Dead Cell and Cell Debris Removal: Protocol for Removing Dead Cells in Cell Culture

Dead Cell and Cell Debris Removal: Protocol and Methods for Removing Dead Cells in Cell Culture

Cell viability is an integral part of studying cell behavior. Cell viability is the number of healthy cells that are still functional after cell separation takes place. Dead or damaged cells do not perform the same way as healthy cells, which could paint an unrealistic picture of how they react in certain situations. Dead cells also release cell debris into a solution that skews downstream results.

What is Cell Debris?

In simplest terms, cell debris is the leftover waste after a cell dies. When a cell dies or has its membrane ruptured, it will release its inner components out into the solution. These cell fragments are often counted as whole cells, which creates false positives in experimental results. To get the most accurate information researchers must carry out the separation of viable and nonviable cells to reduce cellular debris as much as possible. Understanding where dead cells come from is the first step to decreasing cellular debris in a sample.

What Causes Cellular Debris?

Cellular debris can appear in your sample for a variety of reasons. It is a natural part of cells’ life cycles to die. When their membranes rupture or lyse, their intracellular components will disperse into the surrounding solution. Cellular debris from natural cell death does not typically overwhelm a sample with contamination, however, it should still be removed for optimal downstream results.

Another factor that may cause too many dead cells in a sample is the method of cell separation used to manipulate the population. Depending on the separation technique, the cells may be subjected to harsher environments or forces than they are used to. Especially when working with a fragile or rare cell population it’s extremely important to employ a gentle method that will preserve cell health and viability.

Cellular debris can also be caused by mistakes or contaminants. If a sample is contaminated with a harmful chemical or biological substance, the health of the cells could be at risk. When a cell sample is contaminated it’s usually not realistic to try and salvage results. For this reason, the best way to deal with cell debris and dead cells is to prevent them all together.

How to Avoid Cellular Debris

To avoid cell debris, you must avoid cell death, this can be done by keeping your cells as healthy and safe as possible. While a small amount of cell death may be inevitable from the natural cell life cycle, paying close attention to your cell population and separation protocols will help maximize the viability of your sample. Properly sanitize all equipment and use precise measurements to increase efficiency and decrease cell death.

In regard to the method of cell separation you use, a gentler method will reduce the number of cellular debris in a sample. Starting with a safe and easy removal technique is a great way to avoid an overwhelming number of dead cells.

Removing Cellular Debris and Dead Cell Removal

If it’s too late to prevent cell death and you find yourself looking to remove extracellular debris and dead cells from the mixture, you may need to look into removal tactics. There are a variety of techniques that can be used for cell debris removal, including BACS, centrifugation, FACS, and magnetic bead-based.

Separation of Dead and Live Cells by Centrifugation

One of the simplest methods of cell debris removal is density-gradient centrifugation. Density-gradient centrifugation harnesses a device called a centrifuge that spins a heterogenous mixture at high speeds. Depending on the density of the particles in the sample, similar substances will group together when exposed to rotational force. Because dead cells and cellular debris are fractured, they become less dense than living, healthy cells. Adding in certain separation reagents such as Ficoll or Percoll can further purify the sample by acting as a barrier that only one population can pass through.

Beyond the size and cost of the centrifuge, centrifugation also takes longer than some other tactics and can cause more cells to lyse because of the high rotation speeds. Centrifugation is sometimes used as the first dead cell removal method then followed up by a more precise and gentle method for further purification.

Using FACS to Sort Dead Cells

Another cell separation method that can be used for dead cell removal is fluorescence activated cell separation, or FACS. FACS is a modified version of flow cytometry, which uses a complex machine to analyze and sort cells based on their physical characteristics (size, structure, light scatter) with lasers and fluorescent antibody markers. A normal flow cytometer is only used to gather specific data on cell populations within a sample for downstream analysis. When used for FACS, the flow cytometer is equipped with an additional feature that allows it to sort different cells into their own respective groups.

