February 2021 Share
The process of culturing cells involves the introduction of a host population to a growth medium, allowing the population to reproduce quickly in a nutrient-rich environment. After enough cells have spawned to satisfy the requirements of the intended experiment, those same cells need to be removed from the cell culture medium.
The cells are intertwined within the substance in which they multiplied and need to be separated by means of filtration or centrifugation. The process of isolating target cells from a growth medium is called cell harvesting. There are multiple cell harvesting methods that one might choose depending on the context of their experiment.
One of the most common forms of cell harvesting is centrifugation. Centrifugation is the process of spinning a solution continuously until substances separate themselves. By using the density difference between solids and fluids, the process of sedimentation is accelerated, meaning more dense particles move to the outside while less dense particles float toward the center of the sample.
A centrifuge, the device used to spin samples in centrifugation, can accelerate a fluid sample in a fraction of a second. This method works quickly and is very easy to perform. The industrial and continuous nature of a centrifuge also allows it to work perpetually and produce a stream of purified cells. When harvesting large populations of cultured cells, cell harvest centrifugation can help to maximize quantity in shorter periods of time.
While centrifugation is fast and easy, it can pose a risk to the cell population. The high levels of acceleration can shear the membranes of cells. This cell rupture, or lysis, causes organelles and intracellular proteins to leak into the broth. The extra debris damages the purity of the sample and the total number of viable cells that can be used for research. When the goal is to achieve maximum cell extraction efficiency with minimal disruption, centrifugation may not be the researcher’s first choice.
It’s also worth noting that when centrifugation is performed on an industrial scale, it only provides around 90% purification. This requires the secondary step of depth filtration to clarify the sample to a viable level.
Another form of cell harvesting is depth filtration. Depth filtration is a process that uses a cellulose based filter media to suspend particles of different densities in different areas. When using depth filtration, the target cells are trapped in the filter media while the carrying liquid filters through. Cell culture media filtration allows the researcher to collect the desired sample directly from the surface without collecting any of the residual protein-heavy solution.
This process is much gentler than centrifugation. There is no extreme speed or harsh pressures on any of the fragile cells. When using rare cells or cells that were difficult to obtain, it’s beneficial to use some form of filtration to increase the throughput of the overall procedure. This method is also preferred over centrifugation when working with smaller samples, particularly those within a laboratory. The flexibility of being able to buy or save media filters depending on the outcome of a cell culture is preferred to running centrifuges more than once or having to invest in a larger device altogether.
Depth filtration has limits on the size of sample it can properly sort. When harvesting cell cultures above 4000 L, most bio-manufacturers turn to centrifugation for its economy. The purity of a product is extremely important to researchers, but manufacturers and industries tend to place equal value on the savings that can be obtained through mass harvesting.
Another form of filtration used for harvesting cell cultures is microfiltration. As the name implies, microfiltration uses the same tactics of pressure and suspension to separate particles, but on a smaller scale. The pores in the media filter are smaller than a normal depth filter in an effort to strain the liquids out of a sample. This process raises the concentration of target cells within a sample by removing unwanted substances. For the truly ambitious there is also ultrafiltration, which uses pores one-tenth the size of those used in a microfilter.
Microfiltration works very well for liquids of a smaller volume that need to be sorted with more attention. Another reason microfiltration is popular is because its pores are still large enough for proteins to pass through. Microfilters are small enough to effectively remove a majority of the debris while letting recombinant proteins squeeze through to be removed from the sample.
The reason these different levels of filtration exist is because there are different particles that must be separated. Microfiltration will suffice for certain substances, but for others one must use ultrafiltration or risk target cells passing through the filter.
The more precise a filter is required to be, the more expensive it is. Microfilters are more expensive than a typical depth filter, and depth filters are more expensive than a centrifuge at industrial scale.
The cell culture process is used for a variety of different uses — most often research and treatment. The ability to isolate and expand cell populations ex vivo, or outside the body, has drastically increased the potential for advanced cell-based healing procedures. These new forms of treatment involve the extraction of immune cells like T and B cells to be multiplied, altered, and returned into the body.
The first step of culturing immune cells is to cleanly isolate them from other blood components such as plasma and red blood cells. The lymphocytes must then be further isolated from additional peripheral blood mononuclear cells (PBMC) using cell separation.
Multiple techniques are capable of separating the T and B cells from the residual unwanted substances, but some of them can be very expensive and time-consuming. Some of the more traditional methods like magnetic-activated cell separation (MACS) and fluorescent-activated cell separation (FACS) require extensive training and high-tech equipment. Instead of spending a disproportionate amount of resources on techniques that must be done perfectly to avoid the chances of losing purity and throughput, it can be more beneficial to use a newer, quicker method like buoyancy-activated cell separation (BACS).
For procedures that require large samples of similar cells or particles to be sorted into more than two groups, using FACS or MACS might prove worthwhile. However, if the goal is to make cell separation as fast, simple, and affordable as possible, BACS offers the best blend of convenience and efficiency for isolating T and B cells.
BACS uses polymer-shelled microbubbles to gently separate the target cells from the remainder of a sample. This can be done by floating desired cells to the top, or by floating up everything else and leaving the desired cells at the bottom to be collected afterward. The isolation kits can be used in any container that holds liquid, and the only necessary step is to slowly and evenly stir the sample — the bubbles do the rest. This whole process can take less than 15 minutes and eliminate over 99% of residual blood cells. BACS results not only in a pure sample to move forward with population expansion, but also a resource and cost savings that can be allocated to other aspects of the process.
While researchers and patients benefit from B cells being cultured, T cells are currently of special focus in the realm of medical treatment. This is because there are so many different types that can be manipulated to create medicinal vesicles. Adoptive immunotherapy involves the engineering of T lymphocytes to attack specific pathogens they might not normally target (e.g. tumor cells). CAR T cell therapy and engineered TCR therapy are just two examples of newly developed therapeutic approaches that include the activation and insertion of artificial proteins into T lymphocytes before population expansion to create an army of specialized immune cells. Check out our page on TCR vs. CAR-T cell therapy for more information.
T cells are often cultured in different types of interleukins (IL), a group of cytokines that promote growth and differentiation. IL-2, for example, is a catalyst for the development and activation of effector T cells such as helper and cytotoxic. Meanwhile, IL-4, 7, and 15 are reported to be essential for the induction of the memory T cells that help build up long-term immunities.
Altered T cells have unique, artificial proteins in them that can be different shapes and sizes. When isolating an engineered T cell population, these proteins must be factored in and able to pass through the pores in whichever filter is used. There are a number of details that must be considered before making a final decision.
The keys to harvesting a substantial population of viable cells are to set yourself up for success from the onset of the experiment and to remain vigilant. Akadeum’s microbubble technology provides a highly enriched sample using a workflow that is exceptionally gentle on cells of interest, without negatively impacting cell health or physiology. Our T cell isolation kits can deliver fast, easy sample enrichment in only 30 minutes without the need for additional equipment.
If you are reading this, you probably already understand or at least have a very…
Cell isolation—also referred to as cell separation or cell sorting—is the process of isolating one…
“The resulting sample looked perfect after using microbubbles – very happy with the product.” Get…