revealed that the optimal conditions of the device are suitable to effectively separate CTCs from red blood cells (RBCs) within the channel structure, with a high flow rate of 1.5 μL/min, and an electric amplitude as low as 10 Vpp, at the frequency of 1 kHz. The proposed method has shown potential as a simple, easy-to-operate, and low-cost approach enable to enhance the diagnosis systems for cancer detection at early stages.

Microfluidic Separation of Blood Cells Based on the Negative Dielectrophoresis Operated by Three Dimensional Microband Electrodes

A microfluidic device is presented for the continuous separation of red blood cells (RBCs) and white blood cells (WBCs) in a label-free manner based on negative dielectrophoresis (n-DEP). An alteration of the electric field, generated by pairs of slanted electrodes (separators) that is fabricated by covering parts of single slanted electrodes with an insulating layer is used to separate cells by their sizes. The repulsive force of n-DEP formed by slanted electrodes prepared on both the top and bottom substrates led to the deflection of the cell flow in lateral directions. The presence of gaps covered with an insulating layer for the electric field on the electrodes allows the passing of RBCs through gaps, while relatively large WBCs (cultured cultured human acute monocytic leukemia cell line (THP-1 cells)) flowed along the slanted separator without passing through the gaps and arrived at an edge in the channel. The passage efficiency for RBCs through the gaps and the arrival efficiency for THP-1 cells to the upper edge in the channel were estimated and found to be 91% and 93%, respectively.

Numerical investigation into continuous separation of particles and cells in a two-component fluid flow using dielectrophoresis

Separation of microparticles and cells from a heterogeneous mixture is critical in the fields of medicine, engineering, biology, and biotechnology. In this paper, a novel design for continuous separation of three particles/cells in a two-component fluid flow by dielectrophoresis, as the most efficient separation method, is discussed. A new solver is developed in OpenFOAM to simulate the separation of three polystyrene particles, which are only slightly different in size (2 μm, 3 μm, and 3.5 μm), in a circular channel containing a two-component fluid flow. Simulation results showed that for mean velocity U0 = 200 μm/s, electric potentials >25 V are required to separate particles efficiently. Then, separation of three biological cells including red blood cells, white blood cells, and MDA-MB-231 breast cancer cells is investigated. To achieve simultaneously two goals of separating the cells and preventing the two fluids to be mixed, it was found that the effective electric potential should be within the range of 9 V to 12 V.

Continuous Cell Separation Using Microfluidic-Based Cell Retention Device with Alternative Boosted Flow.

The development of a continuous process for cell separation is growing rapidly due to the current trend of cost-effective manufacturing in biological industries. The continuous cell separation process has a significant reduction in capital equipment costs and facility size compared to the conventional batch process. In the study, a multi-layered microfluidic-based device integrated with the porous membranes was fabricated for continuous size-based isolation of the cells based on the mechanism of restrictive cross-flow filtration, allowing the biological sample entered in a single inlet of the device and separated into two outlet streams. One stream which contained the cells returned back to the original sample fluid, while another stream with conditioned medium only was collected for later applications. The membrane fouling issue was overcome by introducing the alternative flow rate consisted of a set of higher and lower flows. The device integrated with the controllable flow restriction allows to increase the permeate flow rate, and alternative boosted flow demonstrates the high permeate flow rate (0.3mL/min), high cell viability (>98%), and increase of cell concentration (48%). As a result, we believe that the microfluidic-based continuous cell separation system is a promising tool for downstream bioprocess.

High-throughput continuous dielectrophoretic separation of neural stem cells.

We created an integrated microfluidic cell separation system that incorporates hydrophoresis and dielectrophoresis modules to facilitate high-throughput continuous cell separation. The hydrophoresis module consists of a serpentine channel with ridges and trenches to generate a diverging fluid flow that focuses cells into two streams along the channel edges. The dielectrophoresis module is composed of a chevron-shaped electrode array. Separation in the dielectrophoresis module is driven by inherent cell electrophysiological properties and does not require cell-type-specific labels. The chevron shape of the electrode array couples with fluid flow in the channel to enable continuous sorting of cells to increase throughput. We tested the new system with mouse neural stem cells since their electrophysiological properties reflect their differentiation capacity (e.g., whether they will differentiate into astrocytes or neurons). The goal of our experiments was to enrich astrocyte-biased cells. Sorting parameters were optimized for each batch of neural stem cells to ensure effective and consistent separations. The continuous sorting design of the device significantly improved sorting throughput and reproducibility. Sorting yielded two cell fractions, and we found that astrocyte-biased cells were enriched in one fraction and depleted from the other. This is an advantage of the new continuous sorting device over traditional dielectrophoresis-based sorting platforms that target a subset of cells for enrichment but do not provide a corresponding depleted population. The new microfluidic dielectrophoresis cell separation system improves label-free cell sorting by increasing throughput and delivering enriched and depleted cell subpopulations in a single sort.

