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Microfluidic biotechnologies for hematology: separation, disease detection and diagnosis
Published in Annie Viallat, Manouk Abkarian, Dynamics of Blood Cell Suspensions in Microflows, 2019
Leukocytes are CD45 expressed cells and each different fraction of WBCs has its own specific surface markers. Techniques targeting these biomarkers can achieve high specificity separation of the desired population of white cells from whole blood. The general method involves employing antibody-attached magnetic beads to label the target WBCs. By applying an external magnetic field, the immunomagnetically labeled cells can be pulled out from the background cells. These MAC based separation techniques have been extensively studied and this concept has been well proven to be effective in retrieving target cells. Inglis et al. developed a silicon substrate chip with nickel magnetic stripes [27]. Leukocytes are labelled with CD45 magnetic beads and their flow direction can be altered by the slanted oriented magnetic stripes. This device achieved a high leukocyte selectivity, however, there is a sim50% cell loss because of cell loss in the device or non-sufficient separation. Moreover, due to the complexity of whole blood, direct labeling and separation of white cells from unprocessed whole blood require a long processing time and the efficiency is always compromised by the large number of RBCs. Besides, the magnetic labelling process could possibly damage the cells inadvertently.
Magnetic Separation in Integrated Micro-Analytical Systems
Published in Nguyễn T. K. Thanh, Clinical Applications of Magnetic Nanoparticles, 2018
Smistrup et al.16 used permalloy micromagnets and an external magnetic field of 50 mT to create a magnetic bead separator. The micromagnets are patterned in stripes and measured l × h × w = 4400 μm × 50 μm × 150 μm in a microfluidic channel sized l × h × w = 13,500 μm × 80 μm × 200 μm. Particles separated are 1 μm fluorescent magnetic beads Spherotec FCM-1052-2. Electroplating was used to fabricate thick micromagnets. They further modified the system with a 2D composed of small alternately arranged Nd–Fe–B permanent magnets, each of which is sized 2 mm × 2 mm × 2 mm to characterize the separation efficiency for 250 nm Nanomag-D plain silica beads.35 Inglis et al.20 used 2-μm-thick nickel stripes to separate continuously flowing Leukocytes (white blood cells). Anti-CD45 Microbeads from Miltenyi Biotech were used. CD45 is a type of protein expressed in all leukocytes. The diameters of the beads were in the range of 20–100 nm, and an average magnetic moment of 1.8 × 105 μB was estimated. In order to create a 2-μm-thick nickel pattern, they first created stripe-shaped grooves on a silicon substrate, and a nickel film deposited on the grooved substrate was chemically and mechanically polished to obtain a flat substrate with patterned magnets. A similar approach of using a stripe patterned magnets has been used by Kim et al.36 for separation of CTCs. Lou et al.37 used a similar stripe-patterned device as Inglis to separate aptamers through systematic evolution of ligands by exponential enrichment (SELEX) process. As will be discussed in Section 11.4, magnetic separation plays an important role in the SELEX process for aptamer selection.
Using co-axial electrospray deposition to eliminate burst release of simvastatin from microparticles and to enhance induced osteogenesis
Published in Journal of Biomaterials Science, Polymer Edition, 2019
Xiaowei Yuan, Mei Zhang, Yilong Wang, He Zhao, Dahui Sun
SD rat BMSCs were used to investigate the in vitro cytocompatibility of the electrosprayed MPs. BMSCs were isolated based on a method modified from that of Zhu et al. [42]. In brief, four 120 g male SD rats were euthanized by cervical dislocation. After the rats were disinfected, the soft tissues, such as muscles and tendons, were carefully disassociated completely from the tibias and femurs using dissecting scissors and a scalpel, to avoid contamination. Bone marrow cavities of the femurs and tibias were slowly flushed with culture media under sterile conditions until the bones become pale. The cells were seeded at 100 cells per cm2 and grown in a 90-mm sterile culture dish with complete culture medium (L-DMEM supplemented with 10% FBS and 1% penicillin-streptomycin solution) at 37 °C with 5% CO2 in a humidified incubator. All samples were processed within 30 min of the animal death to ensure high stem cell viability. The growth medium was changed every two or three days. Cells were subcultured at a split ratio of 1:3 (resuspended in 75 cm2 cell culture flask [Corning, NY, USA]) by trypsin-EDTA solution when they reached approximately 80% confluence. While non-adhesive cells were removed by replacing the media with fresh media, the adhesive BMSCs were cultured to the third-generation. The expression of surface antigens was detected using flow cytometry (Abcam, Cambridge, UK) with CD11b, CD29, CD45 and CD90. BMSCs were plated in triplicate and maintained in adipogenic and osteogenic induction medium for 21 days for adipogenic differentiation and osteogenic differentiation, respectively. The undifferentiated cells were used as controls.