Cell Separation Methods Research Articles

Experimental and numerical study of the effect of the channel curvature angle on inertial focusing in curvilinear microchannels

Passive cell separation methods have attracted great attention due to their superiority over the other methods stemming from their easy fabrication, precise manipulation, cost-effectiveness, sensitivity, and simplicity. The fluid inertia in these methods is the main factor that is affected by the channel design; thus, the channel design parameters should be chosen accordingly. Even though all channel design parameters are well addressed in inertial microfluidics, the curvature angle of the channel has not yet been extensively studied. In this study, three different curvilinear microchannels with curvature angles of 180°, 210°, and 270° were designed, keeping all other remaining parameters the same. The focusing ability of the fluorescent polystyrene microparticles with diameters of 1.1, 3.3, and 9.9 μm was investigated both experimentally and numerically to understand focusing efficiency affected by the curvature angle of the microchannel. The first set of experiments was to determine the effect of the channel curvature and indicated the favorable design as channel C, which showed focusing qualities of 0.85 and 0.92 for 9.9 μm particles at volumetric concentrations of 2% and 5%, respectively. The remaining set of experiments and CFD simulations were conducted to observe the interaction of 3.3 and 9.9 μm particles and reveal the distortion of the focusing line and particulate phase contours for 9.9 μm particles at the flow rates between 0.3 and 0.7 ml/min, which was further confirmed by enriched mixtures containing 1.1, 3.3, and 9.9 μm particles. The study showed that mixtures comprising low diameter particles could not satisfy the focusing criteria, which emphasized the importance of an appropriate particle size and concentration for a single focus line. On the other hand, it was shown that geometric features of the microchannel such as the hydraulic diameter and the curvature angle together with the particle size determine the focusing quality both experimentally and numerically. To sum up, the increment of the channel curvature angle is a determining factor for particle focusing, and a single focusing line was observed on the particles maintaining the focusing criteria even in many particle conditions. While the focusing quality of the particles was reduced by multi-particle interactions, they were proven to be separable achieving the appropriate concentration ratio.

Retinal Layer Separation (ReLayS) method enables the molecular analysis of photoreceptor segments and cell bodies, as well as the inner retina

Understanding the physiology of the retina, and especially of the highly polarized photoreceptors, is essential not only to broaden our knowledge of the processes required for normal vision, but also to develop effective therapies to prevent or slow retinal degenerative diseases. However, the molecular analysis of photoreceptors is a challenge due to the heterogeneity of the retinal tissue and the lack of easy and reliable methods for cell separation. Here we present the ReLayS method—a simple technique for the separation of photoreceptor segments (PS) containing both inner and outer segments, outer nuclear layer (ONL), and inner retina (InR) that contains the remaining retinal layers. The layer-specific material isolated from a mouse half-retina with the ReLayS method was sufficient for protein isolation and Western blotting or RNA isolation and real-time PCR studies. The separation of PS, ONL, and InR was successfully validated by Western blotting and real-time PCR using proteins and genes with known expression profiles within the retina. Furthermore, the separation of the PS from the ONL enabled the detection of light-driven translocation of transducin from the PS to the soma. ReLayS is a simple and useful method to address protein and possibly metabolites distribution in photoreceptor compartments in various situations including development, ageing, and degenerative diseases.

A thermoresponsive cationic block copolymer brush-grafted silica bead interface for temperature-modulated separation of adipose-derived stem cells

