cell isolation and cell isolation methods
- 1. In the name of God 1
- 2. Cell Separation and Cell Isolation Methods
- 3. Introduction • Cell biology is complex, given the multitude of variables that researchers must be aware of and account for in order to obtain meaningful results. Conducting experiments on an isolated population of cells, rather than a heterogeneous mixture of cells, is a common approach to reduce experimental complexity. This allows cell biologists to confidently attribute observed effects and responses to a particular cell type. Thus, mastering the basic techniques of cell isolation is a valuable skill for any cell biologist.
- 4. What is cell separation? • Cell separation, also commonly referred to as cell isolation or cell sorting, is a process to isolate one or more specific cell populations from a heterogeneous mixture of cells. There are a number of cell separation methods available, each with its own pros and cons.
- 5. Why do scientists isolate cells? Conducting experiments on isolated cells allows scientists to confidently answer specific research questions by minimizing interference from other cell types within the sample. Isolated cells have many applications within life science research, allowing scientists to: •Conduct molecular analysis of a single cell type, including RNA expression and epigenetic analysis •Genetically modify and expand a particular cell type of interest for disease modelling or cell therapy research applications (e.g. T cell therapy research) •Directly use purified cells for adoptive cell transfer experiments in various animal models •Increase sensitivity of analytical methods (e.g. cell isolation for HLA analysis, cell isolation for FISH analysis) •Study the in vitro effects of drug candidates on specific cell populations (e.g. hematotoxicity testing) •Fuse enriched plasma cells with myeloma cells to produce hybridomas
- 6. 6 • How do scientists prepare samples for cell separation? • There are many different ways to prepare samples for optimal cell isolation. The method you select depends on your starting sample and may involve removing certain elements from it or simply creating a single-cell suspension. • Cell separation can be performed on a variety of complex biological samples, including: • Whole peripheral blood • Leukapheresis products (e.g. Leukopaks) • Peripheral blood mononuclear cells (PBMCs) • Bone marrow • Cord blood • Spleen and lymph nodes • Other tissues (e.g. skin, liver, lung, fat, brain, tumor, etc.)
- 8. Immunomagnetic Cell Separation Immunomagnetic cell separation is a technique whereby magnetic particles are used to isolate target cells from heterogeneous mixtures. To accomplish this, the magnetic particles are bound to specific cell surface proteins on the target cells via antibodies, enzymes, lectins, or streptavidin. The sample is then placed in an electromagnetic field that pulls on the magnetic particles, bringing the labeled cells with them. The unlabeled cells remain in the supernatant, thus creating a physical separation between target and non- target cells within the sample.
- 9. Magnetic cell separation, also known as immunomagnetic cell separation or magnetic cell sorting, involves targeting cells for selection or depletion using antibodies or ligands directed against specific cell surface antigens. Labeled cells are cross-linked to magnetic particles, also known as magnetic beads, that can be immobilized once an electromagnetic field is applied. Due to its speed and simplicity, magnetic cell separation is one of the most commonly used methods by which scientists isolate highly purified populations of specific cell subsets. Should I use positive selection or negative selection? Negative and Positive Selection in a Column-Free Magnetic Cell Separation Technology
- 10. column-based and column-free magnetic cell separation methods While commonly-used, column-based magnetic cell isolation protocols can sometimes be costly, complicated, laborious, and time-consuming, requiring multiple washes to avoid contamination between separations and the use of new columns for each experiment. In addition, it’s not uncommon for columns to become clogged, risking the loss of precious samples, especially when working with tissue samples that contain a significant amounts of debris.
- 11. Which method should you choose? In general, column-based and column-free technologies are both well-established methods that result in highly purified cells. Both technologies have been used by life science researchers for more than 20 years in a variety of applications and with thousands of citations in peer-reviewed publications. In an increasingly competitive research environment, we recommend choosing the most efficient technologies available to help you complete your cell separation—and, ultimately, your downstream experiments— in less time and with less effort. In our experience, column-free magnetic cell isolation techniques are the most efficient approaches to isolate highly purified
- 12. Due to its speed and simplicity, immunomagnetic cell separation is one of the most commonly used methods by which scientists isolate highly purified populations of specific cell subsets. Immunomagnetic cell separation has several advantages, including: •High purity •Fast protocols •Ease of use •Low equipment cost •Many cells can be isolated at once •Potential for automation •High cell viability
- 13. Fluorescence-activated Cell Sorting • Fluorescence-activated cell sorting (FACS) is a method that uses flow cytometry and fluorescent probes to sort heterogeneous mixtures of cells. Fluorophore-tagged antibodies bind to epitopes on specific antigens on the target cells within a single-cell suspension. After tagging, the flow cytometer focuses the cell suspension into a uniform stream of single cells. This stream is then passed through a set of lasers that excites the cell-bound fluorophores, causing light scattering and fluorescent emissions. Based on the wavelengths produced by the laser excitation, the resulting photon signals are converted into a proportional number of electronic pulses that assign a charge to the droplet that is formed around the cell. As each droplet falls between the deflection plates, its charge causes the droplet to either be deflected into collection tubes or fall into the waste chamber.
