When researchers study a cell population, they must first isolate a target cell culture. Once isolated, they will test those healthy cells to observe and manipulate their behavior. Running a successful experiment involves keeping cells alive and well, or viable. One of the best ways to increase sample viability and reduce uncontrolled cell death is through cell culture maintenance. However, cells do undergo a natural cycle that cannot be prevented which ultimately leads to apoptosis.
The five steps of the cell cycle are typically divided into two major phases: the mitotic phase and interphase. The mitotic phase is when cell division occurs and involves a multitude of smaller steps. The two stages of the mitotic phase are mitosis and cytokinesis, respectively. Interphase is composed of the other three stages: G1, S, and G2.
This is the process by which most cells procreate and multiply. Cells undergo the cell cycle at their own individual rate.
Mitosis is a form of cell division that occurs when one cell divides into two identical daughter cells. The other form of cell division, meiosis, is only applicable during reproductive processes that involve the integration of two unique gene pools. Mitosis occurs in all cells that have the capability to divide and allows them to procreate.
The first stage of mitosis is prophase. In prophase, chromatin within the nucleus coils and condenses to form chromosomes. These chromosomes contain one single piece of highly organized DNA from the host cell.
Pairs of chromosomes link together at a centromere to form sister chromatids. After prophase, these dividing cells will enter the prometaphase step.
In prometaphase, the nuclear envelope which contains the nucleus breaks apart. This breaking releases the sister chromatids so they may begin developing microtubules for their next step. The microtubules extend to each end of the chromatids as the cell moves into metaphase.
The third phase of mitosis is metaphase. Metaphase is the stage in which the chromosomes line themselves up across the middle of the cell. These chromosomes are lined up by the pushing and pulling of the microtubules. Once the chromosomes are aligned along the equator of the cell, the fourth stage of mitosis will begin.
The fourth stage of mitosis is called anaphase. Anaphase is when the mitotic spindle pulls the sister chromatids apart. The mitotic spindle comprises the microtubules which are attached to both the ends of the chromosomes and the ends of the cells. These microtubules tug on the chromosomes to pull apart chromatids at their centromeres.
Anaphase makes sure that both daughter cells will have an identical set of chromosomes after mitosis is completed. The final phase after anaphase is telophase.
Telophase, the fifth stage of mitosis, does not begin until replicated and paired chromosomes have been divided to opposite poles of the cell. During the process of telophase, a new nuclear membrane begins to form around the separated chromosomes to separate the DNA from the cytoplasm. As they become enclosed, chromosomes uncoil and spread out, reverting to their original functional state.
Telophase also combines with the process of cytokinesis to complete the mitotic process. Cytokinesis begins during anaphase but does not finish until the telophase ends. Cytokinesis is the process that shrinks the cell’s plasma membrane inward to divide the cytoplasm into two separate cells, each with its own replicated nucleus.
Mitosis and cell division are the eventful stages of the cell cycle, and they happen quickly relative to the other steps. There are three other phases—G1, synthesis, and G2—that are all categorized under the term interphase. Interphase is when the cell grows and prepares to undergo mitosis again.
After mitosis, the cell will enter an intermediate phase called the first gap, or G1 phase. During this segment of the cell cycle, the new cell will slowly grow and prepare for DNA replication to take place. Different parts within the cell will also begin replicating to prepare for the synthesis phase.
The synthesis phase, also called the “S” phase, is when the cell creates a complete copy of all the DNA in its nucleus and duplicates centrosomes. The replicated DNA will be used during mitosis to create corresponding nuclei for the daughter cells.
During G2, or the second gap phase, the cell continues to grow and produce proteins. The cell will also start reorganizing its organelles to prepare for mitosis.
The important takeaway from the cell cycle is that cells are living things that both reproduce and die. While some specialized cells (such as red blood cells) can no longer undergo mitosis to multiply, all cells will eventually experience death in one way or another.
There are two main forms of cell death—apoptosis and necrosis. Necrosis is unplanned cell death that occurs because of external factors. Apoptosis is a programmed cell death that occurs from the inside of the cell outwards. From a preventative standpoint, apoptosis will always occur in a cell population, whereas necrosis can be avoided.
