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Why is Cell Separation Important?

The ability to study individual cells provides insight into their specific functions and roles within the human body. Knowing exactly what certain cells do allows scientists to harness their abilities and learn from them. Cell separation is a major catalyst in the push for individualized medicines and the ability to treat large populations with effective generalized methods.

What are the Approaches to Isolating Cells?

Cell separation methods typically take one of the three following approaches:

• Positive selection
• Negative selection
• Depletion

The approach you choose should depend on the context of your experiment.

Positive Selection Cell Separation

Positive selection is when the cell type of interest is targeted by the removal mechanism and retained for downstream applications. This approach involves targeting the desired cell population with an affinity molecule specific to a surface marker of the cell, leaving behind unwanted cells in the sample.

Negative Selection Cell Separation

Negative selection is when unwanted cell types are labeled with affinity molecules such as antibodies or proteins that target specific cell markers or populations and then removed, leaving one cell type untouched. The untouched cell sample is then collected for downstream applications.

Positive vs. Negative Selection

Choosing between positive and negative selection will depend heavily on the context of the experiment. If the target cell has a very clear selection marker on its surface, positive selection can provide a higher purity than negative selection. If selection markers are not clear, and you have intentions to perform downstream assays on your isolated cells, negative selection will remove unwanted cells quicker without altering the enriched population.

Cell Depletion

Cell depletion is the third and simplest approach in which a single cell type is removed from a biological sample. This strategy is typically used to remove large quantities of a single common contaminant, such as red blood cells (RBCs) or dead cells. If a sample is heavily saturated with residual RBCs after the cell separation process, RBC depletion kits can be used to further purify the sample.

Cell Separation Methods and Technologies

There are several different technologies used to isolate cell populations. These technologies are usually based on one or more properties unique to the targeted cell type—such as size, density, electric charge, shape, or protein expression—to label those cells for removal.

Buoyancy Activated Cell Sorting (BACS)

Buoyancy activated cell sorting (BACS) is a negative selection process developed by Akadeum Life Sciences that involves sorting cells with buoyant microbubbles. The microbubbles are coated with affinity molecules that attach to target cells and lift them to the surface of the solution. Once the cells are at the top, they can be removed from the sample through vacuum aspiration, leaving behind the enriched sample at the bottom. Microbubbles can also be used for the depletion of RBCs.

Microbubbles allow researchers to increase the scalability of their experiments and expand their diagnostics to rare cell populations. This innovative method can be custom-tailored with a variety of bio-analytes to target specific cell groups while maintaining a high purity, yield, recovery, and viability.

BACS is fast, easy, and inexpensive in comparison to the other methods, and preserves cell health and physiology for downstream applications. It can be used in conjunction with other techniques to further purify a sample or by itself as a standalone isolation method.

Magnetic Based Cell Sorting

Magnetic based cell sorting is a form of immunomagnetic separation that involves binding magnetic particles to target cells through an affinity molecule/surface marker interaction. Then, the sample is subjected to a magnetic field that suspends cells in a liquid solution, letting other cells flow through freely. Depending on the cells being targeted, MACS can be a positive or negative selection method.

Protocol for Cell Isolation Using Magnetic Beads

There are two variations of magnetic cell sorting, column-based and column-free cell sorting. Both strategies use magnetic beads coated with specific molecules that bind to surface markers on target cells in the sample.

In column-based magnetic sorting, the sample is passed between two columns that create a magnetic field. When turned on, this magnetic field catches the labeled cells which are bound to the magnetic beads. When the field is turned off, the labeled cells are released for easy collection or removal.

In column-free magnetic sorting, the sample is placed inside a tube and subjected to a magnetic field that pulls the beads to the side of the container. The unwanted cells are then poured off while target cells are suspended. Then the magnetic field is turned off leaving behind only the desired cells in the tube.

Magnetic Based Cell Separation Equipment

Depending on which strategy you decide to use you may or may not need to purchase magnetic columns. These columns add additional expenses and require more storage space. There are also machines that perform magnetic cell sorting automatically without requiring the experimenter to manipulate the magnetic field. Regardless, the most important piece of equipment necessary is the magnetic beads.

How Much Does Magnetic Isolation Cost?

