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Cell Death and Sample Viability

November 2020

Cell Loss

When performing research, it’s easy to forget that individual cells are living things. Each one has its own role and internal organelles that help it survive from day-to-day. The process of isolating cells for research and removing them from their ideal environment can be harmful.

Significance

With each lost cell the sample becomes less valuable. Often, the goal of isolating cells is to study their structure and function. If a cell is damaged or destroyed, it will no longer perform the same way as a healthy cell. The throughput of working cells is a large consideration when deciding on a separation strategy.

Fluorescence Activated Cell Sorting

Fluorescence activated cell sorting, or FACS, is a flow cytometry technique that encompasses light scattering and cell characteristics to sort a mixture between two or more containers. Antibodies are released into a mixture to bind with the target cell and illuminate them. These samples are then run through a special piece of equipment called a flow cytometer that shines a laser through the stream. The laser measures physical properties such as size and granularity then separates the cells accordingly.

Loss

There is a high risk of cell loss that must be accounted for when working with a flow cytometer machine. Not only does the volume of cells passing through the laser sometimes cause cells to be clumped together or improperly sorted, the movement of liquids through a medium also causes issues. Shearing is a form of hydrodynamic stress that results when the force of the flow ruptures the membrane or causes the cells to collide with the edge of the tube.

Shearing can also be a problem in the centrifugation process associated with flow cytometry. To improve the purity of isolated cells, samples are washed in a centrifuge to remove dead or unnecessary cells. The revolutions can be too intense for cell membranes to handle. FACS and flow cytometry create a constant battle between quality and quantity.

Magnetic-Activated Cell Sorting

Another method of cell separation called magnetic-activated cell sorting, or MACS, uses a magnetic field to separate target cells from unwanted substances. By attaching antibodies to magnetic beads and binding them to the surface level markers placed on the target cells, the desired substance can be pulled to the edge of the tubes for collection.

Loss

One of the main drawbacks of this method is the loss of cells in a sample. Due to the harsh nature of a magnetic field, fragile cells run a higher risk of dying with MACS than other cell isolation processes. This technique is not viable with small samples of uncommon cells.

MACS is also used as a strategy to filter out dead cells. The dead cell removal protocol is the process of attaching microbeads to cells that have died in a sample and drawing them to the side of the tube for extraction. In doing so, even more cells die. The removal of these nonviable cells is necessary to maintain a usable sample. Although harmful to the desired sample, MACS can help to remove dead cells from a suspended mixture, which improves purity in the end.

Solution

There are strategies that can be paired with MACS and FACS to reduce the amount of cell loss in an experiment. The tube model, washing agent, and cell type can all effect the throughput of an isolation process.

When using MACS, it’s best to focus mostly on the type of cell being used. Since the main cause of damage is the magnetic forces, altering the tubes or solution won’t have as significant of an effect. By targeting stronger cells of a higher quantity with magnetic cell sorting, the number of healthy cells that make it through will increase as a simple byproduct of their resilience.

FACS, on the other hand, has many more variables at play. If cells are being sorted improperly, the flow cytometer’s rate can be decreased; if cells are clumping, different reagents can be added to keep them apart; even the tube width and material can affect the number of cells lost to shearing.

Minimizing the cells lost is the same as maximizing the value of an experiment financially and temporally. Producing a larger output of cells in an experiment reduces the chances of having to repeat the process. Lost cells add up fast to create a hidden cost when using traditional strategies.

Microbubbles

Most methods require researchers to make a choice between speed and accuracy, but not all. Buoyancy-activated cell sorting, also called BACS, is a cell separation technique that harnesses the power of microbubbles to quickly and gently isolate target cells. Like other methods, antibodies start by binding to antigens on the surface markers of target cells. Specially designed microbubbles then bind to these antibodies, linking them to the desired substance.

One of the reasons BACS is so effective is the low rates of cell death. There is no harsh magnetic field or fast-flowing liquids. Aside from a gentle stir, the cells are not subject to any impact. The microbubbles are soft and float the target cells slowly to the surface to separate them from the rest of the solution.

In contrast with MACS and FACS, BACS is less expensive, less time-consuming, and less harmful to the desired cell sample. Using microbubbles is also simpler than learning the equipment and procedures for other methods. Even the tiniest mistake in FACS or MACS could mitigate an entire trial and waste all the resources that went into it. With BACS, it’s just mixing and stirring, and if something does go wrong another microbubble pack can make up for it in as little as ten minutes.

Try Akadeum’s Microbubble Technology Today

BACS is quicker and cheaper than its competitors. Its strong but gentle nature makes it a solid candidate for isolating cells. If you’re looking for a simple way to improve the process of cell sorting by minimizing cell loss, try Akadeum’s microbubble products today.

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