Overview

CAR T cell activation and expansion are essential steps in CAR T cell manufacturing. Activation prepares a patient’s collected T cells for genetic modification, while expansion increases the number of engineered cells needed for a therapeutic dose. These processes shape CAR T cell quality, consistency, scalability and clinical performance, making them central to successful cell therapy development and manufacturing.

CAR T cell therapy is a type of cancer immunotherapy that uses a patient’s own T cells to recognize and kill cancer cells more effectively. Because tumors can suppress immune activity or evade recognition, collected T cells are engineered to express chimeric antigen receptors (CARs) that improve tumor targeting. Understanding how CAR T cells are activated and expanded is critical for improving manufacturing efficiency, product quality and therapeutic outcomes.¹

T cells are central to the adaptive immune response, but natural tumor recognition is often limited. In CAR T cell therapy, patient-derived T cells are modified to express receptors that detect tumor-associated targets more effectively. Once engineered, these cells must be activated, expanded and quality tested before they can be infused back into the patient.²

Successful CAR T Cell therapy depends on expanding CAR T cells in the laboratory to produce a large number of copies that can ensure a sustained and effective response in the patient's body.

How are CAR T cells isolated, activated and prepared for transduction?

T cell expansion begins with the isolation and activation of T cells. A technique called leukapheresis separates peripheral mononuclear blood cells (PBMCs), including leukocytes and lymphocytes, from a patient's blood sample. T cells are separated from other white blood cells using cell processors or centrifugation. T cell subsets (e.g., CD4 and CD8) can be isolated using magnetic-activated cell sorting (MACS). Simultaneously, enrichment and depletion techniques remove the remaining red blood cells and platelets.

Isolated T cells are activated by co-incubation with engineered antigen-presenting cells (APCs) that can produce the necessary activation signals. Additionally, magnetic beads coated with antigens, such as CD3 and CD28, combined with the immune-activating cytokine IL-2, can be used to promote T cell activation. This procedure renders T cells suitable for CAR transduction. Nevertheless, the duration of T cell priming must be monitored to prevent prolonged activation, which may lead to T cell exhaustion.

The final step before expansion involves transduction, which integrates the CAR gene into the T cell genome. Viral vectors, such as lentiviruses or retroviruses, are often used to deliver the CAR transgene. These vectors are designed to be replication-deficient, making them efficient and safe for clinical use. During transduction, viral gene delivery must be performed incrementally and the culture media must be routinely monitored to optimize transduction efficiency.

What happens during CAR T cell expansion?

The next step is to expand T cells to produce a large population of functional CAR T cells capable of detecting tumor cells.

What drives CAR T cell growth and proliferation?

Large-scale proliferation is achieved by culturing T cells in a medium containing cytokines, such as IL-2, IL-7 and IL-15 and costimulatory signals to promote cell survival and division.³,⁴

The three most important factors for T cell expansion are the seeding density, interleukin concentration and bead-to-cell (B: C) ratio. T cell proliferation also depends on the conditions of the growth medium, such as oxygen and nutrient concentrations.⁵

Which manufacturing factors affect CAR T cell expansion?

Cell density is one of the most crucial factors in successful T cell expansion, as manufacturers must determine the optimal cell density to ensure optimal growth. While a low cell density may lead to apoptosis due to oxidative stress and insufficient survival signals, extremely high cell densities may cause T cells to compete for resources.⁵ Research by Ghaffari et al. reveals an initial increase in cumulative population doubling as the cell density increases, with the maximum value estimated at 250 × 10^3 cells/mL. Further increases in cell density during the culture step led to a rapid decline in T cell expansion. The research also revealed the importance of increasing the bead: cell ratio to improve expansion rates.⁵

Cytokines, such as IL-2, IL-7 and IL-15, must be added to the culture to promote survival and proliferation. These cytokines also regulate the differentiation of T cells into cytotoxic and regulatory subtypes.³,⁴

Another factor influencing T cell expansion is the presence of serum in the medium. Although fetal bovine serum and human serum can provide the essential growth factors, vitamins, proteins, ions and hormones for cell growth, their composition depends on the donor, potentially leading to batch-to-batch inconsistencies.⁶ Therefore, manufacturers shifted towards serum-free medium containing synthetic and purified ingredients.

Other factors include gas-permeable culture systems to facilitate oxygen and CO2 exchange and metabolic support, such as adequate glucose supply, to ensure that the T cells remain active during rapid proliferation.⁷

How do expansion kits support CAR T cell manufacturing?