Adipose-derived mesenchymal stem cells (ADSCs) have beneficial effects in cell transplantation therapy; these cells are collected from adipose tissue using low-invasive methods. However, to prepare ADSCs for cell therapy, a cell separation method that neither involves modification of the cell surface nor causes loss of cell activity is needed. Here, we aimed to develop ADSC separation columns using thermoresponsive cationic block copolymer brush-grafted beads as packing materials. The block copolymer brush was formed by a bottom cationic segment, poly(N,N-dimethylaminopropylacrylamide) (PDMAPAAm), and an upper thermoresponsive segment, poly(N-isopropylacrylamide) (PNIPAAm), and was grafted in two atom transfer radical polymerization reactions. The copolymer brush-grafted silica beads were packed into a column. An ADSC suspension was introduced into the columns at 37 °C and adsorbed on the copolymer brush-modified beads through electrostatic and hydrophobic interactions with the PDMAPAAm and PNIPAAm segments, respectively. The adsorbed ADSCs eluted from the column by lowering the temperature to 4 °C. In contrast, most Jurkat and vascular endothelial cells eluted at 37 °C, because of the relatively weaker electrostatic interactions with the block copolymer brush compared to ADSCs. Using the prepared column, a mixture of ADSCs and Jurkat cells was separated by changing the column temperature. The recovered ADSCs exhibited cell activity. The developed cell separation column may be useful for isolating ADSCs without cell surface modification, while maintaining cell activity.

Separation of microalgae cells in a microfluidic chip based on AC Dielectrophoresis

AbstractBACKGROUNDMicroalgae is an important natural resource with extensive applications in various fields, such as health products, animal feed, biopharmaceuticals, and clean energy. However, before the research and utilization of microalgae resources, the microalgae cells of interest must be separated from hybrid strains and bacteria. The traditional method of microalgae separation requires manual labor under the microscope that is time‐consuming and labor‐intensive.RESULTSHerein, a novel microalgae cell separation method based on the dielectrophoresis (DEP) technique was proposed. A unique DEP separation chip with a 3D electrode was designed. Chlorella and Closterium were used as the experimental samples to verify the effectiveness of the proposed method because they have similar volumes and very different electrical properties. Within a certain frequency range, Chlorella suffers from negative dielectrophoresis (nDEP) force and Closterium suffers from positive dielectrophoresis (pDEP) force, so their separation can be achieved. The separation efficiency exceeded 90%. In addition, the DEP response of the Chlorella and Closterium were studied, and the DEP spectra of the two microalgae cells were obtained. Moreover, the effects of DEP force on cell viability under different voltages and frequencies were experimentally analyzed.CONCLUSIONThese findings will contribute to DEP technology, to the portable and harmless separation of microalgae, and to research on the dielectric properties of microalgae cells. © 2022 Society of Chemical Industry (SCI).

A high-throughput microfluidic device based on controlled incremental filtration to enable centrifugation-free, low extracorporeal volume leukapheresis

Leukapheresis, the extracorporeal separation of white blood cells (WBCs) from red blood cells (RBCs) and platelets (PLTs), is a life-saving procedure used for treating patients with cancer and other conditions, and as the initial step in the manufacturing of cellular and gene-based therapies. Well-tolerated by adults, leukapheresis poses a significant risk to neonates and low-weight infants because the extracorporeal volume (ECV) of standard centrifugation-based machines represents a particularly large fraction of these patients’ total blood volume. Here we describe a novel high-throughput microfluidic device (with a void volume of 0.4 mL) based on controlled incremental filtration (CIF) technology that could replace centrifugation for performing leukapheresis. The CIF device was tested extensively using whole blood from healthy volunteers at multiple hematocrits (5–30%) and flow rates (10–30 mL/min). In the flow-through regime, the CIF device separated WBCs with > 85% efficiency and 10–15% loss of RBCs and PLTs while processing whole blood diluted with saline to 10% hematocrit at a flow rate of 10 mL/min. In the recirculation regime, the CIF device demonstrated a similar level of separation performance, virtually depleting WBCs in the recirculating blood (~ 98% reduction) by the end of a 3.5-hour simulated leukapheresis procedure. Importantly, the device operated without clogging or decline in separation performance, with minimal activation of WBCs and PLTs and no measurable damage to RBCs. Compared to the typical parameters of centrifugation-based leukapheresis, the CIF device had a void volume at least 100-fold smaller, removed WBCs about twice as fast, and lost ~ 2–3-fold fewer PLTs, while operating at a flow rate compatible with the current practice. The hematocrit and flow rate at which the CIF device operated were significantly higher than previously published for other microfluidic cell separation methods. Finally, this study is the first to demonstrate a highly efficient separation of cells from recirculating blood using a microfluidic device. Overall, these findings suggest the feasibility of using high-throughput microfluidic cell separation technology to ultimately enable centrifugation-free, low-ECV leukapheresis. Such a capability would be particularly useful in young children, a vulnerable group of patients who are currently underserved.