The goal of cell culture maintenance becomes accounting for and adjusting to apoptosis while taking the proper steps to avoid and prevent necrosis.
Cell culture maintenance refers to the protocols and ongoing strategies a laboratory uses to ensure the health and functionality of a cell population. This can include the strategies used to collect and isolate cells, the storage environments for cell populations, and the decontamination processes used to reduce issues.
Cells must be alive and healthy to function properly. If they are not behaving as they should, they cannot be adequately studied by scientists. The viability of a sample refers to the amount of healthy isolated cells which can be manipulated for downstream assays. Whether simply gathering data on a cell population, or performing additional experiments after isolation, the viability is directly conducive to accurate results.
Certain cell separation methods have a difficult time differentiating live and dead cells. This could lead to dead cells being counted as false positives and finding their way into the enriched sample. This can affect the overall behavior of the cells in the enriched sample and skew results.
Cells also release cellular debris upon death. When the nucleus of a cell is lysed, or ruptured, the organelles of that cell are released into the surrounding matrix. This extra debris can interfere with other living cells by taking up space or clumping together and causing a blockage. For cell separation methods that require cells to flow through a sorting medium, cell clumps can create a barrier, which doesn’t allow the target cells to properly pass through.
Beyond inaccuracies in sorting, dead cells typically lead to more dead cells as well. This creates a continuously detrimental cycle if the sample is not cleaned up using some form of dead cell removal.
Different types of cells have their own specific life cycles. For example, red blood cells in a healthy individual live an average of 120 days. Some types of white blood cells can live for hours, while some can live for years, it just depends on the type and its function.
Along with varying lifespans, each cell also has an ideal environment. When isolating cells for immunology research it’s rare that cells will be collected directly from a host and tested; cell populations will typically need to be stored or transported from one place to another. Creating an environment that protects these cells is an important part of cell culture maintenance.
This might mean keeping them at a specific temperature, consistently cleaning potential contaminants, and minimizing the physical forces that act upon them.
On top of harsh environmental conditions, two other potential hazards to sample viability are contamination and human error. Contamination can occur from bacteria, fungi, chemicals, etc. encountering a cell sample. Cell populations are often stored in protected environments that promote cell growth and survivability. For this reason, if a different type of cell culture finds its way into the environment, it will grow rapidly. This new culture will steal resources and space from the original cell population and ultimately render the original sample unusable.
Human error can involve improper precautions, not following protocols, or simply dropping something that shouldn’t be dropped. Each of these situations can have a significant impact on the overall success of an experiment.
Cells naturally die as a result of the cell cycle—they also die unnaturally as a result of ignorance or improper accommodations. To prevent cell death in your sample and maintain high cell viability, cell culture maintenance is a necessity.
One way to prevent cell death is by using a quick and gentle method of cell separation to increase viability. Akadeum Life Sciences in Ann Arbor, MI has developed an innovative cell separation technique that harnesses the natural buoyant properties of glass microbubbles to carefully separate labeled cells. Compared to traditional methods such as fluorescence-activated cell sorting or magnetic-based cell sorting, buoyancy activated cell sorting (BACS) exposes the sample to much less potential harm; there are no harsh magnetic fields or flowing liquids that could damage fragile cells.
BACS also requires no additional equipment aside from the microbubble kit and has a 30–45-minute total workflow. Cell death occurs from external forces and naturally over time. If you can minimize harmful external forces and the amount of time the separation process takes, you can reduce cell death. Akadeum’s microbubbles can help you achieve this goal in a cost-effective, time-effective way.
Cell culture maintenance can also include dead cell removal. Dead cell removal enables you to salvage a cell sample that may have experienced mild damage. By removing dead cells and cell debris, you can save the viability of the remaining cells and potentially still perform an experiment.
Akadeum is currently in the process of developing a dead cell removal kit that uses microbubbles for target sample enrichment. Check out our online webinar highlight videos or downloadable content to see how our technology is expanding to include dead cell removal.
If you’re interested in finding out how our technology can benefit your cell separation efforts, schedule a meeting with a scientist and we’ll answer any questions you may have. We look forward to working with you!