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Culturing T Cells: Primary T Cell Culture and T Cell Expansion Protocol

February 2021


Isolating and Culturing T Cells

One of the best ways to increase the accuracy of research is to increase the sample size that is being studied. The larger the group of subjects, the more representative they may be of the total population. Not to mention experiment security in case something goes wrong. In the field of medical research this increase in population can be carried out by culturing cells. Cell culture is a process by which cells are removed from an organism to be expanded in an artificial environment that is more suitable for their growth; it is also called cell expansion.

Medical researchers generally use cell culture on cells that are less common in the body, like T cells. T cells, or T lymphocytes, are specialized cells that help the immune system target specific foreign particles. Different types of T cells do different things. Culturing T cells allows a more detailed observation of how the immune system naturally responds to harmful pathogens. When attempting to isolate and study T lymphocytes, it’s important to get as ample a sample size as possible.

Why T Cells Need to Be Cultured and Expanded

T cells can be harnessed in a variety of different ways and are mostly used for research purposes. Studying activated populations of T cells can reveal information such as:

  • The way our bodies naturally respond to disease
  • The way we develop immunities
  • The process by which our body learns to recognize a pathogen
  • How to artificially produce different medications
  • The effects that T cells have on general well-being

Learning from nature allows us to build on strategies that already work.

T cells also need to be studied for their individual functions. A helper (CD4+) T cell releases cytokines to dictate cellular response. A cytotoxic (CD8+) T cell actively poisons and destroys unknown substances. These unique roles can be manipulated in different situations for the most optimal treatment. The subtle differences also cause them to be cultured in different ways.

While one of the main functions is research, they can also be cultured for treatment. With a disease like cancer that requires an intense and prolonged immune response, expansion may be necessary. Only specific T cells will be designated to fight against tumor cells. To bolster a strong enough and long enough response, the activated T lymphocytes are sometimes extracted, multiplied, and reintroduced into the body as reinforcements. Before any research or treatment can be done, the T cells need to be isolated from other peripheral mononuclear blood cells.

Traditional methods for isolating T cells require complex equipment, specially trained experts, and patience. Some methods can damage fragile cells with harsh magnetic fields or rapidly flowing liquids. It’s important when isolating T cells to choose a technique that is cost-effective and will provide a large, clean sample.


Culturing T cells can benefit the researcher in a multitude of ways. As previously mentioned, the larger a sample is, the more accurate and representative it will be of the original population. The downstream throughput and result applicability will both increase with a higher number of cellular subjects. Taking the step of generating more from the onset can save scientists from having to run a second experiment.

Even beyond these advantages cell culturing can allow scientists to study an extremely specific variation of cell. If a research study is focusing purely on how immunodeficient human CD8+ T cells react to the COVID-19 virus, only a small sample of these particular cells need to be obtained for expansion. After the cells are properly cultured, there will be a sufficient population to conduct a full study.


Although cell culture has much to offer, there are a few reasons it’s not widely used. The main reason is how costly it can be in terms of both time and money. Animal cells grow slower than contaminants like bacteria, viruses, and fungi. Culturing cells requires highly skilled personnel and strict asepsis techniques to carry out a tedious process with extreme focus. While T cell expansion is sometimes beneficial, unless it’s necessary it can cost more than it’s worth.

Cell Culture Protocol

The human T cell expansion protocol involves a range of steps from beginning to end that generally outline the most effective way to culture cells.

Preparing an Aseptic Environment

Make sure all potential equipment is properly sterilized. Everything that could be used during the procedure and the surrounding area must be cleaned to prevent microbial growth. Any form of contamination could infringe on the desired cell population. The process should be performed in a clean environment or under a fume hood to prevent airborne contaminants from interfering with the culture.

Prepare Cell Growth Medium

Depending on the type of cell that’s being expanded, choosing the correct medium is essential. The culture medium is a viscous substance that is curtailed to maximize the growth of a particular cell. Some media will cause cells to grow faster than others and some will be more reliable, so let the goal of the procedure drive the decision. Make sure the chosen medium is properly sterilized and placed in an aseptic environment. Some cells also require extra chemical coating within the container to amplify cell growth.

Basic medium requirements will suffice for most types of T cells. While it is known that more optimal combinations exist for different subtypes—such as CD8+ T cells or CD4+ T cells—further research is needed to determine exactly what those specifications are. The populations grow at different rates when given the same stimulus and it seems to be linked to their structure and the number of cytokines present. Cytokines are small proteins that help to regulate cell processes. 

Check the Cells

The T cells must be monitored daily to ensure they are healthy and growing. Different reagents can be added to reduce clumping and test different levels within the medium. The cells need to be discarded if they are detaching and look very dark in color, or if they remain in quiescence over time (meaning they don’t appear to be growing).


When a population becomes too confluent, meaning most of the flask surface becomes covered by the cultured cell, the sample must be divided. Overpopulation of the medium can have a negative effect on cell viability and gene expression. When splitting cells, it’s important to prepare another medium ahead of time to minimize loss and damage to a sample. It’s also beneficial to know the rate at which a particular T cell will need to subculture so the extra cells can be used for an experiment and not go to waste.

Changing Media

If the cells have been growing but not to the point that they are confluent then it may be necessary to change the medium. This will help provide fresh nutrients and keep the pH at the correct level. Make sure the new medium is pre-warmed and replace aspirated old media before returning to the incubator.

Optimizing the Growth Setup

The superordinate goal of these steps is to develop the healthiest crop of activated primary T cells possible. The primary T cell culture is a term for those cells directly expanded from the extracted T lymphocytes. A well-rounded primary T cell culture is the key to successful experiments and continual growth. A secondary T cell culture is any group of cells that were expanded from the primary cell culture, and so on.

There is currently no set formula for the perfect T cell medium. Each different subtype requires slightly different factors to achieve optimal growth. There are T cell media recipes that researchers have released to the public and products that come already concocted, but more research is necessary to establish the best method. In general, a T cell expansion medium should include the following:

  • A buffer system
  • A protein
  • Trace elements
  • Vitamins
  • Inorganic salts
  • Energy sources

Many of the recipes for T cell expansion also call for interleukin-2 (IL-2), a cytokine that is known for regulating the growth of white blood cells in the body.

Another integral part of a successful procedure is the isolation process of the T cells. By starting the process with more isolated T cells, the population will grow at an exponential rate. Separating the maximum amount of healthy T lymphocytes from other substances in the body can ease some of the tension and reduce the amount of time researchers must wait before their sample is confluent.

Akadeum Life Sciences

While some methods of cell separation can be harsh and damaging to rare and delicate cell types, Akadeum’s microbubble technology does the job quickly and gently while maintaining cell health and physiology. The microbubbles attach to unwanted cells and carry them to the top of the sample for removal, leaving the enriched population of T cells untouched and ready for downstream processing.

Akadeum’s buoyancy-activated cell sorting kits work in 10-45 minutes and can be used directly in the sample container. No expensive equipment and no rigorous training is needed, just decontaminated T cells ready for culturation.  Explore our line of T cell Isolation kits today, or learn more about microbubble technology by viewing past webinars.

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