February 2021 Share
When working with single-suspension cell cultures in a growth medium, it is not uncommon to experience cell loss in a sample. As cells are ruptured, they release DNA and debris that cause cells to aggregate into large clumps that make it difficult for them to expand. Cell clumping can both lead to and be caused by cell apoptosis, or cell death.
As more cells die and release debris, the problem will continue to worsen. Dealing with cell clumping as fast as possible or avoiding it altogether will benefit the downstream results of an experiment.
The goal of a single-cell suspension medium is to give target cells as many resources to grow as possible. This includes ample nutrients for growth and sufficient space for reproduction. When cells clump together, they restrict each other, reducing the throughput and ultimately compromising downstream results.
Cell clumping also leads to lower recovery of target cells and can interfere with labeling. Certain methods of cell analysis such as flow cytometry require cells to be properly isolated. In flow cytometry, a large volume of cells are passed through a machine in a rapid fluid stream. The machine, called a cytometer, detects the difference in physical characteristics between different cells and uses fluorescent light to label them accordingly. If cells are clustered together when they pass through the tube, the cytometer will struggle to accurately measure their characteristics. This can cause them to be improperly sorted and negatively affect downstream results.
One of the best ways to prevent cell clumping in cell culture is to understand what causes it and take precautions to avoid those events from occurring. Some reasons that cells in a culture may clump include:
While some of these happen through experimental error or are unavoidable given a specific cell sample, there are actions that can be taken ahead of time to lower the risk of cell clumping.
There are measures that can be taken before starting an experiment to prevent cells from aggregating. For example, an endonuclease called DNase I can be mixed into a sample to fragment the DNA from ruptured cells. Breaking up this extra debris helps to keep space open for target cells to grow. However, DNase I should not be used when there are intentions to engineer or change cells downstream because it can affect cell health and physiology.
Cell clumps can also be reduced through proper handling and equipment usage. If a centrifuge is being used to separate or mix a sample, setting it to the correct speed can reduce the chances of buildup. Typically, faster speeds prevent the cells from piling up, but the fragility of cells must also be taken into account when subjecting them to high velocity.
There are also ways to separate cells clumps without causing excessive damage. Certain chemicals called chelators have an affinity to positively charged ions. Depending on the nature of the clumps, chelators can be added to dissolve the bonds between them without harming the cells. Ethylenediaminetetraacetic acid (EDTA) is an example of a chelator commonly used to dissolve calcium bonds.
An alternate method of unclumping cells is trituration. Trituration involves the gentle, repetitive pipetting of a sample to break up weak bonds between cells.
Microbubbles can decrease the number of cells clumping together by reducing overall cell death. By effectively sorting cells in a gentle way that preserves cell health and physiology, there is less residual DNA and debris floating around in the sample. Akadeum’s products provide a fast and efficient method of cell separation that results in a highly enriched sample of the target cells.
While other techniques, such as magnetics or cell sorting, may subject cell populations to harsh physical forces, Akadeum’s microbubbles use gravity and buoyancy to carry unwanted cells to the surface for removal. The workflow is simple, and delivers a highly-enriched pool of the desired cells for downstream applications.
Akadeum’s microbubble kits are exceptionally gentle on delicate immune cells, allowing for fast and easy immune cell enrichment that is designed to maximize yield and deliver reliable, consistent results each and every time.
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