Identification of Leukemia Cells in Microscopy Digital Image Using Improved Canny Edge Detection Method

Leukemia is a blood cancer mutates inside the bone marrow. If unidentified at an early stage, it can lead to severe consequences and later death of a person. To diagnose leukemia, machines and manpower skills are required to identify if the illustrative image is healthy or unhealthy. The manual observation of the large number of images may lead to error in the results. One of the biggest barriers in the result's accuracy is identification of concerned region in the overlapped cells. Motivated by the challenges, framework is developed based on image processing for automatic detection of White Blood Cells in the peripheral blood smear image. In this paper, canny edge detection with Circular Hough Transform is applied to count the number of white blood cells. The outcome is to obtain the classification of the sample images whether the sample is unhealthy or healthy sample image. Total 108 multi-cell Acute Lymphoblastic Leukemia images are considered. It was found that the proposed cell separation method yields an accuracy of 98% in comparison to state of the art segmentation technique.

Acoustophoretic Motion of Leishmania spp. Parasites.

The analysis of cell motion in an acoustic field is of interest as it can lead to new methods of cell separation, isolation and manipulation for diagnosis and treatment of diseases. Studies of the motion of different species of Leishmania parasites during exposure to ultrasonic standing waves in a microfluidic device allowed identification of acoustic responses of these parasites in their promastigote and amastigote forms. Both forms exhibited a positive acoustic contrast factor and were driven toward the pressure node established in the center of the channel by the acoustically induced radiation force (FR). Promastigotes experience calculated FR amplitudes one order of magnitude larger than those experienced by amastigotes because of the measured differences in volume. The aggregates formed at the pressure node have distinct shapes and stability conditions, for both promastigotes and amastigotes.

A novel laser scribing method combined with the thermal stress cleaving for the crystalline silicon solar cell separation in mass production

Nowadays as the wafer sizes of the crystalline silicon solar cells get larger and larger, separating the cells becomes an essential step. However, in mass production, there is still lack of a low scribing loss and broad compatible cell separation method. In this work, a systematic investigation was conducted on the crystalline silicon solar cell separation processes, including the laser scribing and the cleaving process. Firstly, by combining a 1/4 beam splitter with the laser head, we developed a new laser scribing process, which can achieve a smaller groove width-to-depth ratio (24%) with narrower heat affected area (total width of 30 μm) compared to traditional laser optical systems. Then, we explored the minimum required scribing depths for separating four typical crystalline silicon cells by applying the thermal stress cleaving process. The Czochralski-grown substrates need only point scribing at the beginning and the end of the separating path, but if there is a metal electrode in the separation path, the scribing depth has to be about 20% of the cell thickness. At last, we analyzed the electrical and mechanical strength losses caused by different separation processes. The 20% scribing depth may cause about 30%rel mechanical strength loss. It is proved that combining this new scribing method with the thermal stress cleaving is an economical separation solution with less laser scribing loss and better compatibility for different cells. Our results would shed some light on future separating and module design efforts of solar cells.

Proof-of-Concept of a Novel Cell Separation Technology Using Magnetic Agarose-Based Beads

The safety of the cells used for Advanced Therapy Medicinal Products is crucial for patients. Reliable methods for the cell purification are very important for the commercialization of those new therapies. With the large production scale envisioned for commercialization, the cell isolation methods need to be efficient, robust, operationally simple and generic while ensuring cell biological functionality and safety. In this study, we used high magnetized magnetic agarose-based beads conjugated with protein A to develop a new method for cell separation. A high separation efficiency of 91% yield and consistent isolation performances were demonstrated using population mixtures of human mesenchymal stem cells and HER2+ SKBR3 cells (80:20, 70:30 and 30:70). Additionally, high robustness against mechanical stress and minimal unspecific binding obtained with the protein A base conjugated magnetic beads were significant advantages in comparison with the same magnetic microparticles where the antibodies were covalently conjugated. This study provided insights on features of large high magnetized microparticles, which is promising for the large-scale application of cell purification.

