A Comprehensive Review of Cell Isolation Methods
- 1. A Comprehensive Review of Cell Isolation Methods Isolation of one or multiple cell types from a heterogeneous population is an integral part of modern biological research and routine clinical diagnosis and treatment. Purification of specific cells is essential for basic cell biology research, cellular enumeration in specific pathologies and cell-based regenerative therapies. The central principle of separating any cell type from a population is to utilize one or more properties that are unique to that cell type.
- 2. Basic Principle of Cell Isolation A cell isolation procedure can either be a positive selection or a negative selection – the former aims at isolating the target cell type from the entire population, usually with specific antibodies while the latter strategy involves the depletion of all cell types of the population resulting in only the target cells remaining.
- 3. Due to the use of specific antibodies targeting a particular cell type, positive selection yields a higher purity of the desired population. Furthermore, a cell population isolated through positive selection can be sequentially purified through several cycles of the procedure, a benefit that negative selective cannot provide. Positive Selection Negative Selection It is more complicated to design an antibody cocktail to deplete all the non-target cells making negative selection less efficient vis-à-vis purity. Positively selected cells carry antibodies and other labeling agents that may interfere with downstream culture and assays – if that is a concern, it is preferable to use a negative selection method. VS
- 4. Cell Characteristics As The Basis For Cell Separation The physical properties of size and density are commonly used for the bulk recovery of cells; either by sedimentation, filtration or density gradient centrifugation. Cell size and density Specific binding of surface antigens to either antibodies or aptamers can selectively capture cells of the specific surface phenotype. The captured cells are subsequently detected with the help of measurable probes. Surface markers This feature determines the extent of attachment of cells to plastic and other polymer surfaces and can be used to separate adherent cells from suspension/free-floating cells. Surface charge & adhesion Different cell types can be distinguished by shape, histological staining, media- selective growth, redox potential and other visual and behavioral properties which can then be harnessed to isolate those cells. Cell morphology & physiology
- 5. Adherent Cells These cells require a suitable surface to attach to thrive. This attachment occurs through the interaction between the cell adhesion molecules (CAMs) on the cell surface and the corresponding ligands on the culture surface. These cells naturally do not require an attachment surface and occur in suspension in the body, usually in fluids like blood and lymph. Examples include lymphocytes, granulocytes, and other immune cells. Cancer cells that have lost contact inhibition and anchorage dependency. In serum-free conditions without any anchorage facilities, cancer cells can form spheroids and amorphous aggregates. Suspension Cells Cancer Cells
- 6. Cell Isolation Through Adhesion Properties Adherent Culture Suspension Culture Adherent cultures – example: macrophages, fibroblasts, mesenchymal cells Macrophages are routinely isolated from bone marrow or peripheral blood. Right after the isolation of mononuclear cells, they can be seeded on coated polystyrene plates along-with serum and monocyte/macrophage differentiation cytokine cocktail. After 5-7 days, the cells differentiate and form an adherent monolayer while the unwanted cells remain in suspension and are discarded. Suspension cultures – examples: stem cells, embryoid bodies, tumorspheres. Cells naturally growing in suspension and cells that have lost anchorage dependency can be separated from the adherent cells by culturing in ultra-low attachment plates in the absence of serum. The desired cells either grow in a single- cell suspension or aggregate to form floating spheroids. The adherent cells die out without the support of an attachment surface.
- 7. Advantages and Limitations of Adhesion-Based Isolation Adhesion-based methods are technically simple, reproducible and mostly cost-effective which yield a good crop of the cells. The limitation is that the purity of the recovered cells is low and there is always a risk of cross-contamination with other cells as well as with bacteria.
- 8. Cell Density/Size-Based Separation Separation techniques relying on the size and density of cells are frequently used to separate specific cell types from bulk peripheral blood (PB) and bone marrow (BM). The most commonly used methods in this category include density gradient centrifugation and filtration. They are also called ‘bulk sorting’ methods since they help isolate relatively large cell populations in a short duration. Although bulk sorting methods are an essential part of several clinical and biotechnology procedures because of high yields, the purity and homogeneity of the harvested cells are quite low compared to other cell separation procedures.