- 14. 14 A fluorescence-activated cell sorter (FACS)
- 15. Pre-enrich samples prior to FACS Isolating rare cell types by FACS can be time consuming, expensive and can result in low cell recovery. Researchers can pre-enrich their samples for target cells using immunomagnetic cell separation to reduce the sort time and improve purity and recovery. researchers may choose to pre-enrich for other cell types, including CD4+ T cells, CD8+ T cells, B cells, or dendritic cells, prior to sorting for more specific or rare cell subsets.
- 16. 16 Density Gradient Centrifugation Density gradient centrifugation relies on the varying densities of cells within a heterogeneous sample. The sample is layered on top of a density gradient medium before being centrifuged. During centrifugation, each cell type will sediment to its isopycnic point, which is the place in the medium gradient where the density of the cells and medium are equal. Common applications include the fractionation of peripheral blood mononuclear cells, exclusion of dead cells from a cell culture, and separation of plasma from blood cells.
- 17. 17 There are several types of density gradient media: •Lymphoprep™, Lympholyte® , and Ficoll- Paque® are similar media that consist of saccharides and sodium diatrizoate; they have a density of 1.077 g/mL. These media are commonly used to isolate mononuclear cells from peripheral blood, cord blood, and bone marrow. •Percoll® (density: 1.131 g/mL) consists of colloidal silica particles coated with polyvinylpyrrolidone (PVP) and is widely used to separate cells, organelles, viruses, and other subcellular particles. •OptiPrep™ is a medium consisting of iodixanol in water that is used to isolate viruses, organelles, macromolecules, and cells.
- 18. Density gradient centrifugation is an inexpensive cell separation technique but has limited specificity: low purity, and low throughput. In addition, even though it is a common laboratory technique, density gradient centrifugation can be a slow and laborious process that is difficult to master. Scientists typically need to carefully layer their sample over the density gradient medium, centrifuge for 30 minutes without brakes, then carefully harvest and wash the appropriate layer of cells. Technologies like SepMate™ make this method easier and faster. SepMate ™ is a specialized tube that allows users to quickly layer blood over the density gradient medium, prevents the layers from mixing and facilitates fast and easy harvesting of the target cells. With SepMate™, cells can be obtained in as little as 15 minutes. Density Gradient Centrifugation
- 19. Immunodensity Cell Separation 19 Immunodensity cell separation, also referred to as erythrocyte rosetting, is a negative selection method that uses a combination of antibody-based labeling and density gradient centrifugation. With this method, antibodies are added to a whole blood sample, labeling the unwanted cells and cross-linking them to red blood cells. This results in the formation of complexes called immunorosettes that are much denser than the mononuclear cells being isolated. During centrifugation, the unwanted cells pellet with the red blood cells, leaving the target cells in a layer above the density medium. Immunodensity cell separation doesn’t require any specialized equipment beyond a centrifuge, can be easily incorporated into established density gradient centrifugation protocols, and can be used to isolate specific cell subsets directly from whole blood. However, the technique is limited to negative selection, relies on the operator’s blood sample layering technique, and requires a high concentration of red blood cells in the starting sample.
- 20. 20 RosetteSep™ is an example of a commercially available immunodensity cell separation reagent . RosetteSep™ can be combined with SepMate™ PBMC isolation tubes for even faster and easier immunodensity cell separation.
- 21. Sedimentation 21 Sedimentation works on the basis that gravity will cause larger and denser components to sediment faster than materials that are smaller and less dense. The largest and densest components in a sample can be pelleted through an initial low-force centrifugation due to their high rate of sedimentation. The supernatant can then be spun again. Through successive centrifugations, components with an increasingly lower rate of sedimentation can be isolated. Leukocytes are commonly separated from erythrocytes through dextran sedimentation. HetaSep™ is an example of an erythrocyte aggregation agent that is used to separate nucleated cells from red blood cells (RBCs) in whole blood. Sedimentation is inexpensive but generally results in lower purity than other methods.