Microfluidic methods for cell separation and subsequent analysis

Cell is the most basic unit of the morphological structure and life activity of an organism. Learning the composition, structure and function of cells, exploring the life activities of cells and studying the interaction between cells are of great significance for human cognition and control of the life activities of organisms. Therefore, rapid, convenient, inexpensive, high-precision and reliable methods of cell separation and analysis are being developed to obtain accurate information for the study of cytology and pathology. Microfluidic chip is a new emerging technology in recent years. It has a micromanufacturing structure, which can not only realize the precise space-time control of fluid and cells, but also reproduces the three-dimensional dynamic microenvironment of cell growth in the body. In addition, the microfluidic chip has unique microphysical properties and facilitates the integration of microdevices, which provides the possibility of real-time monitoring, continuous culture, separation and enrichment, and even in situ analysis of cells. In this review, we summarized recent advances in the development of different techniques for cell isolation and analysis on microfluidic platforms. Focus was put on biochemical and physical methods for cell separation on microfluidic chips. Subsequent cell analysis depending on fluorescence, Raman, cytomicroscopic imaging, mass spectrometry and electrochemical methods also was remarked. Through analyzing and learning the advantages and disadvantages of different technologies, we hope that microfluidic chips will continue to be improved and expanded for medical and clinical applications.

A novel microfluidic device with tapered sidewall electrodes for efficient ternary blood cells (WBCs, RBCs and PLTs) separation

Separation and counting of blood cells from whole blood is very significant in measurement and data extraction in health care diagnostics and monitoring health parameters. Many techniques are available for blood cell separation out of which dielectrophoresis (DEP) on lab on chip platform is an emerging technique due to its phenomenal advantages that the force in this technique acts directly on cells based on dielectric properties. In DEP more challenges lie in optimization of the electric field at lower operating voltages as higher voltages damage the biological cells via joule heating and the separation process cannot be accomplished. Due to this optimization of the electric field distribution in the microchannel of the cell separation device is very much important and can be achieved via numerical model or design optimization of electrodes shapes and material. In this work, a microfluidic cell separation device is optimized using the finite element method for the ternary separation of blood cells. The design is realized with two inlets, three outlets, and one main channel having tapered sidewall electrodes for cell separation purpose. Inlet velocities are optimized to achieve improved cell focusing before subjecting cells to separation effects by varying the velocity ratio of main inlet velocity to buffer inlet velocity. Results have shown successful separation of platelets, red blood cells, and white blood cells with 96.7%–100% yield and purity with velocity ratio from 1:4 to 1:5 at electrode excitation voltage of 1.5 V.

Analysis of Cell Dielectrophoretic Motion with Microfluidic Device Embedding Multi-electrodes

The motion characteristics of different cells in the multi-electrode array microchip were studied under the dielectrophoretic force, and the two cells with the same external morphology but different internal histone protein were separated. The multi-electrode array microfluidic chip was embedded in the 5 square cross section of the channel, and 8 electrodes were embedded on each side of the cross section. The structure enlarged the size of the microfluidic channel, which was beneficial for the high flow separation of the cells under the action of the dielectrophoretic force. To study the characteristics of cell motion in the microfluidic device, the electric field distribution in a cross-section was calculated, and the optimal electrode combination pattern was obtained, which generated a uniform electric field distribution and a greatest the dielectrophoretic force. After that, the motion characteristics of MRC-5 in human lung fibroblasts in different frequencies and complex electric fields at different frequencies and complex electric fields were analyzed. By analyzing the spectrum of the dielectrophoretic force, the separation frequency of the two kinds of cells of wild type (WT) and histone-GFP type was f=30 kHz, and the separation rate of cell was calculated by adding different proportions of sucrose solution and two cell mixture at two inlets. That was, when the flow-rate ratio of the 2 inlets was 12:1, the separation rate of the two types of cells was 93.5%. The method for cell separation using microfluidic chips provided a basis for the rapid separation of cells in the future.

