Variable Methods of Cell Separation
Immunology is an evolving field of study that constantly requires new equipment and methods to keep up with rising demands. The number of diseases and possible treatments to research only increases as time passes. The immune system is complex, and various immune cells react differently to various pathogens, diseases, and other factors. It is important to understand the contribution of each immune cell type in order to develop more targeted approaches to treatment. Studying individual contributions of cells typically requires cell separation and isolation, especially for low-frequency cell types whose contributions could be masked by cells of greater abundance. Cells become much easier to observe and record when they are isolated.
There are multiple methods of cell separation to choose between that vary in cost and effectiveness. When selecting a process it’s important to consider things like time, equipment, and monetary resources. Whichever separation method is capable of isolating and purifying the most cells in the shortest amount of time (while maintaining cell health and viability) is the most valuable to researchers.
The three most common methods of isolation are fluorescence activated, magnetic-activated, and buoyancy activated cell sorting. Each of these abide by similar principles but vary in the means they use to achieve their goals.
Fluorescence Activated Cell Sorting
The most versatile method that’s currently the standard in most laboratories is FACS. FACS or fluorescence activated cell sorting, uses light scattering to sort particles by their physical characteristics. Target cells can also be marked with antibodies that light up when they flow past a laser and are sorted accordingly by a machine called a flow cytometer. Even though this method uses a flow cytometer, FACS is different than flow cytometry. While this method can be applied to many different types of cells and can sort large, crowded samples all at once, it is an expensive and time-consuming process.
The flow cytometer is a complex, expensive device that must simultaneously run multiple tasks. Even used, outdated machines can run upwards of $35,000. The average used cytometer equipped with standard features will cost anywhere between $80,000 and $150,000. If you’re hoping to purchase a brand new, top of the line device with cutting-edge features, the price can be close to $500,000. Large manufacturers are working to bring down the prices to make adequate equipment more accessible, but for now researchers are heavily limited by their resources. In large institutions with access to shared resources (like a dedicated flow core facility), the cost is determined by the number of hours required for sample processing. In cases like these, advanced sample preparation techniques (like BACS) to enrich the sample prior to processing can help to keep the time and expense to a minimum.
Beyond the price, FACS is also time-consuming and potentially inefficient. The setup alone for a sort lasts 60-90 minutes. Depending on the sample size, the actual process can take hours. With experiments spanning over multiple hours it’s difficult to do more than one process during a workday.
FACS is capable of sorting large samples with up to four separate populations all at once, but a larger sample can translate to more inaccuracies. If the flow volume is too fast, cells can clump together or be incorrectly sorted. The speed of the liquid can also cause loss due to shearing, which is when the cell membranes tear from contact with the fast-moving fluid used to flow the cells through the narrow lines of the cytometer.
Magnetic-Activated Cell Sorting
Another popular method used to sort cells is MACS. MACS, also called magnetic-activated cell sorting, is a separation technique that uses a magnetic field to draw target cells away from other substances in a solution. By attaching magnetic beads to the targeted population’s surface markers, magnetic columns on either side of the solution will lure the desired substance to the tube walls. This allows unwanted cells to pass through and be removed from the sample.
MACS is less complicated than FACS, which has led to more variations of the same process. This means that it’s much easier to find less expensive equipment and there’s not an expectation for how the device is supposed to function. MACS requires the purchase of magnet, and a recurring expense for the magnetic beads used to sort cells – and, in some cases, the purchase of separation-specific columns. Depending on the kit being used, the column may need to be purchased uniquely for each separation. This process requires up-front investment in the magnet itself, as well as the columns and magnetic beads themselves, which are consumables that need to be purchased on an ongoing basis.
The true cost of using MACS has less to do with money and more to do with sample quality. The magnetic pull can be too powerful for cells with a gentle membrane, resulting in cell death. Using rare and fragile cell populations puts researchers at risk of not having a pure enough throughput to properly study the desired substance. While faster and cheaper than FACS, the resulting quality is not as high.
Buoyancy Activated Cell Sorting
The final cell separation method is called buoyancy activated cell sorting, or BACS, and relies on microbubbles to bind and isolate target cells. First, antibodies are released into the solution to bind to surface level antigens on the target cells. Microbubbles are then stirred into the mixture to bind to those antibodies. Finally, the buoyant properties of the bubbles will pull the desired substance to the top to be collected or removed.
BACS is a very simple process — it only has three steps — mix the microbubbles into the sample, allow them to rise, then remove the microbubble layer. Since the entire process can be done in virtually any container, the only thing to pay for is the product itself. A microbubble kit ranges from $300 to $800 depending on the population size and cell type. Although BACS can’t handle samples as large as FACS, multiple trials can be run simultaneously since there’s no need for expensive equipment.
BACS also sorts samples with a very high purity. The microbubbles are gentle enough to leave fragile cells unharmed. The accuracy, speed, and low cost of this separation technique allows for sample prep that takes minutes without requiring additional equipment like magnets or columns in an efficient workflow that is gentle on cells.
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