August 2022 Share
Cell separation is an integral step in various research areas including, but not limited to, immunology, cell therapy, and single cell sequencing. The isolation of a specific cell population allows the behaviors of the individual cell type to be studied without interference from other cells. The cellular isolation strategy varies depending on the cell subset of interest. Deciding on an isolation strategy is also dependent on the sample matrix. Cell samples can be isolated from whole blood, whole tissues such as spleens, or biopsies. When performing cell-based assays it is important to invest in a cell separation strategy that offers a multitude of products for varying cell populations.
Akadeum Life Sciences uses silica microbubbles to float unwanted cells to the top of a sample for removal. Microbubble kits can come with premade antibodies to sort specific cell types while others can be individualized to a researcher’s specific needs. This versatility is a benefit of using streptavidin microbubbles in combination with biotinylated antibodies specific to the individual researcher’s needs.
Cell markers are molecular identifiers used for the classification of cells. They are uniquely expressed – either internally or externally – on specific cell subsets at distinct times to signal the differentiation and growth of cell populations. Some external markers are also called surface antigens because they are typically found on the plasma membrane of a cell. A variety of surface markers can be used in combination to identify specific cell subsets. Beyond classification, cell markers can enable the cell to perform essential tasks. These cell markers can be used to help diagnose and serve as treatment targets.
Antibodies are immunoglobin proteins meant to seek out and bind to specific antigens. The antibody-antigen relationship enables scientists to target various cell populations by using antibodies specific to an antigen on the cell’s surface. Some cell separation methods use this strategy to extract cells of interest. Other methods use negative selection, targeting all undesired cells in a sample, leaving only the cells of interest behind.
Using antibodies specific to an antigen on a cell’s surface can also be used to differentiate between B cell markers and T cell markers. Cell surface markers can be expressed broadly on various cell subsets such as CD45. CD45 is expressed on all leukocytes and is often used as a positive cell marker to identify cells as such. Cell marker expression can also be unique and exclusive to specific cell subsets such as CD3 expression on T cells, or CD20 expression on B cells. Using cell surface markers that are uniquely expressed on a given cell subset allows scientists to differentiate between cell types and target specific cell subsets.
Naïve cell markers express a variety of surface antigens. Of these, some are broad and are expressed on a variety of cell types while others are very specific. The most common identifier for human naïve T cells is the selective expression of CD45RA protein isoform. This specific antigen is expressed on unactivated T cells. Researchers may have a difficult time targeting this exact cell type. Due to this, it may be more beneficial to focus on the cell markers unique to non-naive T cells that might be found within a given sample. For example, if the naïve T cells are being isolated from a whole blood sample or leukopak, scientists could label red blood cells for removal leaving the naïve T cells untouched.
Extracting immune cells from humans is a costly and complex task, so commercial leukopaks are often purchased for human cell separation experiments. Leukopaks are a leukocyte-rich product that has been extracted from a patient via apheresis. Rich concentrations of desired immune cell populations can be obtained through leukopak processing and further cell separation.
Human naïve T cells are the precursor to effector T cells and can be studied to understand the T cell development process. Human effector T cells are some of the most commonly known and studied cell subsets. This includes activated cells which have specific effector functions in the immune system, such as CD4+ helper T cells, CD8+ cytotoxic T cells, and memory T cells. These cells are often isolated to be used in experimentation with pathogens to understand how they function to protect the body.
B cells have many responsibilities including the production of antibodies and building long-term immunities. B cells expand in response to a wide variety of foreign substances and pathogens. Due to this, there are times when antigen-specific B cells are more abundant. B cell surface marker expression varies depending on different types of stimuli, which can make isolation more complex. It is critical to use a gentle isolation process to maintain cell properties and function while minimizing cellular death.
When isolating both T and B cells from human tissues RBC contamination is inevitable. The consistent composition of red blood cells make them easier to target than white blood cells. For this reason, RBCs are often depleted, or removed, from a sample to allow researchers to study the function of the white blood cells. Akadeum’s Human RBC Depletion Microbubbles remove 99% of RBC contamination in three simple steps and 10 minutes to complete, resulting in a cleaner, purer sample.
Human cell isolation can be an expensive, complicated, and time-consuming process. One frequent alternative to research using human cells is the use of mouse models. Mouse cells are more widely available and more cost effective than human cells while still sharing many of the same characteristics. Mouse cells cannot be used as a perfect substitution for human cells; they can provide a reference for how human cells may function under similar circumstances.
The protocol for mouse cell isolation is slightly different than human isolation because tissues, such as spleens, that have significantly higher lymphocyte concentrations are more accessible. Because of their small size and low blood volume, mouse spleens are often collected to isolate white blood cells. Mouse spleen cells, or splenocytes, provide a substantial number of T cells for isolation and further downstream experimentation.
Like in humans, mouse naïve T cells are a rare cell population that require a gentle cell isolation method to obtain the highest recovery. Magnetic fields can rupture mouse naïve T cell membranes, causing cell death. Harsh physical forces such as centrifugation or the swift moving currents of FACS can cause cells to lyse. These cells are at a disproportionately high risk of dying with traditional cell separation methods.
As with other mouse cells, mouse B cells are used to infer how human B cells will behave. B cells are more accessible in mice than in humans which makes mouse B cell isolation an important part of immunology research.
Similar to in humans, mouse red blood cells contaminate freshly isolated splenocyte samples. Removing these contaminants will improve the cell separation process, resulting in a higher sample purity, and can improve the reliability of the experimental data. Many laboratories will perform mouse red blood cell depletion before and after cell separation to ensure the sample is as clean as possible.
One of Akadeum’s primary products harnesses the use of the biotin-binding protein streptavidin to allow target cell customization. The streptavidin conjugated microbubbles can be used to isolate a variety of cells of interest using known cell surface markers and antibodies specific to those targets. While most of the other Akadeum products come ready made to separate specific cell types (CD4+, mouse naïve, red blood cells, etc.), the streptavidin microbubbles come ready to be modified to your specific needs.
Buoyancy activated cell sorting, or BACS, is an innovative isolation platform that is faster and more reliable than traditional methods without sacrificing cell health or physiology. BACS is a negative selection cell separation method, meaning it isolates desired cell populations by removing all other cell populations. Most kits come with an “antibody cocktail” that labels the unwanted cells by targeting their cell surface markers.
Once the unwanted cells are labeled, the streptavidin covered microbubbles will bind to the unwanted cells and gently lift them to the top of the sample. These unwanted cells and microbubbles can then be removed by aspiration, while the cells of interest sink to the bottom. The enriched sample is then collected from the bottom of the tube to be used in downstream applications.
Comparatively, BACS outperforms other cell separation methods. Through the use of antibody-antigen targeting the microbubbles can precisely bind to the unwanted labeled cells and float them to the top. The gentle nature of the bubbles preserves the viability of the sample for downstream applications unlike other, harsher separation methods. Cell separation methods like FACS and magnetic-based cell separation require expensive equipment and can damage cells. BACS also doesn’t require any specialized equipment, allowing for multiple samples to be run simultaneously—even whole leukopaks can be processed for separation.
Akadeum Life Sciences is always looking for new partners to help commercialize our products. If our technology can benefit you, reach out to speak with one of our scientists about how you can get involved.
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