Density Gradient Medium for Cell Separation



The isolation of viable mononuclear and polymorphonuclear cells from blood serves as the starting point for a broad spectrum of immunology and cell biology studies. Unlike red blood cells and platelets, all white blood cells are nucleated and can be classified by the structure of their nucleus as mononuclear or polymorphonuclear cells. Lymphocytes and monocytes are the two main categories of mononuclear white blood cells with single-lobed nuclei in the immune system to defend the body from infection, cancer, and other foreign invaders. In humans, lymphocytes make up the majority (70-90%) of the peripheral blood mononuclear cell (PBMC) population, followed by monocytes (10-30%) and a small percentage of dendritic cells (1-30%). 2 %). Unlike mononuclear leukocytes, polymorphonuclear leukocytes have variable shapes of the nucleus, which is usually lobulated into three segments. Neutrophils are the most abundant polymorphonuclear leukocytes, and the other types (eosinophils, basophils, and mast cells) have much lower populations.

Isolation of mononuclear cells for research use only

The optimized LSM produced by MP Biomedicals has a unique formula in which sodium diatrizoate is successfully substituted for sodium metrizoate. LSM allows the separation of lymphocytes not only from human peripheral blood but also from bone marrow and umbilical cord blood. It has a density of 1,077–1,080 g/mL consisting of 6.2 g Ficoll and 9.4 g sodium diatrizoate per 100 mL solution. LSM was designed to ensure maximum mononuclear cell yield with greater than 96% lymphocyte cell viability in single-step centrifugation. (The separation principle used by LSM to isolate lymphocytes from other blood constituents is summarized. This tried and tested separation medium has been cited in over 2,200 scientific publications.

Examples of LSM applications include the following isolates:

  • Isolation of human peripheral blood mononuclear cells: Liu et al. monocytes isolated from the peripheral blood of patients with axial spondyloarthritis and healthy controls to study the differential regulation of tumour necrosis factor alpha-induced protein 3 (TNFAIP3) in blood-derived macrophages. This study demonstrated that monocytes isolated with LSM retain their differentiation abilities and can produce functional macrophage colony-stimulating factor-derived macrophages for use in immunological studies.
  • Isolation of mononuclear cells from rat spleen cells: LSM has also been used successfully to isolate lymphocytes from rodent species despite the fact that rodent lymphocytes have a slightly higher average density than humans. In a recent study, the paracrine effects of exosomes derived from mesenchymal stem cells (MSCs), after acute myocardial infarction, on angiogenesis and anti-inflammatory activity were examined. In this case, lymphocytes were isolated from rat spleen using LSM to perform T-cell proliferation assays used to assess the angiogenic potency of MSC-derived exosomes.
  • Isolation of parasites from stool or blood: LSM has been shown to be effective in isolating parasites from peripheral blood samples. Lymphatic filariasis is a mosquito-borne tropical disease caused by filarial nematodes, such as Brugia malayi. Microfilariae (Mf) or first-stage larvae are eaten by mosquitoes that can infect humans through mosquito bites. Schröder et al. successfully used LSM to isolate Mf from the blood of infected gerbils. Mf was cocultured with human umbilical vein endothelial cells to study the mechanism of Mf sequestration in the lungs of infected individuals.

Separation of lymphocytes for in vitro diagnosis

LymphoSep Lymphocyte Separation Medium is based on the highly optimized Bøyum formulation, which was originally designed for the in vitro isolation of lymphocytes from diluted whole blood. It has a density of 1.077 g/mL. LymphoSep has been validated for in vitro diagnostic (IVD) use and designated by the US Food and Drug Administration (FDA) as a Class I medical device which, as a means of separating lymphocytes, is exempt from the FDA’s premarket notification requirements (21CFR864.8500). Like LSM, LymphoSep provides a high yield of lymphocyte cells with greater than 96% cell viability in single-step centrifugation.

Defibrinated or heparinized blood samples are first diluted with physiological saline or balanced salt solution in a 1:1 ratio, layered on the density gradient medium for cell separation, and centrifuged at low speed about 400 g for 15 to 30 minutes. . During centrifugation, the blood constituents migrate differentially in several layers.

When LymphoSep is used as the separation medium, the layers that are formed are like those that are formed when LSM is used. They are, from top to bottom, as follows: blood plasma and other constituents; the so-called buffy coat (containing cells such as lymphocytes and monocytes); separation medium; and a precipitate (in the form of a ball at the bottom of a conical tube) containing granulocytes and erythrocytes (red blood cells). To isolate PBMCs, the buffy coat is removed, washed with a buffered salt solution, and then centrifuged, allowing cells to be recovered in high yield in a small volume. The supernatant, containing platelets, separation medium, and plasma, is removed, leaving a pellet of purified PBMC. These cells can then be used in life science applications such as flow cytometry, cell sorting, cell culture, and sequencing procedures.

Isolation of polymorphonuclear cells

Often, researchers want to do more than just isolate the mononuclear cell population. When it is necessary to separate mononuclear and polymorphonuclear cells from blood, a monopoly resolution medium (M-PRM) can be used. It is specifically designed for the optimal separation of mononuclear lymphocytes and polymorphonuclear leukocytes from contaminating erythrocytes in a single-step centrifugation. M-PRM is a solution composed of a polysaccharide (Ficoll 400) and a radiopaque contrast medium (Hypaque) in a specific ratio to produce a density of 1.114 ± 0.002 g/mL. Undiluted anticoagulant-treated blood is layered onto M-PRM prior to centrifugation. Differential migration during centrifugation allows the resolution of mononuclear and polymorphonuclear leukocytes into two distinct bands that are relatively free of erythrocytes.

Typical results from various laboratories have shown that M-PRM blood separation can achieve >90% leukocyte recovery with >99% viability as assessed by the trypan blue dye exclusion method. Generally, the upper band of leukocytes (fraction 1) contains 94 to 98% mononuclear cells (lymphocytes and macrophages), while the second (fraction 2) contains 96 to 99% polymorphonuclear leukocytes.

Both mononuclear and polymorphonuclear leukocytes isolated by this system have been shown to retain functional properties. In particular, polymorphonuclear cells fractionated using M-PRM have better functional capabilities than those prepared by other methods. For example, M-PRM has been used for the isolation of neutrophils from the blood for use in a wide variety of assays for immunological research. These phagocytic granulocytes, which act by ingesting and/or releasing enzymes that kill microorganisms, are the first line of defence against infection. They are an essential part of the innate immune system.

Isolation of mononuclear and polymorphonuclear cells is often the first and often the most critical step in specific cell sorting and separation procedures. It can provide a viable population of cells for subsequent steps, which typically employ flow cytometer or magnetic bead-based systems. Targeted separation is more efficient and cost-effective if the starting material is a highly enriched fraction rather than whole blood. Since the cell subpopulations of interest are extremely rare with limited sample size and volume, it is of the utmost importance to obtain the highest yield of viable cells, without functional degradations, from the sample you choose.

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