Cell Separation

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Cell Separation

Cell separation isolates cells according to their properties. Current researchers isolate cells to study the characteristics or biological processes of a certain cell population. Other applications, such as cell therapy, are also under rapid development. Several cell separation approaches have been developed.

Fluorescence-Activated Cell Sorting (FACS)

Traditional FACS separates cells based on their fluorescent characters. Cell mixtures are funneled single-file through a narrow opening that ends in a nozzle, after which, cells are broken into individual cell droplets. As the droplet falls, a laser (or several lasers) is applied to recognize the cells one by one based on their characters. The scatter of the laser light and the fluorescent signal tell the system which population each cell belongs to. Then, the identified droplet passes through an electrostatic deflection system and the computer directs the instrument to send the droplet to the appropriate cell collector.

Schematic illustration of fluorescence-activated cell sorting

Figure 1. Schematic illustration of fluorescence-activated cell sorting

Organic fluorescent dyes, such as rhodamine and fluorine, are the most common fluorescence tags employed in FACS. Various organic fluorescent dye-labeled antibodies have been commercialized and are readily available in the market. However, in some studies, photoblinking and photobleaching have become big obstructers while manipulating these fluorophores. Therefore, quantum dots and upconverting nanoparticles stand out by their robust resistance to photoblinking and photobleaching.

Quantum Dots (QDs)

QDs are nano-sized semiconductor particles with conspicuous optical and electronic property differences from those of larger particles. Similar to organic fluorescent dyes, many types of QDs emit light of specific frequencies once they are excited by a light of shorter wavelength. This fluorescence property can be precisely tuned by changing the QDs’ size, shape and material, which attracted great interest in research applications. Meanwhile, compared to traditional organic fluorescent dyes which can photobleach after absorbing 105–106 photons, CdSe/ZnS QD can undergo up to 108 photon absorption and exhibit 100 times longer lifetime.

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Quantum Dots
Functional Quantum Dots
Quantum Dots Labeling & Conjugation Kits

Upconverting Nanoparticles (UCNPs)

Opposite to traditional fluorescent dyes, UCNPs exhibit photon upconversion. Two or more incident photons of relatively low energy are absorbed and converted into one emitted photon with higher energy, resulting in an absorption in the infrared and an emission in the visible or ultraviolet regions of the electromagnetic spectrum. Their excitation and emission wavelengths can also be changed by tuning their size, shape and material. Therefore, UCNPs can be applied in various applications, such as FACS.

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Upconverting Nanoparticles

Immunomagnetic Cell Separation

FACS classifies and sorts sequentially, which is precious but time-consuming. Especially when it comes to bulk samples, the protracted separation process using FACS may cause cell clumping, losing viability and even cell death. Thus, immunomagnetic cell separation stands out by its high-speed separation ability, which can sort cells simultaneously.

In immunomagnetic cell separation, magnetic particles were characterized with various cell binding ligands, which can recognize a specific cells population of interest. By incubating these particles, the target cells can be magnetically labeled. Therefore, by the application of a magnetic field, these cells can be displaced and separated from mixtures.

Several immunomagnetic cell separation strategies have been developed, such as positive cell separation and negative cell separation. Notably, sorting cells using magnetic particles is appropriate when cells are separated according to only oneseparation criterion or characteristic, rather than several. Multitarget immunomagnetic cell separation requires multiple rounds of beads incubation, which may be even more time consuming than FACS.

Strategies for Cell Separation

  • positive cell separation

Positive cell separation isolates the cells of interest. Magnetic particles bind to the target cells and the whole particle-cell complexes are removed rapidly and mildly from mixtures by the application of a magnetic field.

  • negative cell separation

Negative cell separation isolates the unwanted cells from the mixtures. In this approach, negative cells are magnetically labeled, separated and discarded, leaving behind a purer sample of the cells of interest. No beads are left in the final product.

Schematic illustration of immunomagnetic cell separation process

Figure 2. Schematic illustration of immunomagnetic cell separation process

To recognize the cells of interest, several surface-modified magnetic particles have been developed. Antibody and streptavidin immobilized magnetic particles are the two types of particles that are commonly used cell separations.

Primary Antibody Immobilized Magnetic Particles

Several cell surface receptors have been identified as the biomarkers of a certain cell population. Thus, their corresponding antibodies are conjugated to particles to magnetically label these cells.

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Immobilization of Antibody on Beads

Secondary Antibody Immobilized Magnetic Particles

Secondary antibodies have the ability to capture the primary antibodies. By pre-incubating cells with primary antibodies, cells of interest can be subsequently labeled with secondary antibody immobilized magnetic particles and thus be isolated from mixtures in the presence of a magnetic field.

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Avidin/Streptavidin-biotin Interaction

The streptavidin-biotin binding is the strongest known non-covalent interaction between a protein and ligand and has been exploited for use in many bioseparation processes. A variety of biotinylated primary antibodies have been commercialized and readily available in the market. Avidin/streptavidin-modified magnetic particles can bind to biotinylated antibody-pretreated cells and isolate the target cell by the application of a magnetic field.

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Supplementary Reading

Immunomagnetic cell separation: size matters

Current market offers a variety of magnetic particles with their size ranging from 50nm to several micrometers. A debated on which size range should be chosen for cell separation rises.

Some magnetic particle researchers insisted that nano-sized magnetic particles should be a proper choice for cell separation. They suggested that nano-sized particles offer a mild magnetic field force to separate cells, which causes low damage to the target cells. Meanwhile, nano-sized particles have been widely used and highly published.

On the other hand, another group of scholars argued that the nano-sized particles may be internalized by the cells during incubation. These internalized particles can enter the cell nuclei and influence cell biological processes, leading to an altered cell nature status and even cell death. Whereas, micro-sized particles, since they are roughly the same size as mammalian cells, are less likely to be taken up by the cells. Thus, micro-sized particles should exhibit less influence on the nature status of cells. Meantime, larger particles have a higher magnetic mobility and are capable of separating cells from viscous mixtures.

The table below compared nano- and micro-sized particles in the application of cell separation.

Table 1. Magnetic microparticles vs nanoparticles

Magnetic microparticlesMagnetic nanoparticles
ShapeSpherical, highly uniformedIrregular, variable size
Commercial kits availableFewA lot
PublicationFewer publicationsHighly published
Magnetic field forceStrongerWeaker
Equilibration periodShortLong
Influence on cell nature statusLow influenceHigh influence
Elute from cellsEasyHard
SeparationMore efficientLess efficient

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