Due to the simplicity of magnetic sorting in comparison to FACS, there is a wider variety of less expensive alternatives available. The most expensive part of the magnetic sorting process is purchasing the magnet, whether it be a column, or another device used to magnetize a field surrounding the sample container. Beyond this expense, magnet-based sorting also requires you to continually purchase kits with microbeads in them which are specific to the cells you’re sorting. While these seem relatively cheap at first, they begin to add up over time because they must be purchased on an ongoing basis.

Magnetic Sorting Advantages and Disadvantages

Cell separation with magnetic beads has been around for a longer time than some other methods, for that reason, magnetic beads are currently capable of targeting more cells than some newer technologies. Magnetic sorting is also cheaper and faster than FACS.

However, the magnets employ a harsh magnetic field which can damage and rupture cells. Overall cell recovery is typically lower which may require a larger starting population. Magnetic cell sorting also has hidden costs associated with the devices, storage, and training as mentioned above.

Fluorescence Activated Cell Sorting (FACS)

Fluorescence activated cell sorting (FACS), or FACS analysis is a specialized type of flow cytometry that involves labeling targeted cells with fluorescent markers and running the sample through a flow cytometer device. Then, cells are identified and sorted one by one based on the color of their markers into isolated cell populations.

The Difference Between FACS and Flow Cytometry

FACS is a form of flow cytometry. Flow cytometry is a cell analysis technique that measures the metrics of individual cell populations in a heterogeneous sample. A flow cytometer device is used to record data about cells as they move through the machine.

The major difference between flow cytometry and FACS is the extra step of cell separation. Flow cytometry analyzes a sample to provide information then disposes of the cells. With FACS, the cells are sorted into separate populations for downstream applications.

FACS Machine and Equipment for Cell Separation

When it comes to FACS, the flow cytometer does a majority of the work. The flow cytometer is a complex machine made up of three systems:

• Fluidics — Transports and organizes cells
• Optics — Lenses, lasers, filters, and other components that generate a photocurrent to detect cell characteristics
• Electronics — Digitizes data for processing

Fluorescent dye specific to certain cell types can also be purchased to assist in sorting certain populations.

How Much Does FACS Sorting Cost?

Depending on the features you want in your FACS machine, the price can vary significantly. In facilities where a large flow cytometer is shared between multiple laboratories, the cost is determined by the hour required for sample processing.

On top of the device cost, FACS also has other hidden costs associated with it. Experiments span over several hours, the large samples can lead to inaccuracies, and extra money must be spent to store the cytometer and train personnel to use it properly. If used incorrectly, it could easily corrupt the results of an experiment.

FACS Advantages and Disadvantages

FACS is one of the more commonly used cell separation techniques because it can be applied to diverse cell populations. Other methods are typically used to sort out one individual cell type. In a situation where you want to extract two or more cell types from a complex solution, FACS is the most efficient option.

However, if the goal is single-cell or single-cell type isolation, FACS is relatively expensive, time-consuming, and difficult to perform. Depending on your cell separation goals, FACS may not be the most practical choice.

Cell Separation by Centrifugation

Centrifugation . As the sample is spun, more dense particles will automatically move to the outer edges of the mixture while less dense objects will group together further in. A biological sample is centrifuged until the cell types are isolated into layers.

Centrifugation Applications and Methods

The most popular form of centrifugation used for cell separation is density gradient centrifugation. Density gradient centrifugation separates cell populations based on their respective densities with the help of a gradient medium.

Another form of centrifugation is differential centrifugation, which separates particles based on size. This allows particles which are the same density to be separated by different qualities by running the sample through the centrifuge multiple times without the density gradient.

Centrifugation can be useful for large scale sorting depending on the size of your centrifuge. The ability to work consistently can increase overall yield. However, a centrifuge can be fairly expensive, and the cell viability can be damaged by the machine’s high speeds.

Additional Cell Separation Methods

A multitude of other tactics are used to separate cells from heterogeneous mixtures. While FACS, MACS, centrifugation, and BACS, typically show the best results, certain cell populations work best with a specific technique. Here are a few examples of less popular cell separation methods.

Cell Separation by Adhesion

Different cell types have different adhesion properties, which determine the cells they attach to. Depending on the environment used for a cell culture, you can control whether or not cells will adherently separate from other suspended cells. A good example of cells that can be isolated this way are macrophages.

Cell Separation by Filtration

Filtration is a cell isolation technology based on size. Using a filtration device, targeted cells are captured while other cells pass through the device. Leaving the experimenter with one population at the bottom of the filtration device or medium, and the other population caught before or within it.