Applying model approaches in non-model systems: A review and case study on coral cell culture.

Model systems approaches search for commonality in patterns underlying biological diversity and complexity led by common evolutionary paths. The success of the approach does not rest on the species chosen but on the scalability of the model and methods used to develop the model and engage research. Fine-tuning approaches to improve coral cell cultures will provide a robust platform for studying symbiosis breakdown, the calcification mechanism and its disruption, protein interactions, micronutrient transport/exchange, and the toxicity of nanoparticles, among other key biological aspects, with the added advantage of minimizing the ethical conundrum of repeated testing on ecologically threatened organisms. The work presented here aimed to lay the foundation towards development of effective methods to sort and culture reef-building coral cells with the ultimate goal of obtaining immortal cell lines for the study of bleaching, disease and toxicity at the cellular and polyp levels. To achieve this objective, the team conducted a thorough review and tested the available methods (i.e. cell dissociation, isolation, sorting, attachment and proliferation). The most effective and reproducible techniques were combined to consolidate culture methods and generate uncontaminated coral cell cultures for ~7 days (10 days maximum). The tests were conducted on scleractinian corals Pocillopora acuta of the same genotype to harmonize results and reduce variation linked to genetic diversity. The development of cell separation and identification methods in conjunction with further investigations into coral cell-type specific metabolic requirements will allow us to tailor growth media for optimized monocultures as a tool for studying essential reef-building coral traits such as symbiosis, wound healing and calcification at multiple scales.

Development of a primary skeletal muscle cell culture model from human muscle biopsies

Purpose: This work aimed to develop and validate a culture model of primary human skeletal muscle cells. Methods: Muscle biopsies were obtained post-mortem from the vastus lateralis. The biopsies were enzymatically digested with collagenase 1A (1mg/mL) and dispase II (4,8mg/mL) for 60 minutes at 37°C in order to isolate fibroblasts and satellite cells. Then, the satellite cells (CD56+) were separated from fibroblasts (CD56-) by two methods: pre-plating or immunomagnetic cell sorting using an anti-CD56 antibody, a transmembrane glycoprotein expressed on the surface of satellite cells. Indeed, some human muscle biopsies contain a high proportion of fibroblasts that can contaminate the myoblasts cell culture if the separation method is not efficient. The performance of these cell separation methods was evaluated by FACS. We also compared the effects of two culture media on myoblasts proliferation (10%FBS vs. 10%FBS + 2%Ultroser G (UG)) using cell doubling time as parameter. Two differentiation media (2%FBS vs. 0.5%UG) were tested on the myogenic index, which corresponds to the proportion of cells that have fused to form myotubes. This index was determined through the detection of the myosin heavy chain (MYH) by immunofluorescence. Finally, we studied the expression of several muscle cell markers during myogenesis by qRT-PCR and immunofluorescence. Results: The cells obtained from the enzymatic digestion of the biopsy contained a varying proportion of CD56+ satellite cells (86,15 ± 12,09%; 95%CI:73,45 to 98,84). The cell population obtained after immunomagnetic cell sorting method contained more than 92% of CD56+ cells (96,11 ± 3,27%; 95%CI:92,68 to 99,54). In comparison, the ratio of CD56+ obtained by the preplating method was lower (84,19 ± 9,94%; 95%CI:73,76 to 94,62), but no significant difference was observed between both methods. The cell doubling time was significantly shorter with culture media containing 10%FBS and 2%UG than with the media containing only 10%FBS (25.70 ± 3,96h vs. 70.64 ± 21,68h, p = 0.0313). The myogenic index was slightly higher with the 0.5%UG than 2%FBS differentiating medium (62,75% ± 8,57% vs. 56,39 ± 16,45%). Finally, the increase of MyoD1, myogenin and MYH, three markers of differentiation, indicated the phenotypic transition of myoblasts to a myogenic phenotype. Conclusions: Immunomagnetic cell sorting allows better satellite cells and fibroblasts separation than the conventionally used preplating technique. Ultoser G serum substitute accelerates cell proliferation and improves the differentiation of myoblasts into myotubes. The markers MyoD1, myogenin and MYH are good markers of the myogenic differentiation of myoblasts. This in vitro model makes it possible to purify efficiently muscle satellite cells from human muscle biopsies. These cells can be used in in vitro studies to study the pathophysiological mechanisms of muscle disease and the mechanisms of action of treatments.