Blood Particle Separation Using Dielectrophoresis in A Novel Microchannel: A Numerical Study.

Objective We present a four-branch model of the dielectrophoresis (DEP) method that takes into consideration the inherent properties of particles, including size, electrical conductivity, and permittivity coefficient. By using this model, bioparticles can be continuously separated by the application of only a one-stage separation process. Materials and Methods In this numerical study, we based the separation process on the differences in the particle sizes. We used the various negative DEP forces on the particles caused by the electrodes to separate them with a high efficiency. The particle separator could separate blood cells because of their different sizes. Results Blood cells greater than 12 μm were guided to a special branch, which improved separation efficiency because it prevented the deposition of particles in other branches. The designed device had the capability to separate blood cells with diameters of 2.0 μm, 6.2 μm, 10.0 μm, and greater than 12.0 μm. The applied voltage to the electrodes was 50 V with a frequency of 100 kHz. ConclusionThe proposed device is a simple, efficient DEP-based continuous cell separator. This capability makes it ideal for use in various biomedical applications, including cell therapy and cell separation, and results in a throughput increment of microfluidics devices.

Precise Size-Based Cell Separation via the Coupling of Inertial Microfluidics and Deterministic Lateral Displacement.

We report here a novel two-stage i-DLD sorter through coupling inertial microfluidics with deterministic lateral displacement (DLD), allowing for precise, continuous, and size-based cell separation. The first stage spiral inertial microfluidic sorter is responsible for removing the overwhelming majority of background blood cells at a high-throughput manner. The precise and flow-rate insensitive DLD sorter with triangular posts serves as the second stage sorter which further removes the residual blood cells for obtaining high-purity tumor cells. After demonstrating the conceptual design, we characterize the performances of our two-stage i-DLD sorter for the separation of differently sized particles and cells. The characterization results show that a 100% complete separation of 15 and 7 μm particles was achieved, whereas a separation efficiency of over 99.9% and a target sample purity of 93.59% was realized for the separation of differently sized cells. Finally, we successfully apply our sorter for the separation of rare tumor cells from the diluted whole blood or WBCs at good performances. Our two-stage i-DLD sorter offers numerous advantages of label- and external field-free operation, high-efficiency and high-reliability separation, and high-throughput processing without clogging, and is promising as a potential tool for precise cell separation in low-resource settings.

Continuous Cell Characterization and Separation by Microfluidic Alternating Current Dielectrophoresis.

A novel alternating current (ac)-dielectrophoretic (DEP) microfluidic chip for continuous cell characterization and separation is presented in this paper. To generate DEP forces, two electrode-pads are embedded in a set of asymmetric orifices on the opposite sidewalls to produce the nonuniform electric fields. In the vicinity of a small orifice, the cells experience the strongest nonuniform gradient and are drawn toward it by the positive DEP forces, while the cells experiencing a negative DEP force are repelled away and move toward the large orifice. The DEP behaviors of yeast cells in suspending media with different ionic concentrations, i.e., different electrical conductivities, and over a large range of the ac electric field frequency were investigated. Furthermore, the lateral migrations of yeast cells as a function of the ac frequency were measured. The trends of measured lateral migrations of yeast cells are similar to the corresponding Clausius-Mossotti (CM) factors. In addition, by adjusting the frequency and strength of the ac electric field, the continuous separation of live and dead yeast cells as well as the yeast cells with targeted diameter and dielectric property can be easily achieved. This is the first time that the measurement of ac-DEP lateral migration of yeast cells in solutions with different electrical conductivities as a function of the applied frequency in a microfluidic chip was reported. This ac-DEP system provides a method to characterize the crossover frequency of the specific cells and manipulate the targeted cells.

Viscoelastic Separation and Concentration of Fungi from Blood for Highly Sensitive Molecular Diagnostics

Isolation and concentration of fungi in the blood improves sensitivity of the polymerase chain reaction (PCR) method to detect fungi in blood. This study demonstrates a sheathless, continuous separation and concentration method of candida cells using a viscoelastic fluid that enables rapid detection of rare candida cells by PCR analysis. To validate device performance using a viscoelastic fluid, flow characteristics of 2 μm particles were estimated at different flow rates. Additionally, a mixture of 2 μm and 13 μm particles was successfully separated based on size difference at 100 μl/min. Candida cells were successfully separated from the white blood cells (WBCs) with a separation efficiency of 99.1% and concentrated approximately 9.9-fold at the center outlet compared to the initial concentration (~2.5 × 107 cells/ml). Sequential 1st and 2nd concentration processes were used to increase the final number of candida cells to ~2.3 × 109 cells/ml, which was concentrated ~92-fold. Finally, despite the undetectable initial concentration of 101 CFU/ml, removal of WBCs and the additional buffer solution enabled the quantitative reverse transcription (RT)-PCR detection of candida cells after the 1st concentration (Ct = 31.43) and the 2nd concentration process (Ct = 29.30).