Cell Separation by Sedimentation

Sedimentation relies on the same properties as filtration and centrifugation on the basis that gravity will help denser components sediment faster than less dense components. When the larger materials move toward the exterior of the sample, the cells left behind can be collected.

Sedimentation is relatively cheap compared to other methods, but results in a lower overall purity.

Applications of Cell Separation

There are many cell separation methods, but the methods are vastly outnumbered by the potential uses of isolated cells. Healthy, purified cell samples can benefit a multitude of different scientific disciplines. Below are some of the applications that can be performed or carried out with cell separation or isolated cells.

Blood Separation – PBMC Isolation from Buffy Coat

When dealing with whole blood samples, a very small percentage of cells are white blood cells (WBCs) and platelets. After blood is centrifuged, these cells gather into a thin layer called a buffy coat that can be separated out for research. The high concentration of peripheral blood mononuclear cells (PBMCs) in the buffy coat make it ideal for studying how the body responds to infectious diseases and harmful pathogens.

PBMCs consist of different immune cells that can be isolated for research or medical treatment. An individual can receive PBMCs in a transfusion to bolster their immune system.

RBC Depletion in a Cell Sample

When isolating cells from whole blood the most abundant contaminant is residual RBCs. These cells do not function the same as WBCs and can interfere with research as to how the immune cells behave if left in the sample. Cell separation can be used to remove these cells with ease and clean a sample for downstream analysis and applications.

T Cell Separation for Research and Practical Application

Using cell separation techniques to isolate T cells opens a world of possibility for research and treatment in the field of immunology. Studying the different types of immune cells in the human body can provide insight into the immune response and guide medical research.

Isolating CAR T Cells for CAR T Cell Therapy

Chimeric antigen receptors (CARs) are the receptors found on the surface of cancerous cells. Not all T cells are capable of recognizing these specific antigens. Cell separation allows scientists to do two things:

• Isolate the cells that can target cancer cells, culture them, and reintroduce them back into the body in higher numbers
• Isolate T cells, genetically engineer them to detect the cancerous cells, then reintroduce them in high numbers

Both of these strategies can help an individual’s immune system fight off specific types of cancer and depend on large, highly purified cell samples.

CTC Enrichment for Cancer Research

Cells that break off from a cancerous tumor and float through the bloodstream are called circulating tumor cells (CTCs). These cells can be isolated and studied in a laboratory to gain insight on how cancer cells respond to different treatments or environments. Procuring highly concentrated CTC samples allows for non-invasive cancer research that assesses potential outcomes without putting a patient at risk.

Purifying Cell Samples for Protein Therapy

Another form of cell engineering is found in protein therapy. Protein therapy involves the replacement, replenishment, or reprogramming of specific cells to produce specific proteins. When an individual’s cells are damaged or incomplete, scientists can actually repair or replace proteins to fix the broken cell.

COVID-19 Immune Response Research

Isolating T cells allows researchers to perform a wide range of tests on infectious diseases. Being able to study cells involved in the SARS-CoV-2 virus and COVID-19 disease can provide insights on how to combat them. Infected cells are less abundant and require gentle, accurate cell separation methods to extract high volumes of viable cells.

B Cell Separation

Along with T cells, B cells can also be isolated through cell separation. B Cells create antibodies for fighting off infectious diseases and help to combat harmful substances in the body.

Antibody Research and Manufacturing

Certain B cells receive signals from T cells to manufacture specific antibodies that fight off harmful pathogens. Cell separation allows researchers to study the individual behavior of these B cells.

Developing an in-depth understanding of B cells reveals how the immune system eliminates pathogens. Without the antibody-producing B cells, we would have an incomplete picture of the immune response. Learning how we can mimic antibody production through the use of medicinal drugs and distribute treatment to precise locations will shape individualized medicine in the future.

Stem Cell Separation Technology and Procedure

Certain cells are very valuable when isolated. Stem cells, for example, can be studied and manipulated for a variety of purposes. From medical treatment to developmental and cancer research, cell separation of stem cells is constantly evolving and making more things possible.

Single Cell Analysis Techniques

Cell separation can also take place on a smaller scale, analyzing single cells as opposed to cell type populations. This can be extremely useful when isolating more complicated things, such as DNA or RNA.

Techniques like BACS specialize in preparing samples for single-cell analysis. Through the high purity removal of contaminants and gentle workflow, BACS helps to optimize enriched cell samples for downstream assays.

