Whether in immunology, oncology or regenerative medicine - wherever cells are used, cell separation plays a decisive role. Blood and tissue samples contain a large number of different cell types, which together form a complex biological system. For research purposes, however, it is often necessary to isolate specific cells or cell populations in a targeted manner. This is the only way to precisely analyse their properties, functions and interactions.
However, not every method is equally suitable. The choice of the right technique depends on the issue at hand - and also on the purity, viability and yield required. It is not just a question of know-how, but also of the right tools and reagents that enable standardised and reproducible separation.
Basics of cell separation
Cell separation is the separation of specific cells or cell groups from a heterogeneous mixture, such as occurs in blood (whole blood), bone marrow or tissue. The aim is to obtain the desired cells as unchanged and functional as possible.
There are different approaches to this:
- Physical properties: size, density or electrical charge
- Biological characteristics: Surface markers, receptors or antigens
- Mechanical separation: filtration through special sieves or membranes
The challenge lies in finding a balance between purity, speed and cell protection. While some methods deliver a high level of purity, they can also place a greater strain on the cells. Other methods are particularly gentle, but separate less specifically.
Purity of a cell population
describes in cell biology the proportion of the desired cell population in relation to other, unwanted cell types after an isolation process. Purity thus indicates how "clean" the isolated cell fraction is in terms of its cellular composition.
- Analytical accuracy: high purity is crucial to ensure that measurement results (e.g., gene expression analyses, proteomics) are indeed attributable to the target cell population.
- Reproducibility: impure samples can lead to variable or contradictory results.
- Functional experiments: contaminating cell types can influence the physiological responses or signaling pathways of the target cells.
- Flow Cytometry: Most common method; cells are characterized based on specific surface markers.
- Microscopic evaluation: Morphological assessment, often supplementary.
- Molecular analyses: Detection of cell-specific genes or proteins to verify homogeneity.
Viability (life capability) of the isolated cells
refers to the proportion of intact, metabolically active, and viable cells within a cell population after isolation. Viability indicates how many of the isolated cells are still alive and functional, in contrast to cells that have been damaged or have died during the isolation process. Thus, viability defines the percentage of living cells after isolation and is a key indicator of the quality and suitability of the isolated cell population for further analyses or cultures.
- Mechanical stress (e.g. excessive pipetting or centrifugation)
- Chemical influences (e.g. unsuitable buffers, temperature deviations)
- Too long or aggressive processing processes
- Unfavorable storage conditions before or after isolation
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The viability can be measured using various methods, e.g.:
- Trypan blue exclusion method: Dead cells take up the dye, living cells do not.
- Fluorescent dyes such as Propidium iodide (PI), 7-AAD, or Annexin V/PI in combination with flow cytometry.
- Automated cell counters (e.g., Countess, NucleoCounter).
Yield in Cell Isolation
In cell biology, it refers to the total number of successfully obtained target cells after an isolation procedure in relation to the originally present cell count in the starting sample. The yield indicates how many of the desired cells could actually be isolated, in relation to the theoretically possible maximum, and is a central efficiency parameter in cell isolation. It provides information on how effectively a method is able to obtain the desired cell population with minimal loss and in a targeted manner.
The yield in % is calculated by dividing the number of isolated target cells by the number of target cells in the starting sample and then multiplying by 100. If a blood sample contains approximately 1×10^6 CD4⁺ T cells and after isolation 8×10^5 CD4⁺ T cells are present, the yield is 80%.
- the starting samples are limited (e.g. biopsies, rare cell types),
- large amounts of cells are needed for subsequent analyses,
- or the isolation process is to be validated (e.g., in clinical studies or diagnostics).
- Efficiency of the separation method (e.g., density gradient centrifugation, magnetic separation, FACS)
- Cell losses due to adhesion, filtration, or pipetting
- Cell aggregation or clumping
- Storage time and sample quality before isolation
Methods of cell separation
Various methods are available for the separation of cells. Each method has its own strengths, limitations and typical areas of application.
Cell sieves or sieve cloths are used to separate cells according to size or shape. This method is particularly practical for the preparation of single cell suspensions from tissue or for the purification of samples prior to subsequent analyses.
Advantage: quick, easy, gentle
Disadvantage: lower specificity compared to marker-based methods
Density gradient centrifugation is a classic technique that is primarily used to isolate peripheral blood mononuclear cells (PBMCs) from whole blood. The blood is layered onto a medium with a defined density (e.g. PBMC Spin or Ficoll) and centrifuged. The cell types accumulate in different layers according to their density so that the desired fraction can be easily extracted.
Advantage: reliable, cost-effective
Nachteil: zeitaufwendig, weniger spezifisch als Marker gestützte Methoden
The pluriBead technology enables the targeted enrichment or removal of specific cell types from complex samples such as blood (whole blood) or tissue. pluriBead is a combination of biological specificity and mechanical separation. Using specific antibodies, the desired cells are bound to small monodisperse beads and then selectively separated using filter processes - completely without a magnetic field. The process is fast, gentle and flexible: it is suitable for different cell types, can be easily scaled and preserves the functionality of the cells. Ideal for immune cell analyses, preparations for single cell studies or other applications where high purity cell populations are required.
Advantage: high specificity, good purity, fast
Disadvantage: more expensive reagents, marker dependence
Magnetic Activated Cell Sorting (MACS) uses magnetic particles that bind specifically to cell surface markers. The labelled cells can then be specifically enriched or removed with the aid of a magnetic field. This technique is particularly useful when high-purity cell populations are required, e.g. for immune cell studies. This method is suitable for both positive and negative cell separation.
Advantage: high specificity, good purity
Disadvantage: more expensive reagents, marker dependence
Fluorescence-Activated Cell Sorting (FACS) is a method in which cells can be individually analysed and sorted based on their surface markers and fluorescence signals. This technique not only allows separation, but also provides comprehensive data on each individual cell.
Advantage: high specificity, good purity, time-consuming
Disadvantage: more expensive reagents and devices, marker dependency