Cell suspension culture of Rhizoma zedoariae in a two-stage perfusion bioreactor system for β-elemene production

We presented a two-stage combined bioreactor system consisting of a stir-tank and an airlift column, and challenged with Rhizoma zedoariae cell suspensions for β-elemene production. Two-stage culture was initiated when the cell concentration in both vessels was maintained at an appropriate density. The cells were proliferated in stirred-tank with the maximal growth rate of 0.17 d−1 to present enough cells for β-elemene synthesis. In the airlift column, continuous cell separation from culture medium was achieved by using a cell retention device based on centrifugal and gravity settling when the system was performed in perfusion mode. The results indicated that additives can efficiently promote the accumulation of β-elemene in R. zedoariae cells. In addition, the β-elemene content showed higher levels in cell lines of overexpressing 3-hydroxy-3-methylglutaryl coenzyme-A reductase, Farnesyldi phosphate synthase, and ST02C genes.

Dielectrophoretic separation of microalgae cells in ballast water in a microfluidic chip.

The composition of the ship's ballast water is complex and contains a large number of microalgae cells, bacteria, microplastics, and other microparticles. To increase the accuracy and efficiency of detection of the microalgae cells in ballast water, a new microfluidic chip for continuous separation of microalgae cells based on alternating current dielectrophoresis was proposed. In this microfluidic chip, one piece of 3-dimensional electrode is embedded on one side and eight discrete electrodes are arranged on the other side of the microchannel. An insulated triangular structure between electrodes is designed for increasing the inhomogeneity of the electric field distribution and enhancing the dielectrophoresis (DEP) force. A sheath flow is designed to focus the microparticles near the electrode, so as to increase the suffered DEP force and improve separation efficiency. To demonstrate the performance of the microfluidic separation chip, we developed two species of microalgae cells (Platymonas and Closterium) and a kind of microplastics to be used as test samples. Analyses of the related parameters and separation experiments by our designed microfluidic chip were then conducted. The results show that the presented method can separate the microalgae cells from the mixture efficiently, and this is the first time to separate two or more species of microalgae cells in a microfluidic chip by using negative and positive DEP force simultaneously, and moreover it has some advantages including simple operation, high efficiency, low cost, and small size and has great potential in on-site pretreatment of ballast water.

Isolation of cells from whole blood using shear-induced diffusion

Extraction of cells of interest directly from whole blood is in high demand, yet extraordinary challenging due to the complex hemodynamics and hemorheology of the sample. Herein, we describe a new microfluidic platform that exploits the intrinsic complex properties of blood for continuous size-selective focusing and separation of cells directly from unprocessed whole blood. The novel system only requires routinely accessible saline solution to form a sandwiched fluid configuration and to initiate a strong effect of shear-induced diffusion of cells, which is coupled with fluid inertia for effective separation. Separations of beads and cells from whole blood have been successfully demonstrated with high efficiency (89.8%) at throughput of 6.75 mL/hr (106–107 cells/s) of whole blood. Rapid isolation of circulating tumor cells (CTCs) from peripheral blood sample of hepatocarcinoma patients is also shown as a proof of principle.

Microfluidic cell sorting: Towards improved biocompatibility of extracorporeal lung assist devices

Extracorporeal lung assist technology is one of the last options in critical care medicine to treat patients suffering from severe oxygenation and decarboxylation disorders. Platelet activation along with the consequent thrombus formation is a potentially life-threatening complication of this technique. To avoid platelet-dependent clot formation, this study aims at developing a microfluidic cell sorting chip that can bypass platelets prior to the membrane oxygenator of the extracorporeal lung assist device. The cell sorting chips were produced by maskless dip-in laser lithography, followed by soft lithography replication using PDMS. Citrated porcine whole blood with a clinically relevant haematocrit of 17% was used for the cell sorting experiments involving three different blood flow rates. The joint effects of flow focusing and hydrodynamic lifting forces within the cell sorting chip resulted in a reduction of up to 57% of the baseline platelet count. This cell sorting strategy is suitable for the continuous and label-free separation of red blood cells and platelets and is potentially applicable for increasing the biocompatibility and lifetime of current extracorporeal lung assist devices.