Manual preparation of culture media and components for CAR T cell production can be cumbersome. Life sciences and biotechnology companies assist manufacturers by developing commercial expansion kits that streamline T cell expansion protocols and workflows. These kits comprise standardized activation reagents, such as antibody-coated magnetic beads, cytokines and nutrients to support growth and regulate differentiation. Expansion kits include serum-free, GMP-compliant formulations to enable consistent, reproducible workflows. Thus, they help manufacturers develop CAR T cells that meet regulatory requirements and are suitable for clinical use.

CAR T cell activation vs. expansion: key manufacturing components compared

What are the real-world applications of CAR T cell activation and expansion?

Beyond the laboratory, CAR T cell activation and expansion have direct implications for translational research, process development and clinical manufacturing. In early-stage development, researchers use activation and expansion workflows to evaluate construct design, optimize cytokine combinations and compare culture conditions that may influence potency or persistence. During process development and scale-up, manufacturers focus on reproducibility, closed-system compatibility and media strategies that can support GMP-compliant production. In clinical settings, these upstream decisions affect turnaround time, product consistency and the ability to deliver a therapeutic dose for patients with aggressive disease. As CAR T development expands into solid tumors and next-generation constructs, robust activation and expansion strategies remain central to improving both feasibility and therapeutic performance.

How is quality control managed during CAR T cell manufacturing?

Thorough quality control is essential for ensuring the safety and functionality of CAR T cells. The following tests are implemented to assess T cell expansion:

• Cell viability: Viable cell counts help estimate proliferation rate and detect premature apoptosis.
• Cell size and volume: Size and volume are critical indicators of the activation state of T cells. Changes in size and volume are monitored via label-free methods during isolation, activation, transduction and expansion stages.
• Aggregate detection: This test is implemented during packaging and before administration to ensure safety and functionality.
• Gene expression: Flow cytometry can be used to determine the gene expression levels of the CAR vector. Furthermore, it can help measure surface markers to confirm and quantify T cell subsets.
• Immunophenotyping: Flow cytometry and standardized dry antibody panels can be used to characterize and quantify T cell populations. This method is useful for assessing purity after isolation.

Challenges in CAR T Cell Expansion and Therapeutic Delivery

Despite the clinical success of CAR T cell therapy, manufacturing and clinical delivery remain constrained by variability, scale, timing and safety considerations that can affect both product quality and patient access.

• T cell quality can vary significantly depending on the patient's age, prior treatment and disease burden, impacting CAR expression, expansion efficiency and therapeutic performance.¹
• Manufacturing bottlenecks include contamination risk, inconsistent starting material, suboptimal expansion, labor-intensive workflows and the time required to generate a patient-specific product. These constraints can delay treatment and complicate scale-up across sites. Stringent in-process monitoring is needed to maintain cell purity, viability and reproducibility throughout isolation, activation, transduction and expansion.
• Even when expansion is successful, cell fitness can decline if activation is too prolonged or culture conditions drive exhaustion, differentiation shifts or metabolic stress. This can reduce persistence and antitumor activity after infusion.

Clinical limitations also remain important. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) can complicate delivery after infusion. At the same time, tumor biology, antigen escape and the immunosuppressive microenvironment may limit durable responses in some settings.

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References

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2. Sun L, Su Y, Jiao A, Wang X, Zhang B. T cells in health and disease. Signal Transduct Target Ther 2023;8(1):235.
3. Zhou Y, Husman T, Cen X, Tsao T, Brown J, Bajpai A, et al. Interleukin 15 in cell-based cancer immunotherapy. Int J Mol Sci 2022;23(13):7311.
4. Kim J-H, Lee K-J, Lee S-W. Cancer immunotherapy with T-cell targeting cytokines: IL-2 and IL-7. BMB reports 2021;54(1):21.
5. Ghaffari S, Torabi-Rahvar M, Aghayan S, Jabbarpour Z, Moradzadeh K, Omidkhoda A, et al. Optimizing interleukin-2 concentration, seeding density and bead-to-cell ratio of T-cell expansion for adoptive immunotherapy. BMC Immunol 2021;22(1):43.
6. Eberhardt F, Hückelhoven-Krauss A, Kunz A, Jiang G, Sauer T, Reichman A, et al. Impact of serum-free media on the expansion and functionality of CD19. CAR T-cells. Int J Mol Med 2023;52(1):58.
7. MacPherson S, Keyes S, Kilgour MK, Smazynski J, Chan V, Sudderth J, et al. Clinically relevant T cell expansion media activate distinct metabolic programs uncoupled from cellular function. Mol Ther Methods Clin Dev 2022;24:380-393.
8. Xiao X, Huang S, Chen S, Wang Y, Sun Q, Xu X, et al. Mechanisms of cytokine release syndrome and neurotoxicity of CAR T-cell therapy and associated prevention and management strategies. J Exp Clin Cancer Res 2021;40:1-23.