March 2021 Share
Research is undertaken step by step, and cutting corners provides inaccurate results. It’s important to follow proper protocol from beginning to end to ensure the highest quality findings possible. When working with cells of any kind (immune cells, tumor cells, etc.) the first step is always to collect the desired cell sample from the host tissue. Dissociation, sometimes called disaggregation, involves the breaking down of a cell culture to acquire a small group.
Isolation from tissue protocol requires the cells to be transferred into a single cell suspension medium for a flow cytometer to function properly. After cells are dissociated from their host tissue, they are further isolated from residual substances and run through a flow cytometer machine that uses fluorescent lights to analyze their characteristics. They are then either sorted into groups for further testing, or disposed of if researchers received the information they sought.
Dissociation is a necessary technique that allows researchers to target and isolate the types of cells they are attempting to study. Depending on the characteristics of cells, however, different variations of dissociation may be more or less beneficial to the experiment. Currently, there are three methods of cell dissociation: Mechanical, enzymatic, and chemical.
Mechanical dissociation is the simplest of the three, breaking down tissue with physical force, such as cutting, crushing, or scraping. This method typically uses a mortar and pestle style tool to rupture and destroy the extracted sample, leaving behind fragmented pieces. Among these fragments the desired cells should be loosely floating for further separation.
Mechanical dissociation of cells is beneficial when working with loosely associated samples because it’s fast. Physical separation occurs quickly and doesn’t require a waiting period.
However, mechanical dissociation has also shown inconsistent results between users. The cell yields and cell viabilities can vary widely when comparing one trial to the next.
This process is preferred when working with bone marrow, lymph nodes, spleens, and other tissues that are not bound too tightly.
Another method of cell dissociation is enzymatic dissociation. Enzymatic dissociation uses specific proteins to disaggregate cell culture samples. The process applies enzymes like trypsin or collagenase that digest pieces of tissue to release the target cells. The type of enzyme depends on the type of tissue, and finding the right combination leads to optimal results.
When the correct cell dissociation enzyme is used, this process is extremely efficient. Performing ample research is the ideal way to optimize cell yield.
Partly due to the research and to the enzymes themselves, this method is more time consuming than mechanical dissociation. The enzymes can also inadvertently modify proteins on the surface of target cells, altering their function or how the flow cytometer will identify them.
Enzymatic dissociation is used for more compact tissues which may have a large quantity of debris. Using enzymes can reduce the amount of fibrous connective tissue, resulting in a higher yield of cells from organs such as the liver.
The third technique used to dissociate cells is chemical dissociation. This process takes advantage of the cations that hold together intracellular bonds. Cations are positively charged atoms, and adding a chemical compound with an affinity to them causes bonds within the tissue to dissolve. EGTA, or egtazic acid, is an example of a compound with this property.
Chemical dissociation does not alter the surface of a cell like enzymatic dissociation can, making it a very safe and gentle alternative. It also produces a high viability and cell number post-expansion because the cultured cells are typically healthy.
This process can take a long time, and similar to enzymatic dissociation, yields inconsistent results. The type of chemicals used, the amount, the concentration, and the environment can all have an effect on the overall functional ability of this process.
This method is typically used for more gentle forms of dissociation, like that of embryonic cells.
There are three primary factors to consider when choosing which method would be the best for an experiment: Time, population size, and sample species. In the essence of time, mechanical cell dissociation is the fastest and simplest technique to break down tissue. If there is a huge host population or sample, crushing the cells isn’t very risky. However, if working with rare cells that are less abundant, it may be beneficial to use something gentle like chemical dissociation. Preservation of uncommon cells is a top priority if it would be difficult or expensive to obtain another sample. The last consideration is what type of tissue is being dissociated. Enzymatic dissociation is one of the most applicable methods to a variety of different cells, and therefore might be the only option to properly break things down.
The ultimate goal of tissue dissociation is to gather as many viable cells as possible without negatively affecting cell viability or function. This may require a combination of methods and a large amount of research to succeed. This research is made difficult by the inconsistencies in data across different laboratories and the lack of an industry-wide standardized cell isolation from tissue protocol.
Cell tissue dissociation and disaggregation are vital steps in the beginning of a wide range of research applications, including research into developing cancer treatments, the creation of vaccines, and cell expansion. An optimized protocol for dissociation could save time and money while also delivering a highly-enriched population of healthy cells of interest for downstream processing and analysis.
Following the initial tissue dissociation steps, the researcher is often left with a mixed sample of unwanted substance floating amongst their desired cells. It’s frequently necessary to employ an extra cell separation step to further purify the sample. While there are multiple useful methods for further isolation, one stands out for its ability to deliver a highly-enriched sample while maintaining cell physiology — BACS. BACS, or buoyancy-activated cell sorting, harnesses the natural buoyancy of tiny microbubbles to float unwanted, contaminating cells to the top of a sample for removal, leaving the untouched cells of interest ready for further processing. The microbubble protocol is fast and easy, but more importantly, is exceptionally gentle on rare and delicate cells of interest.
The Akadeum microbubble enrichment protocol can be performed directly in the sample container for quick and easy sample preparation that does not require additional equipment or expensive consumables like magnets or columns. The microbubble approach is exceptionally gentle on delicate cells and eliminates the need to expose cells to harsh chemicals like lysis buffers or external forces like magnetic gradients from rare earth magnets.. The T cell isolation kits work in less than 30 minutes, and the red blood cell (RBC) depletion kits remove >97% of RBCs in less than 10 minutes, delivering a highly-enriched sample of healthy target cells for downstream processing and analysis.
If you’re looking to reduce isolation time and cell loss during the process of dissociation to a viable cell sample, explore our line of cell isolation kits or contact our team to discuss your specific application and whether microbubbles could help you overcome existing barriers in sample preparation.
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