A Triplet Parallelizing Spiral Microfluidic Chip for Continuous Separation of Tumor Cells

Inertial and deformability- based particles separations gradually attract more significant attentions. In this work, we present a hybrid chip by combining the advantages of inertial and deformability –based principle. The chip is a triplet parallelizing spiral inertial microfluidic chip interconnected with numerable tilted slits (Spiral-Slits Chip) for continuous separation of circulating tumor cells. Utilizing the inertial lift and viscous drag forces, different sized particles achieve different equilibrium at distinct streamlines of the spiral microchannel. Numerable tilted slits are organized along the flow direction. They frequently transport segregated streamline particles into a paralleled smaller microchannel. These frequent dragging results in the amount of certain sized particles in the original microchannel gradually and dramatically reduced. Inertial separation of distinct sized particles could be achievable. Two arrays of numerable tilted slits function as bridges. This Spiral-Slits Chip could substitute for Red Blood Cells Lysis (RBCL) and is most effective for ultra-high throughput. The overall arrangement of this triplet parallelizing spiral inertial microfluidic reflects stable streamlines distribution in the first main microchannel. Combining with Ellipse filters, robust and reproducible capture of CTCs could be achieved at high flow rates. Optical absorption detection has been tentatively tested, and this could simplify the process.

Continuous cell flocculation for recombinant antibody harvesting.

BACKGROUNDIntegrated continuous production technology is of great interest in biopharmaceutical industry. Efficient, flexible and cost effective methods for continuous cell removal have to be developed, before a fully continuous and integrated product train can be realized. The paper describes the development and testing of such an integrated continuous and disposable set‐up for cell separation by flocculation combined with depth filtration.RESULTSScreening of multiple flocculation agents, depth filters, and conditions demonstrated that the best performance was obtained with 0.0375% polydiallyldimethylammonium chloride (pDADMAC; a polycationic flocculation agent) in combination with Clarisolve® depth filters. Using this set‐up, a 4‐fold decrease of filtration area was achieved relative to standard filtration without flocculation, with yields of ≥97% and DNA depletion of up to 99%. Continuous operation was accomplished using a simple tubular reactor design with parallelization of the filtration. The reactor length was selected to allow a 13.2‐min residence time, which was sufficient to complete flocculation in batch experiments. Continuous flocculation performance was monitored on‐line using focused beam reflectance measurement. Filter switch cycles based on upstream pressure were controlled by in‐line pressure sensors, and were stable from one filter to the next.CONCLUSIONIt was demonstrated that stable and efficient continuous flocculation associated with depth filtration can be easily accomplished using tubular reactors and parallelization. Continuous cell separation is essential for the development of fully continuous integrated process trains. This cost‐efficient disposable design run in continuous mode significantly reduces facility foot print, process costs and enables great flexbility. © 2017 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Rapid and effective enrichment of mononuclear cells from blood using acoustophoresis

Effective separation methods for fractionating blood components are needed for numerous diagnostic and research applications. This paper presents the use of acoustophoresis, an ultrasound based microfluidic separation technology, for label-free, gentle and continuous separation of mononuclear cells (MNCs) from diluted whole blood. Red blood cells (RBCs) and MNCs behave similar in an acoustic standing wave field, compromising acoustic separation of MNC from RBC in standard buffer systems. However, by optimizing the buffer conditions and thereby changing the acoustophoretic mobility of the cells, we were able to enrich MNCs relative to RBCs by a factor of 2,800 with MNC recoveries up to 88%. The acoustophoretic microchip can perform cell separation at a processing rate of more than 1 × 105 cells/s, corresponding to 5 µl/min undiluted whole blood equivalent. Thus, acoustophoresis can be easily integrated with further down-stream applications such as flow cytometry, making it a superior alternative to existing MNC isolation techniques.

Continuous Separation of White Blood Cells From Whole Blood Using Viscoelastic Effects.

White blood cells (WBCs) are the only cellular constituent containing genetic materials, and, hence, are candidate biomarkers for a host of diseases. However, conventional methods for WBC separation tend to have low sample purity and separation efficiency, which will have adverse implications on downstream polymerase chain reaction (PCR) analyses. In this study, we introduce a two-stage microfluidic device which harnesses the elastic property of a non-Newtonian fluid for size-based separation of WBCs from whole blood. The device displayed high resolution and efficiency in separating polystyrene particles and blood cells of different sizes up to a flow rate of 150 μL/min in polyvinylpyrrolidone solutions. We performed a separate parametric study to evaluate the effects of the fluid elasticity and flow rate on the separation performance. The hematocrit of the blood sample was varied from 0.01% to 20% to investigate the effect of increased intercellular interactions on the separation efficiency. An optimized set of parameters was selected to demonstrate the applicability of the device to the separation of WBCs from diluted whole blood, with excellent efficiency and purity (>90%). This microfluidic device will be especially useful for blood fractionation applications requiring high sample purity and speedy processing. Additionally, the apparent flow-rate insensitivity of the separation allows for its potential use in point-of-care applications.