32.1:
Overview Of Cell Separation And Isolation
Researchers can isolate cells from a tissue and separate them to obtain a population containing only a single type of cells.
Other than blood, all tissue samples must first be minced into pieces and incubated with proteolytic enzymes to release individual cells.
These cells can then be further isolated based on their physical and biochemical properties.
Density gradient centrifugation uses an inert medium, such as Ficoll, to create a density gradient and separate cells into layers based on their density upon centrifugation.
Other techniques, such as cell affinity chromatography and fluorescence-activated cell sorting or FACS, use affinity interactions of cell surface molecules to isolate cells.
Cell affinity chromatography uses ligands, such as antibodies, immobilized on a stationary matrix to separate target cells that express a specific receptor while the rest of the cells flow through.
Similarly, FACS uses fluorescence-tagged antibodies to sort cells from a mixture based on their cell-surface antigens.
The homogeneous cell populations obtained from cell separation methods can be grown in the lab to establish cell cultures or used directly for analysis.
32.1:
Overview Of Cell Separation And Isolation
Cell separation was first achieved in 1964 by S. H. Seal, who separated large tumor cells from the smaller blood cells using filtration. Two years later, Pohl and Hawk performed experiments on how cells respond differently to a nonuniform electric field based on the cell type. Such observations were the inception of cell separation methods, which allow isolating a single cell type from a heterogeneous sample.
Commonly used cell separation techniques include filtration, density gradient centrifugation, affinity-based separation using antibodies, and flow cytometric cell sorting. Additional specialized techniques include aptamer technology, buoyancy-activated cell sorting, and laser capture microdissection. The choice of separation method is based on the characteristics of the cell type to be isolated, such as surface charge, cell size, density, morphology, physiology, and surface markers. Usually, one or more of these characteristics are employed to isolate the particular cell type efficiently.
Applications of Cell Separation and Isolation
Isolated cells can be grown in vitro to produce cell lines, which have numerous applications in areas like pharmacology, immunology, and stem cell therapy. For example, the in vitro effects of drugs can be analyzed on specific cell populations. Cell separation is also essential in screening for the appropriate B-cells for monoclonal antibody production. Single-cell analysis, such as studying gene expression patterns and epigenetic effects, also relies on the isolation of specific cells to study. Many oncological studies that contribute to our understanding of cancer cells require isolating particular tumor cells from the tissue. Thus, cell separation and isolation methods are used virtually in all major fields of modern biology.
Challenges of Cell Separation Methods
Cell separation techniques face a high noise-to-signal ratio, with a small number of specific cells (target) against a large number of varied components that form the tissue. Various factors affect the purity of the final isolate; hence, the protocols require standardization for each target cell type and application. For example, the efficiency of cells dissociating from the tissue decides the yield of isolated cells. Excess digestion increases the presence of dead cells in the isolate, whereas incomplete digestion results in cell type contamination. Cell type contamination can also occur in affinity-based cell separation. While labeling a particular cell type with specific antibodies, non-specific binding may occur, introducing different cells into the preparation. Additionally, the population size of the target cell type in the tissue is also a limiting factor — low-abundance cells often need tedious protocols to yield sufficient numbers in the isolated cell populations.
Suggested Reading
- Mattanovich, Diethard, and Nicole Borth. "Applications of cell sorting in biotechnology." Microbial cell factories 5.1 (2006): 1-11.
- Nicodemou, Andreas, and Lubos Danisovic. "Mesenchymal stromal/stem cell separation methods: concise review." Cell and Tissue Banking 18.4 (2017): 443-460.
- Aaheli Roy Choudhury., A comprehensive review of cell isolation methods. MATER METHODS 2017;7:2260. //dx.doi.org/10.13070/mm.en.7.2260.
- Atajanov, A., Zhbanov, A. & Yang, S. Sorting and manipulation of biological cells and the prospects for using optical forces. Micro and Nano Syst Lett 6, 2 (2018). https://doi.org/10.1186/s40486-018-0064-3
- Gross A et al. (2015) Technologies for single cell isolation. Int J Mol Sci 16(8): 16897-919.