Primary T Cell Culture and Expansion

Updated on Nov 29, 2023 By Dominique Badea, PhD Share

T Cells are a critical component of the immune system. They play a pivotal role in the body’s defense against infections, cancer, and a variety of other diseases. To harness the full potential of these immune warriors, scientists have worked hard on perfecting the art of T cell culturing. Research and development of new immunotherapies requires robust techniques to purify and culture T cells.

This article serves as a primer to understanding the nuances of T cell culture, from isolation to activation, while explaining some of the tools and techniques that optimize the process. Moreover, we describe methods for isolation of T cells from blood followed by activation and expansion using Akadeum’s Activation and Expansion Kit.

What Is T Cell Culture?

T cells, a class of lymphocytes, are critical to the adaptive immune system. They safeguard against infection and malignancy, making them an attractive target for immune cell-based therapies.

T cell culture refers to the laboratory technique of cultivating and maintaining T cells outside of the human body. This controlled environment allows researchers to manipulate, study, and expand T cells for various applications, including immunotherapy, adoptive cell therapy, and research studies on autoimmune diseases.

Why Is T Cell Culture Important?

Culturing T cells is an essential component of several scientific and medical applications, including:

• Research: T cell culture allows scientists to investigate T cell behavior, function, and responses to various stimuli, such as antigens or cytokines. There are different types of T cells, such as cytotoxic T cells (CD8+) and helper T cells (CD4+), and each type can be targeted for optimized growth in cell culture.
• Therapeutic applications: For several immunotherapy techniques, such as adoptive cell therapy for cancer, T cells are isolated from a patient or donor, engineered to target specific diseases, and cultured to expand the number of cells before being reinfused back into the patient.
• Vaccine development: T cell culture plays a vital role in the development of vaccines by understanding and studying T cell-mediated immune responses to inactivated pathogens or other vaccine components.
• Understanding of disease mechanisms: Culturing T cells helps in studying the pathogenesis of various diseases, including HIV and autoimmune disorders.

Isolating T Cells

To obtain T cells, blood is collected from donors and leukapheresis is performed to isolate and extract peripheral mononuclear blood cells (PMBCs). Isolation is a crucial step that ensures the purity of the T cell population and minimizes contamination with other cell types.

Techniques for T Cell Isolation

Several techniques can be employed to isolate T cells, including:

• Density gradient centrifugation: Density gradient centrifugation is the separation of cells based on the density as they pass through a density gradient under a centrifugal force This allows T cells to be separated from other blood components.
• Magnetic-activated cell sorting (MACS): MACS separates cells by using magnetic beads coated with antibodies against a particular surface antigen. When the cell suspension is placed into a magnetic field, magnetically labeled cells bind to column consumables and are isolated, while unlabeled cells can be removed.
• Fluorescence-activated cell sorting (FACS): Flow cytometry-based sorting isolates T cells based on specific surface markers that are labeled with fluorophores for identification.
• Microbubble isolation: Functionalized microbubbles bind to target cells and float them to the surface. Buoyancy activated cell sorting (BACS™) is Akadeum Life Sciences’ line of products that offer a gentle and efficient method for T cell isolation, preserving cell viability and purity.

T Cell Sources: Donors and PBMCs

T cells circulate throughout the bloodstream and are often acquired for research and clinical purposes by collecting blood from a donor. Scientists and clinical researchers typically process whole blood into peripheral blood mononuclear cells (PBMCs), a collection of primary immune cells that include T cells, B cells, monocytes, and dendritic cells. PBMCs are used for downstream research, clinical diagnostics, and therapeutic applications—including further isolation of T cells.

Primary T cells for cancer therapies are sourced from either autologous (the patient) or allogeneic (not the patient) donors. Autologous samples are standard practice in adoptive cell treatments like CAR-T therapy to help avoid autoimmune reactions with non-self antigens when infused back into the patient. Culturing and expansion of the engineered T cells are vital in these situations because the number of native T cells is typically reduced in cancer patients.

Allogeneic T cells are easier to source since they may be collected from any healthy individual, but require additional considerations related to immune compatibility. Therefore, allogeneic T cells are not currently used for many therapies.

Culturing T Cells

Once isolated, T cells are placed into specialized growing conditions. The type of culture media for successful cultivation can vary depending on the type of cell or subset of cell you require.This includes strictly controlled nutrient liquids, called culture medium, that have various serums and reagents added to them to optimize T cell growth while staying compatible with the type of downstream clinical applications.

The T Cell Culture Medium

Choosing the right culture medium is essential for the growth and maintenance of T cells. At a minimum, T cell media includes a buffer system, proteins, trace elements, vitamins, inorganic salts, and energy sources. The most widely used medium for culturing T cells in research laboratories is RPMI 1640, which provides the necessary nutrients and support for cell growth. Serum, such as fetal bovine serum (FBS), is often added to culture media to provide essential growth factors and proteins but must be considered seriously when cells are intended for human therapy.

Buffers are included to maintain the desired pH level. Sodium bicarbonate is one popular buffer used for culturing cells. Having a reliable buffering system is critical for primary cell culture because T cells are particularly sensitive to pH imbalance. Depending on the application, various energy sources, trace elements, antibiotics, and antimycotics may also be used.