- 9. Density Gradient Centrifugation Filtration The biological sample is centrifuged in a suitable gradient medium at the appropriate speed till the different cell types are fractionated into different layers or phases depending on their respective densities. Filtration-based cell separation is a size- based method wherein cells smaller than the pore size of the specific filtration device pass through and larger cells are trapped. Advantages and Limitations Advantages and Limitations The procedure is technically simple and cost-effective. It can be scaled up or down as per requirement with minimal adjustments. The yield of cells obtained, especially for blood samples, is high. The purity of the different cell fractions obtained is low, especially when fractionating blood. The procedure is time-consuming and low throughput. Simple, easy to perform and reproducible. Filters containing the captured cells can be directly used in downstream assays. Poor specificity and purity – rare cells are frequently lost, and false positives are common. The surface phenotype of some cancer cells is lost during the process, which affects downstream assays.
- 10. A specific cell type can be selected over unwanted cell populations by culturing them in a medium that provides some selective advantage to the desired cell type. In routine cell culture, a particular cell type can be enriched by adding specific growth factors and cytokines – typical examples include enriching populations of a specific lineage and differentiation stage. Methods Based on Cell Physiology and Morphology
- 11. This strategy can be best explained by the HAT medium selection often used in hybridoma technology. HAT stands for hypoxanthine-aminopterin- thymidine: aminopterin blocks de-novo DNA synthesis and hypoxanthine and thymidine provide the raw materials for the alternative ‘salvage pathway’. The key enzyme needed for the salvage pathway is HGPRT – hypoxanthine guanine phosphoribosyltransferase and only cells expressing the HGPRT gene can survive in the HAT medium. Metabolic/Biosynthetic Enzymes If the transfected cells express an antibiotic resistance gene, the specific antibiotic is added to the medium at a concentration that is lethal to the cells which do not express the resistance gene. Common antibiotics used for mammalian cell selection include bleomycin, puromycin and hygromycin. Antibiotic Resistance The Principle of Selective Growth
- 12. The time and labor required for the expansion and maintenance of the relevant clones The emergence of spontaneous resistant clones that do not carry the gene of interest The high costs of some reagents and specialized media Reproducibility Relative ease of performance Adequate cell yield The Advantages and Limitations of Selective Media Isolation Advantages Limitations
- 13. Laser capture microdissection (LCM) was developed to isolate pure cell populations from heterogeneous tissues on the basis of their natural morphology or specific histological/immunological staining. Laser Capture Microdissection A variety of source materials can be utilized for LCM technology: examples include live cells from cell cultures, formalin-fixed paraffin embedded tissues, frozen tissues, fresh tissues, metaphase spreads, and blood smears. Tissue sections (frozen or paraffin embedded) are the most commonly used samples and for optimal capture and analysis.
- 14. The Advantages of LCM Depending on the laser voltage and tissue structure, as many as several thousands of cells can be collected in a short period A wide range of tissues can be subjected to LCM LCM microscope and platforms are easy to operate and can be easily synergized with other assays LCM collected cells can be successfully used for downstream assays requiring functionally active nucleic acids and proteins. Due to the high precision laser beams, the damage to adjacent tissues is minimal, and one tissue section can be probed several times
- 15. For removal of selected cells post laser targeting; the tissue sections cannot be coverslipped. Without mounting medium and coverslip, the refractive index of the tissue is altered. Most LCM platforms employ lasers whose minimal spot size is 7.5µm, and this is not precise enough to isolate single cells. Another technical challenge that results due to tissue drying (especially frozen tissues) is the lack of adherence of the dissected tissue onto the film or inverted cap. The Limitations of LCM
- 16. Website: www.creative-bioarray.com E-mail: info@creative-bioarray.com Cell Surface Marker- Based Techniques Cellular surfaces are populated with a multitude of ligands – proteins, carbohydrates, and glycoproteins - which perform various biological functions and give every cell a unique surface phenotype. Antibody-mediated cell detection and isolation techniques became commonplace with the discovery of the cluster of differentiation (CD) markers – surface receptors usually involved in cell signaling and adhesion. The unique CD profiles of different cell types are used to define and separate different populations within a tissue, isolate rare cells like adult stem cells, cancer stem cells, etc. and differentiate between control and treated cells depending on the experimental design.
- 17. Fluorescence-Activated Cell Sorting (FACS) The Fluorescence Activated Cell Sorting (FACS) was invented by Bonner and Herzenberg for sorting viable cells with the help of fluorescent probes. The populations stained with different fluorophore- tagged antibodies can be separated by the different fluorescent signals that they generate – in terms of wavelength and intensities. FACS is used exclusively for the positive selection and isolation of cells.