Dead cells are capable of nonspecific binding, which may give rise to inaccurate results if not addressed during the sorting process. This could negatively impact the purity of a sample when using FACS for dead cell removal. When using FACS you must also be sure to invest in specific dye that illuminates dead cells. Since dead cells have an incomplete or nonexistent membrane, a dead cell assay that cannot pass through a live membrane will reveal all dead cells in a sample. FACS with the help of a dead cell assay can remove cellular debris from a healthy cell population with high accuracy.

There is also risk of the cells shearing when flowing through the flow cytometer. Shearing occurs when the force of fast flowing liquids is too much for a cell membrane and it bursts, releasing more cellular debris. Like centrifugation, FACS is sometimes used as a first step before sorting more precisely with another method. This technique is not very accessible to laboratories with limited funding.

Dead Cell Removal Kit Protocol

Several dead cell removal kits use a tactic called MACS, or magnetic activated cell sorting. MACS uses magnetic beads with antigen-specific antibodies on them to link to target cells. Once linked, the researcher can turn on a magnetic field to suspend the magnetic bead-bound cells and let other cells pass through, effectively sorting the solution into two groups.

When using a MACS kit for dead cell removal, the magnetic beads target dead cells, cell debris, and dying cells. These cells are bound to magnetic beads and suspended while the live cells are removed for further separation or analysis.

The disadvantages of MACS are primarily in the throughput. The use of a harsh magnetic field can damage and rupture fragile cells. When working with rare populations it may be risky to involve the magnetic forces necessary for this method.

BACS For Cell Debris Removal and Cell Separation

Akadeum has developed a  Buoyancy Activated Cell Sorting (BACS™) microbubble approach for depleting dead cells from biological samples using a fast, easy, and exceptionally gentle workflow that maintains the health and viability of analytes of interest while effectively and efficiently depleting dead and dying cells. Depletion of dead cells is achieved through the selective capture of cells with exposed phosphatidylserine (PS) using Annexin V-conjugated microbubbles. Once mixed with the sample, the microbubbles capture PS+ dead and dying cells and float them to the sample surface for removal, leaving behind the healthy and untouched cells.

This process is exceptionally gentle, as the live cells remain untouched and ready for downstream use, while the PS-expressing dead cells are easily removed. This process enables straightforward dead cell removal while maintaining the health and physiology of the remaining sample, including the live cells and other analytes of interest.

Akadeum’s microbubble platform is uniquely well-suited for the targeted depletion of dead and dying cells while maintaining the health of live cells and other delicate analytes of interest. Not only is Akadeum’s microbubble depletion workflow fast, easy, and exceptionally gentle, it is also uniquely scalable in both fluidic volume and number of samples being processed concurrently, eliminating common constraints of traditional approaches that require the use of magnets, columns, and other volume-limiting and time-intensive processing steps.

Akadeum’s Dead Cell Removal Microbubbles effectively remove PS+ cells on-par with both column-free and column-based magnetics processing, while providing significantly better recovery of target cells using a fast and easy workflow that saves time. Akadeum’s microbubble approach to dead cell removal requires no additional equipment to perform, with removal occurring directly in the sample container – no harsh chemicals, no shear forces, and no exposure to magnetic gradients from rare earth magnets. Microbubbles harness the power of gravity (they float) for separation, maintaining the health and physiology of the viable cells during the depletion process and resulting in an enriched population of healthy cells for downstream applications.

How Can Akadeum’s Microbubbles Benefit You?

To learn more about Akadeum’s revolutionary approach to dead cell removal, you can see the latest data and developments in our webinar highlights video or access the full webinar library to learn more about Akadeum and our microbubble technology.

If you’re working with rare or fragile cell populations in your laboratory, you can download our app note about Combining Microbubbles and Cell Sorting for Quick & Gentle Isolation of Unique Cell Populations to learn more about how a sample preparation step using Akadeum’s microbubbles can decrease sort times 18-fold while maintaining the health and physiology of delicate cells of interest.

If you’re simply curious about cell separation and how our technology can benefit your efforts, schedule a meeting with one of our scientists. We look forward to hearing from you and helping you overcome long-standing headaches in sample preparation.

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