Optimizing Culture Conditions

Maintaining optimal conditions is critical for successful T cell culture. These conditions include:

1. Temperature: T cells thrive at 37°C, which mimics the body’s natural conditions.
2. CO2 levels: Culture incubators are frequently set to 5% CO2. The gas is not necessary for cellular metabolism as much as for creating a physiological pH environment.
3. Nutrient supply: Regularly replenished or exchanged culture medium ensures an adequate supply of nutrients. Serum can be a valuable component of the medium.
4. Contaminant control: Cell cultures are easily contaminated by bacteria, fungi, and viruses—especially if scientists conduct other experiments in shared lab space. Frequent decontamination of supplies and culture inspection are key to keeping unwanted pathogens at bay.

Activating T Cells in Cell Culture

T cells require specific signals for growth and survival, which are contingent on a range of various cytokines for further proliferation and activation. In the body, they rely on cytokines, antibodies, and cell stimulation to proliferate and remain active. Cell culture activation mimics their natural response to pathogens or antigen stimulators and is typically achieved through:

• Antigens: Adding specific antigens to the culture can activate T cells with known antigen-specific receptors.
• Mitogens: Mitogens are potent stimulators of T-cell activation and proliferation. Mitogens can be either endogenous or exogenous.
• Monoclonal antibodies: Treatment of T cells with monoclonal antibodies (anti-CD3/CD28) provides a co-stimulatory signal that engages the TCR, which can be used for antigen-induced activation.

T Cell Expansion and Proliferation

In many applications, a small number of T cells may not be sufficient. T cell expansion allows researchers to generate a large scale of T cells to maximize the therapeutic potential. Specific cytokines, such as interleukin-2 (IL-2) or IL-7/IL-15, are often added to the culture to support T cell expansion and survival. These cytokines are vital to promoting T cell proliferation.

Repeated antigen exposure to activate T cells can also drive expansion, particularly when antigen-specific T cells are desired in large quantities. However, repeated activation should be used with caution, as it induces T cell exhaustion and reduction in functionality.

The duration of T cell culture can vary based on factors like the initial cell concentration, the degree of expansion required, and the experimental design. In some cases, researchers may achieve substantial T cell expansion in an average time frame of 10-14 days. However, for long-term research applications, T cells may need to be cultured for several weeks.

Maintaining T Cell Viability

T cell viability is critical for the success of any T cell culture experiment or therapy. Factors that influence viability include:

• Cell density: Avoid overcrowding in culture systems, which can lead to competition for nutrients and space.
• Medium composition: Ensure the culture medium, including serum components, provides essential nutrients and growth factors.
• Contamination: Maintain sterile conditions to prevent contamination, which can compromise the culture.

A Note on Primary T Cells

Primary T cells are derived directly from donors and are highly relevant for immunotherapy and cell therapy. However, they present unique challenges in culture:

• Limited lifespan: Primary T cells have a finite lifespan in culture compared to “immortalized” research strains and may require periodic re-isolation. A specially tailored growth medium can help support their proliferation.
• Cryopreservation: Cryopreservation techniques that freeze cells can extend the lifespan of primary T cells, making them available for future experiments. Serum-containing cryopreservation solutions are often used.

Functional Assays for T Cells

Once a sufficient number of T cells are cultured, assay tests are used to assess phenotype, functionality, and viability:

• Cytotoxicity assays: These measure the ability of T cells to kill target cells, such as cancer cells or infected cells.
• ELISpot assays: Enzyme-linked immunosorbent spot (ELISpot) protocols quantify the secretion of specific cytokines, providing insights into T cell function.
• Flow cytometry: This allows for the analysis of surface markers and intracellular proteins in T cells. Antibodies are essential tools in flow cytometry.
• Proliferation assays: These tests measure the ability of T cells to divide and expand in response to stimuli.

After the successful expansion and activation of T cells in culture, these potent immune cells can be harnessed for many applications, including research and cancer therapy.

The Role of Akadeum’s Microbubble Products

Successful T cell culturing and expansion start with clean cell isolates. While some methods of cell separation can be harsh and damage rare and delicate cell types, Akadeum’s microbubble buoyancy activated cell sorting (BACSTM) technology does the job gently and quickly while maintaining cell health and physiology. Our negative selection kits have microbubbles that attach to unwanted cells, carrying them to the sample’s top for removal, leaving the enriched population untouched and ready for downstream processing.

Akadeum’s microbubble products are designed to simplify and enhance the isolation of various cell types, including T cells. These microbubbles offer several advantages:

1. Efficiency: Microbubbles efficiently isolate T cells in 10-45 minutes, minimizing the risk of contamination and maximizing yield.
2. Gentleness: The gentle isolation process ensures high cell viability, preserving the functionality of your T cells.
3. Versatility: Akadeum’s microbubble products can be customized to various cell samples for isolation and enrichment. In our negative selection kits, the isolated cells are untouched and untagged, leaving more options open for clinical research applications.

Are you ready to elevate your T cell culture game? Discover the power of Akadeum’s microbubble products and experience a new level of efficiency and consistency in your T cell isolation and culture.

Contact us today to learn more about how our innovative technology and optimized T cell culturing can accelerate your research in immunology and cell therapy. Unlock the full potential of your T cell culture with Akadeum!