- 18. FACS is a highly sensitive and high throughput procedure for isolating cells from heterogeneous populations. It is the ideal method of simultaneously sorting multiple populations based on just their immuno-phenotype. FACS is a highly versatile technology that can separate cells based not only on surface markers but also cell size and granularity, cell cycle status, intracellular cytokine expression, metabolic status, etc. The Advantages of FACS
- 19. FACS raises the problem of ‘spillover’ of the different fluorophores into non-specific channels. The recovery of most FACS sorters is around 50%-70% making it necessary to begin the sort with a high number of cells. As the sophistication and precision of FACS instruments increase, so does the probability of technical errors. Since FACS requires single-cell suspension, information regarding the tissue architecture and inter-cellular interactions are not available. The Challenges of FACS
- 20. Immuno-magnetic separation of cells is based on the deflection of cells in a magnetic gradient field. Based on the type of magnetic particles used, immunomagnetic separation are mainly of two kinds – Dynabeads®-based and MACS™ technologies. Dynabeads® can be coupled with antibodies or other ligands to targeting of specific cells or immunoprecipitation reactions. The target – or non-target cells depending on the selection strategy – are labeled with 50nm magnetic microbeads conjugated antibodies and then subjected to a magnetic field. Dynabeads® MACS Immuno-magnetic techniques can be used for both positive and negative selection of cells. Magnetic Separation
- 21. MACS is a high throughput, selective and rapid method for isolating target populations or removing undesired cells. Advantages Advantages MACS can be scaled up or down depending on the desired yield and the downstream applications. Advantages It can be extended to a wide range of cells and over various platforms including microfluidic devices. Advantages The MACS columns help isolate rare cells with high specificity. The Advantages of Magnetic Separation
- 22. In case sorting with Dynabeads®, the latter need to be eluted out owing to their large sizes as it may affect downstream assays. Separating cells using magnet separators is not very efficient as draining out the unbound cells. For delicate cells, binding of the magnetic particles may cause mechanical shear. MACS columns should be used instead of separators to minimize the shear. The Limitations of Magnetic Separation
- 23. Aptamers are single-stranded oligonucleotides – DNA or RNA – that can bind to highly specific targets based on structural conformation. Aptamers were first generated using a procedure called SELEX – Systemic Evolution of Ligands by Exponential enrichment – which involves the sequential binding of the oligonucleotides to target molecules. Aptamer Technology
- 24. Direct binding Target induced structural switch Sandwich binding Target induced dissociation It is the simplest assay where aptamers labeled with a single moiety of a fluorophore or a luminophore directly bind to the target and the signal can be easily read out with a suitable detector. In the absence of the target, the fluorophore-labeled DNA aptamer forms a partial duplex with another (non-specific) aptamer labeled with a quencher. When the target is introduced, the aptamer binds to the target, thereby releasing the quencher from the fluorophore and triggering the latter’s signal. The specific unlabeled aptamers immobilized on a solid phase first capture the target cells, which is followed by the addition of the biotinylated version of the same aptamers. The signal is generated through further binding of streptavidin-conjugated HRP to the biotin molecules as in ELISA. DNA aptamers coiled with gold nanoparticles (AuNPs) are used for this assay. As soon as the aptamers see the target, they uncoil and bind to the target cells and release the AuNPs. The latter are precipitated in a salt solution bringing about a color change. Aptamer Technology
- 25. The preparation of aptamers is completely in vitro and thus obviates the use of live animals. The repertoire of specific aptamers that can be synthesized for targeting any cell is virtually limitless. The chemical synthesis of aptamers and the scale-out PCRs are easy to perform and cost- effective. The aptamer assays are highly reproducible, and variability is generally very low between different batches.
- 26. Advantages of FACS Due to the vast number of aptamers generated, there is always the risk of non-specific binding. Therefore several clones need to be tested for specificity which can be time-consuming. The safety and bio-compatibility concerns of aptamers have not been addressed so far, therefore it not yet approved for clinical applications. The cell yield is very low which does not make it an ideal method for the isolation of rare cells.
- 27. Certain cell isolation techniques make use of more than one cell characteristic (as classified above) and are therefore the combination of at least two different techniques. The objective is to either combine the advantages of both techniques or overcome the limitations of one of them. The combination is usually that of an immuno-label-based technique with a label-free technique utilizing a cell property like density, size, morphology, etc. Combination Isolation Techniques
- 28. USA Address: SUITE 115, 17 Ramsey Road, Shirley, NY 11967, USA Tel: 1-631-626-9181 Fax: 1-631-614-7828 info@creative-bioarray.com Europe Tel: 44-208-123-7131 info@creative-bioarray.com Email: info@creative-bioarray.com Email: info@creative-bioarray.com Contact Us