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Miniaturized Sorters: Optical Micro Fluorescence Activated Cell Sorter
Published in Frances S. Ligler, Jason S. Kim, The Microflow Cytometer, 2019
Kamlesh D. Patel, Thomas D. Perroud
Recently reported magneto- and dielectrophoretic sorting strategies have demonstrated impressive metrics for throughput, purities, and enrichment. These sorting methods are highly specialized techniques for limited applications, and thus, are not as general as optical pFACS. Both MACS (magnetic-activated cell sorting) and DACS (dielectrophoretic-activated cell sorting) strategies require that each target cell be covalently bound to either a magnetic or dielectrophoretic tag through antibodies (typically a 1 pm polystyrene bead). External labeling not only prevents sorting cells based on intra-cellular events, but also can interfere with the cell's physiology (e.g. phagocytosis of the particles). These particles have either high superparamagnetic or dielectric properties, which interact with the electromagnetic field generated by preprinted electrodes on the surface of the microfluidic channel. These sorters operate passively by guiding tagged cells along transverse field lines, and, as a result, can achieve very high throughputs and enrichment factors. However, caution must be used when comparing their metrics with optical pFACS. The guidelines used to report the metrics with optical pFACS are more stringent since the work is focused on sorting mammalian cells. Indeed, 1 pm E. coli bacteria, as reported with these techniques, has a smaller Stokes drag than 10 pm mammalian cells making it easier to sort. It remains to be seen if magnetic or dielectrophoretic forces represent a viable approach for sorting large mammalian cells.
Biophotonics
Published in Mohammad E. Khosroshahi, Applications of Biophotonics and Nanobiomaterials in Biomedical Engineering, 2017
Preparation of cell suspensions. Human osteosarcoma cell line (G 292, NCBI-C565), which was initiated from a primary bone tumor osteosarcoma, purchased from national cell bank of Pasteur institute of Iran. Normal osteoblast cell (HOB) were extracted by MACS (Magnetic activated cell sorting) method and measurements were made on cells having passage numbers of 30 or less. The G 292 cells were grown in Dulbecco modified Eagle medium (DMEM, GIBCO 116-12800) supplemented with 10% fetal bovine serum (FBS, GIBCO 106-10270). The HOB cells were grown in DMEM and HAM'S F12 (Sigma-Aldrich N6658) in ratio of 1:1, supplemented with 12% FBS. Both cells supplemented with 1% Antibiotic-Antimycotic Solution (PAA, P11-002), and then cells were incubated at 37ºC with 5% CO2. Upon reaching confluence for G 292 cells and pre-confluence for HOB cells (generally 3 days after passage), these cells were collected from culture flasks by trypsinization to yield a suspension and washed with phosphate-buffered saline (PBS) (GIBCO 18912-014). After washing, the cells were resuspended in a volume of 2 mL PBS. The cell suspensions pipetted into a cuvette with 1 cm path length for LIFS analysis. Cell concentration was determined by counting the number of cells per milliliter (cells/mL) manually in a standard manner with a hemocytometer from Neubauer and light microscope. The average cell viability determined with a manual viability count after addition of Trypan Blue 0.25% in PBS. The measurements were repeated independently three times for each sample.
Use of Nanotechnology for Viable Applications in the Field of Medicine
Published in T. S. Srivatsan, T. S. Sudarshan, K. Manigandan, Manufacturing Techniques for Materials, 2018
Mazaher Gholipourmalekabadi, Mohammad Taghi Joghataei, Aleksandra M. Urbanska, Behzad Aghabarari, Aidin Bordbar-Khiabani, Ali Samadikuchaksaraei, Masuod Mozafari
Separation of pure population of stem cells from heterogeneous cells is an essential part and represents a major contribution in tissue engineering. For example, islet 1+ as cardiac progenitor cells exists in the human heart and mouse heart in very small population and is surrounded by differentiated cells of adult heart tissue [149]. A robust and high efficacy method needs to be developed to separate a pure population of rare islet 1+ cell line. In some cases, stem cells should be differentiated in vitro before transplantation, especially in using ESCs. Then, target differentiated cells should be purified and transplanted. Cell-sorting techniques separate cells based on size, density, or affinity (chemical, electrical, or magnetic). Conventional cell separation techniques such as size-based cell sorting, fluorescence-activated cell sorting, and magnetic-activated cell sorting have some disadvantages in terms of cost and time [150]. In modern medicine, miniaturized cell separation systems (microsized/nanosized) have been developed and possess many advantages compared to conventional cell-sorting systems, such as (1) small sample volume, (2) low cost, and (3) portability [150]. Using NPs such as superparamagnetic NPs serves to enhance the resolution. For example, in a study [151], leucocytes (CD 45+ cells) were separated from red blood cells using magnetic-activated cell sorting. After exposure of cells to anti-CD45 conjugated superparamagnetic NPs, CD45+ cells carried about 5000 NPs [151]. Development of a perfect cell-sorting technique for the purpose of separation of rare cells from undesired cells with advantages of high efficacy, low cost, and time is strongly sensed in stem cell biology, and the use of microscale/nanoscale devices enables us to enhance these techniques.
A method for separation and purification of mouse splenocytes by density gradient centrifugation
Published in Preparative Biochemistry & Biotechnology, 2021
Yao Lu, Chenghao Fu, Chao Xia, Shiliang Ma
Spleen is an informative and easy-accessible tissue with complex immunocyte composition. Therefore, character and condition of spleen requires accurate analysis by isolating specific cells. In the process of achieving this, cell separation is a key first step before further biological assays. Traditionally, some cell types can be separated by culturing according to different cellular adhesion and growth property. It is a label-free way to separate suspension cells from adherent cells. This method is easy-operating but has low efficiency and low throughput. Currently, the methods for separating cells mainly include magnet-activated cell sorting (MACS), fluorescence activated cell sorting (FACS) and density gradient centrifugation. MACS and FACS can isolate cells with high specificity by labeling cells exogenously. Clinically, the sorted cells are avoided to be activated by antibodies, which may trigger a series of cell changes. Therefore, MACS especially negative selection has been applied in clinical research[20,21]. Although a large percentage of non-labeled cells can be obtained, there remains a small population of uninterested cells in the target cell suspension and adversely influence the subsequent studies[22]. FACS has also been widely used in biological research. While, the serious limitation of FACS is its expensive price and the damage to cells which may trigger the cell signaling by combining cell surface receptors. Studies also have shown that cell viability and amplification rate were significantly decreased by using FACS[23]. Density gradient centrifugation allows separating cells from a large initial cell sample. And the separation solutions are also gentle on cells. Therefore, it is widely used for clinical cell separation. Besides, it can perform experiments without removing red blood cells in advance, which greatly reduces analysis time. While the current problem of this method is a high purity cannot be obtained under the situation where cell density differences are not significant.