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- 23. Adhesion 23 The unique adhesion profiles of different cell types can be used to separate target cells from heterogeneous populations. By choosing suitable growth factors and cell culture plates to selectively favor or inhibit adhesion, adherent cells can be separated from cells in suspension. Macrophages are inherently adherent and often isolated from peripheral blood and bone marrow by adhesion. Mononuclear cells can be cultured with serum and a differentiation cocktail, promoting the formation of an adherent monolayer of macrophages. After removing the supernatant containing unwanted cells, the macrophages can be isolated.
- 24. Microfluidic Cell Separation 24 Microfluidics is an umbrella category of cell separation methods. Designed to manipulate fluids on a microscopic level to facilitate single-cell isolation, microfluidic technologies are frequently built onto microchips and are commonly known as "lab-on-a-chip" devices. These devices have several advantages, including the smaller volumes of samples and reagents required for use. Lab-on-a-chip devices are also portable, making them particularly useful as field-based diagnostic tools. Microfluidic methods can be divided into active and passive systems. Active microfluidic systems involve external forces, whereas passive microfluidics make use of the cell’s density and mass in combination with gravity. These methods can also be classified by the presence or absence of cell labeling; although some methods involve labeling cells with antibodies, most methods are known for being label-free.
- 25. Several different microfluidic methods used for cell isolation, including: •Acoustophoresis •Aqueous two phase systems •Biomimetic microfluidics •Cell affinity chromatography •Electrophoretic sorting •Field flow fractionation •Gravity and sedimentation •Magnetophoresis •Microfiltration and Optical sorting
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- 27. Other Cell Separation Techniques 27 Aptamer Technology Aptamers are single-stranded RNA or DNA oligonucleotides that form structures that can bind to highly specific targets. Through systematic evolution of ligands by exponential enrichment (SELEX) technology, aptamers can be screened and synthesized to target any cell type. These aptamers have high affinity and specificity toward their targets, and can be labeled with fluorochromes or magnetic particles to facilitate cell separation. The main advantage of aptamers is that they lack immunogenicity. Fluorophore-labeled aptamers have been used to sort mesenchymal stem cells from bone marrow and RNA aptamers have been used to isolate mouse embryonic stem cells.
- 28. 28 Buoyancy-Activated Cell Sorting Buoyancy-Activated cell sorting is a cell separation technique that utilises glass microbubbles labeled with antibodies specific to the target cells. When mixed into the sample, the microbubbles bind to the target cells. Due to the augmented buoyancy force, the microbubbles float to the surface, separating the target cells.
- 29. Laser Capture Microdissection 29 Laser capture microdissection (LCM) is a technique that uses a narrow laser beam to cleave target cells or areas from mostly solid tissue samples. Through microscopic visualization, LCM can isolate cell populations from heterogeneous mixtures using cell morphology or specific histological and immunological staining. LCM is particularly useful when working with small sample sizes. Schematic view on laser capture microdissection (LCM) methods. (a) Contact-based via adhesive tapes; (b) Cutting with a focused laser followed by capture with a vessel. Cut-out section extracted by gravity; and (c) Cutting with a focused laser followed by pressure catapulting with a defocused laser pulse.
- 30. Immunoguided Laser Capture Microdissection 30 Immunoguided laser capture microdissection combines immunostaining with laser capture microdissection. This allows immunophenotypes to be used, in addition to morphology and tissue location, to identify and isolate target cells from the tissue sample. This technique employs immunohistochemistry or immunofluorescence to guide the dissection process for isolating cells expressing a specific molecular marker, and is particularly useful when histological stains do not recognize certain cell
- 31. Limiting Dilution 31 Limiting dilution involves isolating single cells through the dilution of a cell suspension. This technique can be carried out with standard pipetting tools and is commonly used to produce monoclonal cell cultures and single cell cultures for single-cell analysis.
- 32. Micromanipulation 32 Micromanipulation, a form of manual cell picking, is a cell isolation technique involving the use of an inverted microscope and ultra-thin glass capillaries connected to an aspiration and release unit. The system moves through motorized mechanical stages, allowing the operator to carefully select a specific cell and apply suction via micropipette to aspirate and isolate the cell.
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Editor's Notes
- #9: DNA nucleotides, the building blocks for the new DNA Template DNA, the DNA sequence that you want to amplify Primers, single-stranded DNAs between 18 and 30 nucleotides long (oligonucleotides) that are complementary to a short region on either side of the template DNA DNA polymerase, a heat stable enzyme that drives, or catalyzes, the synthesis of new DNA