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magnetic activated cell sorting

Magnetic activated cell sorting for whole blood cell separation

Cell sorting is widely used in research and clinical therapy. The latest advances in stem cell therapy, tissue engineering and regenerative medicine show the potential of cells derived from different tissues. Sorting cells from a heterogeneous population enables the study of the different isolated types, but also allows for the introduction of enriched cell populations to a patient. The use of highly selective separation procedures is also critical to improve cell-based treatments on stem cell therapy, tissue engineering and regenerative medicine.

Free PDF guide: "Basic guide to Magnetic Bead Cell Separation"

Magnetic activated cell sorting is an alternative to centrifugation, columns, filtration and precipitation. It eliminates undue cell stress and reduces the risk of negative impact on cell function and phenotype.

In a typical process, magnetic beads are added to a cell sample, which is then incubated. When the magnetic activated cells are placed with the entire mixed-cell population into a Magnetic Bead Separation system, the targeted cells are pulled by magnetic force, separating them from the cell culture with the attached beads.

The growing demand for cell sorting protocols is putting a lot of pressure on scientists, both in academy and industry. Great efforts are made to select the right markers and magnetic beads to ensure their correct attachment to the desired cells. However, most of the tools and protocols are developed for low viscosity media (water, plasma etc) and new applications usually require working on whole blood or other dense suspension.

How could this affect the Magnetic Bead Separation process? It would depend on the magnetic separation rack being used. The first approach is to use the same magnetic separation device.

If you are interested in using Magnetic Bead Separation for isolating cells, check out our latest free guide: The Basic guide to magnetic bead cell separation:

Magnetic activated cell sorting using a classical magnetic separation rack: the cause of the problems when the cells are in whole blood

In common magnetic separators, the magnetic force decreases with distance. As a result the farthest beads experience less force than those that are closer. Since the force depends on the distance to the magnet, the separation time increases exponentially with the diameter of the tube/vessel. This is a well known limitation in the use of classical separation racks for large volumes of sample (see, for example “The Basic Guide for Scaling Up Magnetic Bead Separation Processes”).

In magnetic activated cell sorting, sample volumes are usually small. Therefore, it is generally believed that performing the process in the same commercial separators used for small volume in immunoassays or protein purification should not be problematic. However, this hypothesis does not resist the reality check. Even when the separation time is increased, the efficiency of classical separation racks is very low when used with whole blood, leading to the loss of a significant fraction of the labeled cells.

The reason is simple: the separation speed is the result of the competition between the magnetic force and the drag force. The latter is directly proportional to the viscosity of the media, and whole blood has a viscosity 3-4 times that of water. As the magnetic force is the same but the drag force is 3-4 times higher, the separation speed at each point would be 3-4 times lower in whole blood than in water. The effect would be similar to using a tube vessel that was 3-4 times larger: the separation time would need to be about 50 times longer to collect the same amount of magnetic labelled cells.

How to use magnetic activated cell sorting with homogeneous magnetic force

The physics of the process is the same for advanced Magnetic Bead Separation systems. However, in these modern devices the magnetic force is homogenous: i.e. the value of the force does not depend on the distance to the retention area. This means that the separation time is linearly dependent on the viscosity: the magnetic force is the same at all points, as is the speed.

As a result, speed is constant during the whole magnetic activated cell trajectory. Thus, the separation time is proportionally longer, by a factor of between 3 and 4. In the same way as homogenous Magnetic Bead Separation systems can be used for scaling-up process to larger volumes (up to tens of liters), the constant value of the force over the whole working volume allows the use with viscous media, such as blood.

The figure shows an example of the separation vs. time of the same concentration of magnetic activated cells in two different suspensions. The thin lines (the fast curves) show the time needed for 100% recovery when the buffer is water. As expected, the inhomogeneous magnetic separation rack –grey line- separates the first fraction faster (force is higher close to the retention area) but takes considerably longer to collect the last third of the magnetic activated cells. The homogenous Magnetic Bead Separation system –orange line-, collects 100% of the cells much faster, although its effect on the first fraction is slower due the gentler retention force (see ‘Does it matter what magnetic separation rack I use in my cell sorting processes?’ for further details).

As we change to whole blood (thick lines), the effect on the homogenous Magnetic Bead Separation system (this can be a SEPMAG® A or a SEPMAG® LAB) is simply a change in speed, inversely proportional to the viscosity increase. However, if a classical separation device is used, the time necessary to collect all the magnetic labelled cells increases exponentially. Note that if there is no risk of damaging the cell with the initial excessive force, classical magnetic separation racks can be an option for very small volumes in low viscosity media. Homogenous systems, however, have the additional advantage of achieving more consistent results, as small variations in the position of the tube do not affect the separation time, which would not be true in inhomogeneous devices.

advanced magnetic activated cell sorting

Advanced Magnetic Bead Separation systems, having a very well defined force, also allow precise definition of magnetic cell sorting conditions. If a shorter separation time is necessary, the magnetic force can be increased by designing a customized system. In the example, we may design a Magnetic Bead Separation system with a magnetic force 3-4 times higher than a given value. Therefore, the magnetic separation time of the labelled cells would be 3-4 times faster: we can design a tailored system to obtain the same separation time for whole blood samples as that achieved by ‘standard’ advanced Magnetic Bead Separation when water suspension is used.

As in most Magnetic Bead Separation processes, magnetic cell sorting requires a great deal of interdisciplinary knowledge. It takes a lot of talent, work and investment to find the right marker to efficiently and selectively label the target cells. Finding the right magnetic beads and developing the protocol for coupling the biomarker to them would also imply a significant effort. However, a third step also needs to be considered: an efficient magnetic activated cell sorting process requires choosing the Magnetic Bead Separation conditions carefully.

In the preliminary quick tests, when the team is still focused on the biomarkers and how to couple them to the beads, most of the tests are done in small volumes and low viscosity media. If the results are good enough, the inefficiencies are attributed to lack of optimization of the biochemistry part. Even if this is sometimes true, neglecting good and well controlled Magnetic Bead Separation conditions implies a large project bottle neck when the biochemical part is successfully completed. As soon as the volume increases a little or the experiments start to use whole blood, not having the right Magnetic Bead Separation conditions can jeopardize the whole project.

Please, check our page for FREE eBooks on Magnetic Bead Separation and find out how to avoid most of these problems. If you need help and you think our technology can help you, just contact us!

FREE Download: Basic guide to magnetic bead cell separation

Lluis M. Martínez | SEPMAG Chief Scientific Officer

Founder of SEPMAG, Lluis holds a PhD in Magnetic Materials by the UAB. He has conducted research at German and Spanish academic institutions. Having worked in companies in Ireland, USA and Spain, he has more than 20 years of experience applying magnetic materials and sensors to industrial products and processes. He has filed several international patents on the field and co-authored more than 20 scientific papers, most of them on the subject of magnetic particle movement.

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