Rapid and precise tumor cell separation using the combination of size-dependent inertial and size-independent magnetic methods.

Circulating tumor cells (CTCs) play a significant role in cancer diagnosis and treatment monitoring. One of the major challenges in isolating and detecting rare CTCs from blood is that white blood cells (WBCs) have a size overlap with the target CTCs. To address this issue, we constructed a three-stage i-Mag device integrated with passive inertial microfluidics and active magnetophoresis, enabling rapid and precise separation of tumor cells from blood. The first-stage spiral inertial sorter was applied to rapidly remove small-sized red blood cells (RBCs), and then the second-stage serpentine inertial focuser and the third-stage magnetic sorter were used for removing the magnetically labeled WBCs size-independently, to significantly purify the captured tumor cells. Then, the separation performance of our i-Mag device was explored. The results indicated rapid and precise separation of breast cancer cells from diluted whole blood at a high separation efficiency of 93.84% and at a high purity of 51.47%. The purity of the collected tumor cells could be further improved to 93.60% when the blood dilution ratio was increased. We also successfully applied our i-Mag device for the isolation and detection of trace tumor cells. Our i-Mag device has numerous advantages, such as enabling high-throughput processing and high-precision separation, requiring easy manufacturing at a low cost, and providing tumor antigen-independent operation. We believe that the i-Mag device has great potential to act as a precise tool for separating various bioparticles.

Fundamentals of integrated ferrohydrodynamic cell separation in circulating tumor cell isolation.

Methods to separate circulating tumor cells (CTCs) from blood samples were intensively researched in order to understand the metastatic process and develop corresponding clinical assays. However current methods faced challenges that stemmed from CTCs' heterogeneity in their biological markers and physical morphologies. To this end, we developed integrated ferrohydrodynamic cell separation (iFCS), a scheme that separated CTCs independent of their surface antigen expression and physical characteristics. iFCS integrated both diamagnetophoresis of CTCs and magnetophoresis of blood cells together via a magnetic liquid medium, ferrofluid, whose magnetization could be tuned by adjusting its magnetic volume concentration. In this paper, we presented the fundamental theory of iFCS and its specific application in CTC separation. Governing equations of iFCS were developed to guide its optimization process. Three critical parameters that affected iFCS's cell separation performance were determined and validated theoretically and experimentally. These parameters included the sample flow rate, the volumetric concentration of magnetic materials in the ferrofluid, and the gradient of the magnetic flux density. We determined these optimized parameters in an iFCS device that led to a high recovery CTC separation in both spiked and clinical samples.

A polymer-film inertial microfluidic sorter fabricated by jigsaw puzzle method for precise size-based cell separation

A polymer-film inertial microfluidic jigsaw (PIMJ) sorter with trapezoidal spiral channels using the jigsaw puzzle method was proposed to realize precise and high-throughput rare cell separation. The PIMJ sorter was fabricated by assembling laser-patterned polymer-film layers of different thicknesses. After illustrating the conceptual design and fabrication process, the effects of the cross-sectional dimension, particle size, and operational flow rate on particle focusing were systematically explored under a broad flow rate range. Then, the separation performances of the PIMJ sorter were characterized using the binary particle mixture and the blood samples spiked with four types of tumor cells. The results indicated that the complete separation of the binary particles with a minimum size difference of 2 μm was successfully realized at a high throughput up to 3000 μL/min. A high recovery ratio of 90.57%-94.14% and a high purity of 48.67%-79.05% were achieved for the separation of rare tumor cells from white blood cells (WBCs). Finally, the PIMJ sorter was successfully employed for separating rare circulating tumor cells (CTCs) from the metastatic breast and lung cancer patients with a capture ratio of 7–226 CTCs per 5 mL sample. The results proved the high sensitivity and high reliability of the PIMJ sorter. The PIMJ sorter is expected to be a potential device for precise CTC separation towards the clinical applications.

