To properly study a cell population, scientists must isolate large quantities of a specific type of cell. That quantity must be viable and behave in the same way it would within the system in which it was extracted. T cells isolated from whole blood should behave the same way they would in the human body. This allows researchers to understand how those cells work in the real world, and how those functions can be harnessed for the well-being of humans.
Many problems can occur between sample extraction and cellular experiments that skew results and negate findings. By paying attention to these potential issues before they occur and proceeding in a way that takes them into consideration, most can be fixed or avoided. Solving these problems before they happen to increase sample purity and viability is one of the primary functions of cell preparation.
Preparing a cell culture involves any steps taken before and during the cell separation process which will ultimately improve the quality, accuracy, or efficiency of an experiment. Cell sample preparation includes following protocols, cleaning up unwanted cells, choosing appropriate cell sorting methods, etc.
The goal of cell separation is to isolate as many viable, desired cells from a heterogeneous mixture as possible. Whether isolating for downstream assays that require cells to behave normally or isolating simply for data collection, a clean starting sample will provide more accurate results down the line.
Cell culture contamination occurs when unwanted chemical or biological substances or microorganisms infiltrate a biological mixture and damage cells in some way. Biological microorganisms can steal resources that other cells need to grow and survive. Chemical substances can rupture cell membranes or affect the cell’s typical behavior.
Both forms of contamination can cause cell death and reduce sample viability. Cell culture contamination can also cause issues during cell sorting. Sometimes invasive cells can be misinterpreted, resulting in false positives. This can skew experimental results and sometimes void the entire procedure. To prevent having to dispose of all your hard work, certain sample preparation steps can be taken to avoid contamination.
When it comes to cell contamination, the best solution is simply prevention. Addressing an issue before it becomes one will save your lab time and money while resulting in healthy cells. Following a few simple rules can also save your results from being thrown out altogether.
One of the most effective ways to prevent contaminants from infiltrating your cell population is to use the proper sanitization techniques. Simple tasks, such as washing your hands often and constantly cleaning surfaces, can make a significant difference in protecting your cell population from contaminants. Be sure to use chemical reagents when cleaning that does not damage or alter the cell type you’re working with.
Many of the contaminants that trouble researchers come from small mistakes such as improper handling, among other factors.
Depending on the cell separation method and the specific products you use, the directions for how to successfully complete cell isolation will differ. Different companies use different biomarkers, solution quantities, and equipment to optimize their individual separation kits. Performing separation according to the standards and instructions that they suggest will aid in reducing contamination, mistakes, and cell death.
Dead cells are not necessarily chemical or biological contaminants, but they can have the same effect on a cell population. Dead cells can be misattributed during cell counting, block healthy cells from being sorted properly, and cause other cells to rupture and die. Many contaminants will cause cells to die at a rapid rate. If you notice a high quantity of dead cells in your cell population, you may be dealing with some form of contamination.
Recovering the projected number of cells requires viability maximization. Maximizing viability means minimizing the amount of dead and unwanted cells in a sample, while successfully isolating the highest number of healthy, desired cells. This can be done by purifying a sample through removal, paying attention to certain aspects of cell separation, and choosing an isolation method that takes efficiency and viability into account.
One of the most effective ways to improve sample purity and viability is by removing dead cells. In situations where cell death could not have been prevented, there are products available for dead cell removal. Apoptosis, the natural process of cellular death, leads to characteristic changes in the dead or dying cell. One such change involves the relocation of phosphatidylserine (PS)—a phospholipid normally found on the inner surface of the plasma membrane—to the extracellular surface. The exposed PS is then able to engage with a number of molecules, including Annexin V. This PS-Annexin V interaction can be leveraged to target and remove dead and dying cells from the sample, allowing downstream applications to continue.
Aside from dead cells, there are other cells that can contaminate a sample or skew downstream results. A variety of residual blood cells found in biological samples like peripheral blood mononuclear cell (PBMC) preparations can interfere with the measurement of other target populations.
For example, if a scientist is attempting to study the behavior of CD8+ cytotoxic T cells they will first need to isolate a viable population of this cell type. In a traditional PBMC prep, around 30% of the total PBMCs are CD8+ T cells—making a cleanup step to enrich the sample for the cells of interest a critical first step in order to obtain a highly enriched population of the cells of interest. Sample preparation with Akadeum’s Human T Cell Isolation Kit can remove contaminating, off-target cells to deliver a highly enriched population of happy, healthy T cells for downstream processing.
One way to improve research outcomes is to choose a gentle cell separation method that preserves cell health and physiology. Some traditional methods, such as magnetic-based cell sorting and centrifugation, subject cells to harsh external forces during the isolation process. Akadeum’s buoyancy activated cell sorting (BACS) uses functionalized microbubbles to gently sort desired samples through gravity-powered floatation, allowing for buoyant separation that enables cell isolation with high viability and throughput without sacrificing recovery.
.Apoptosis will naturally occur over time, and the longer that cell preparation takes the more of these cells will die. Choosing a cell separation method with a fast and gentle workflow will help to maintain cell physiology and prevent excessive cell death before and during the cell sorting process.
When cells die, they release extracellular debris that naturally clumps together within the solution. This clumping can clog up certain equipment and ruin cell separation efforts. It’s crucial to remove clumps when preparing cells for FACS because the cell clumps can form a barrier within the flow cytometer that prevents cells from sorting properly.
Cell clumps can also consequently cause more cell death, making them larger. Allowing this cycle to continue for too long makes it nearly impossible to get accurate results.
Whether you are preventing cell death from occurring or cleaning dead cells from a mixture before cell sorting, cleaning a cell culture to improve purity is beneficial in a variety of ways. By reducing unwanted cells, you can improve the efficiency and accuracy of your cell sorting assays. By improving efficiency and accuracy, you will save valuable resources that can be delegated to other areas of the research process. Cell culture cleanup is one of the easiest ways to optimize your sample for downstream processing and research.
Akadeum’s microbubble approach to cell separation aims to maximize yield, reduce processing time, and maintain the health and physiology of immune cells—all while eliminating the need for extra equipment like magnets and columns using a fast, easy workflow that takes place directly in the sample container.
The microbubbles are gently mixed into the sample where they engage their targets before isolating those targets through floatation-based separation. It’s that simple! We’ve taken technology that traditionally requires additional equipment to accomplish this separation and put it into a single container where it’s self-separating.
Our buoyant approach to separation eliminates many of the existing hurdles that are limiting improvements or effectiveness of processes today. Unlike magnetic bead-based separation, microbubbles do not have the same volume and equipment restrictions.
Learn more about RBC Depletion for PBMCs and Dissociated Tissue Samples
Learn more about Cell Enrichment for Immunology Workflows
Learn more about Optimizing Sample Prep for Flow Cytometry
Our microbubble kits are exceptionally gentle on delicate cells, allowing for fast and easy immune cell enrichment that is designed to maximize yield and contribute to reliable, consistent results each and every time.