Single Cell DNA Extraction

DNA is the genetic material that can be found within every living thing. When studying these fragile strands, it’s important not to damage them in any way. Single-cell analysis allows scientists to carefully isolate smaller samples without damaging the cell viability.

Single Cell Isolation Methods

There are two methods typically used for single-cell isolation:

• Manual Picking – Manual picking techniques involve the use of an inverted microscope and a multitude of micro-pipettes that purposefully select the desired cell population.
• Microfluidics – Microfluidic techniques allow researchers to manipulate cell micro-environments during cell separation. This can be very useful for analyzing or separating cells from smaller samples.

Both categories contain more specific strategies that depend on context and goals.

Evaluating the Performance of Cell Separation Methods

There are a variety of different cell separation methods to choose from when isolating a specific population. Making the right decision becomes much easier when you know what characteristics you should be looking for.

Cell Purification and Cell Sample Purity

Cell sample purity refers to the ratio of isolated cells of interest to undesired cell types. This value is typically represented as a percentage equal to the desired cells out of the total number of isolated cells. If the purity of a sample is 80%, then 80 out of 100 cells are the cell type of interest, while the remaining 20 are undesired cell types.

The goal of cell separation is to isolate a specific cell type for independent analysis, making purity one of the most important factors to consider when choosing a separation method.

Cell Yield and Recovery During Isolation of Cells from Cell Culture

Cell yield is the number of desired cells that were successfully isolated after cell separation. Even if the purity of a sample is high, it won’t matter unless the cell yield is sufficient for downstream applications.

Another important statistic is cell recovery, this is the term used to describe the percent of cells that were isolated from the total number of target cells. This can be found by dividing the cell yield by the total number of cells and multiplying by 100.

If a cell separation method has a high cell yield, recovery, and purity, it will be useful in the process of isolating cells for downstream applications or experiments.

Cell Sample Viability for Downstream Research Applications

On top of these factors, the last thing that should be considered in research that requires more steps post-isolation is cell viability. This refers to the amount of healthy, living cells that survive the separation process.

When cells are damaged or destroyed, they do not function properly and can negatively affect the results of a downstream assay. Viability is calculated by staining dead or damaged cells and subtracting the number of stained cells from the total sample, then calculating the percentage of healthy cells out of the total.

Some of the mechanisms involved in traditional cell separation methods such as magnetic fields or harsh physical forces can decrease cell viability.

Speed of Cell Separation Process

When working with fragile cell populations, the amount of time a cell spends being moved around or exposed to external forces has an effect on its ability to survive. For this reason, the efficiency of an isolation process should be considered when determining the proper technique for your experiment.

Additionally, the longer you have to spend completing the cell isolation protocol, the less time you have for other lab tasks or processing more samples. Rapid and easy protocols can maximize efficiency and throughput.

Choosing the Right Cell Separation Technique

The cell separation technique you should choose depends heavily on your situation. If you’re part of a large organization or laboratory with immense funding and rigorous cell sorting demands, it may be alright to spend extra time and money for a more complex set of machinery.

Some cell populations can only be separated with certain methods or are easier to separate with one method as opposed to another. Doing research on the best product for your specific needs can save a major headache when it comes to preserving cell viability for downstream applications.

When it comes to the most cost-effective and time-efficient method for single cell type isolations, the cheapest and quickest method that maintains cell health with a high throughput is Akadeum’s BACS.

BACS vs. Traditional Cell Separation Methods

When comparing BACS to other separation methods such as FACS, MACS, and centrifugation, BACS triumphs in almost every category. Microbubbles have a shorter, simpler workflow that can take place directly in the sample container. They cost less than alternative methods and don’t require any complex machinery. Anybody can perform cell separation with BACS kits, and they can perform as many as they want simultaneously. The bubbles are gentle enough to preserve even the most fragile cells, but strong enough to carry dense cells to the top of the sample with ease.

Whether you are looking to further purify your sample after using another method or perform simple cell separation procedures in the most efficient way, BACS is the best option for speed, ease, and maintaining cell health and physiology.

Akadeum’s BACS kits have already helped a multitude of research efforts. To see what’s happening now with Akadeum, visit our Latest News page.

Checkout Akadeum’s microbubble products or contact us to find out more about how BACS can benefit your cell separation efforts. Our company is always looking for new partners to commercialize microbubble-based protocols.