Development and Study of a Method for Cell Separation During White Blood Cell Segmentation on Images of Bone Marrow Preparations in Information and Measurement Systems for Diagnostics of Acute Leukemia

The issues of using information and measurement systems based on processing of digital images of microscopic preparations for solving large-scale tasks of automating the diagnosis of acute leukemia are considered. The high density of leukocyte cells in the preparation (hypercellularity) is a feature of microscopic images of bone marrow preparations. It causes the proximity of cells to each other and their contact with the formation of conglomerates. Measuring of the characteristics of bone marrow cells in such conditions leads to unacceptable errors (over 50%). The work is devoted to segmentation of contiguous cells in images of bone marrow preparations. A method of cell separation during white blood cell segmentation on images of bone marrow preparations in conditions of hypercellularity of the preparation has been developed. The peculiarity of the proposed method is the use of an approach to segmentation of cell images based on the watershed method with markers. Key stages of the method: the formation of initial markers and building of the watershed lines, threshold binarization, shading inside the outline. The parameters of the separation of contiguous cells are determined. The experiment confirmed the effectiveness of the proposed method. The relative segmentation error was 5 %. The use of the proposed method in information and measurement systems of computer microscopy for automated analysis of bone marrow preparations will help to improve the accuracy of diagnosis of acute leukemia.

A method for separation and purification of mouse splenocytes by density gradient centrifugation

Spleen is an information-rich and easy-accessible peripheral lymphoid organ. It has complex cell composition because of the immunocytes maturity and settle down. Changes of the composition and function of these immunocytes are critical to body immune response. To understand the cell behaviors, specific cell subpopulations are required to be separated without heterogeneity. Density gradient centrifugation is one of the cell separation methods with high throughput. However, the greatest defect of this method is its low cell purity. In this study, the separation conditions of tumor-bearing mouse splenocytes were optimized by separation solutions with different density gradients. After separation, lymphocytes were located at the second layer with the proportion of 84.9%, monocytic-like myeloid-derived suppressor cells (Mo-MDSCs) were located at the fourth layer with the proportion of 54.2% and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) were located at the sixth layer with the proportion of 85.5%. Cells in different layers were further determined by verifying the gene expression pattern of some chemokine receptors on cell surfaces. Furthermore, this method was also used to separate healthy mouse splenocytes. Therefore, this method will be highly useful to separate mouse splenocytes and has laid a foundation for further research on the changes and roles of immunocytes during the development of cancer.

Thermoresponsive Cationic Block Copolymer Brushes for Temperature-Modulated Stem Cell Separation.

Recently, cell separation methods have become important for preparing cells for transplantation therapy. In this study, a thermoresponsive cationic block copolymer brush is developed as an effective cell separation tool. This brush is prepared on glass surfaces using two steps of activator regenerated by electron transfer-atom transfer radical polymerization (ARGET-ATRP). The cationic segment is prepared in the first step of the ARGET-ATRP of N,N-dimethylaminopropylacrylamide (DMAPAAm). In the second step, the thermoresponsive segment is prepared, attached to the bottom cationic segment, through ARGET-ATRP with N-isopropylacrylamide (NIPAAm). The cell adhesion behavior of the prepared thermoresponsive cationic copolymer, PDMAPAAm-b-PNIPAAm, brush is observed using umbilical cord-derived mesenchymal stem cells (UCMSC), fibroblasts, and macrophages. At 37 °C, all three types of cells adhere to the thermoresponsive cationic copolymer brush. Then, by reducing the temperature to 20 °C, the adhered UCMSC are detached from the copolymer brush, whereas the fibroblasts and macrophages remain adhered to the copolymer brush. Using this copolymer brush, UCMSC can be purified from the cell mixture simply by changing the temperature. Therefore, the prepared thermoresponsive cationic copolymer brush is useful as a cell separation tool for the purification of mesenchymal stem cells.