CAR T Cell Protocol: Step-by-Step Laboratory Techniques

Chimeric Antigen Receptor (CAR) T cell therapy has transformed cancer treatment by leveraging the patient’s immune system. This approach involves genetically modifying T cells to recognize and destroy tumor cells, showing remarkable success in treating blood cancers. Producing CAR T cells requires precise laboratory techniques to ensure safety, efficacy, and consistency.

This article details the key laboratory steps in CAR T cell production, from initial preparation to cryopreservation.

T Cell Source Preparation

CAR T cell therapy begins with obtaining T cells from either the patient (autologous) or a healthy donor (allogeneic). Peripheral blood mononuclear cells (PBMCs) serve as the primary source, collected through leukapheresis—a process that isolates white blood cells while returning other blood components. The efficiency of leukapheresis depends on factors such as lymphocyte counts, anticoagulant use, and processing parameters, all of which influence T cell yield. Optimizing leukapheresis conditions, such as maintaining a CD3+ T cell count above 0.5 × 10⁹ cells per liter, improves downstream manufacturing success (Hartmann et al., 2021, Nature Reviews Drug Discovery).

After collection, PBMCs undergo refinement to enrich the T cell population. Density gradient centrifugation separates mononuclear cells from granulocytes and red blood cells. Magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS) then isolates CD3+ T cells. MACS, which uses antibody-coated magnetic beads, is scalable and cost-effective, while FACS provides higher precision by distinguishing between CD4+ and CD8+ subsets.

The decision between bulk CD3+ T cell isolation and subset selection has clinical implications. Some protocols maintain a balanced CD4:CD8 ratio to enhance persistence and efficacy, while others emphasize CD8+ cytotoxic T cells for rapid tumor clearance. A study by Turtle et al. (2016, Journal of Clinical Investigation) found that a defined 1:1 CD4/CD8 ratio improved expansion and durability in patients with B-cell malignancies. Additionally, selecting naïve (Tn) and central memory (Tcm) T cells enhances proliferative capacity and longevity, with CCR7 or CD45RA-based sorting enabling their enrichment.

Vector Delivery Approaches

CAR gene introduction requires an efficient delivery system to ensure stable expression and functionality. The choice of vector impacts transduction efficiency, genomic integration, and therapeutic performance.

Viral Vectors

Viral vectors are widely used due to their high transduction efficiency and stable gene expression. Lentiviruses and gamma-retroviruses are the primary vectors in clinical CAR T cell manufacturing. Lentiviral vectors integrate into both dividing and non-dividing cells, reducing the risk of insertional mutagenesis compared to gamma-retroviruses, which preferentially integrate near transcription start sites (Milone & O’Doherty, 2018, Molecular Therapy).

Lentiviral vector production involves transient transfection of HEK293T cells with plasmids encoding the CAR transgene, packaging proteins, and envelope glycoproteins. The viral supernatant is harvested, concentrated, and purified before transduction. Optimizing the multiplicity of infection (MOI) is crucial, as excessive viral load can cause cytotoxicity, while insufficient MOI reduces transduction efficiency. Despite their advantages, viral vectors are costly and require stringent quality control to prevent replication-competent virus formation.

Non-viral Vectors

Non-viral methods reduce insertional mutagenesis risks and simplify manufacturing. Plasmid-based transfection, particularly electroporation, introduces the CAR transgene into T cells without viral components. However, plasmid DNA often results in transient expression unless integrated using transposon systems like Sleeping Beauty or PiggyBac. These transposons use transposase enzymes to insert the CAR construct into the genome (Singh et al., 2020, Frontiers in Immunology).

The Sleeping Beauty system, derived from a reconstructed Tc1/mariner transposon, has been explored in clinical trials. It requires co-delivery of a transposon plasmid carrying the CAR gene and a transposase plasmid for integration. While scalable and cost-efficient, integration site preferences and lower efficiency compared to viral vectors remain challenges. Optimizing electroporation conditions is necessary to balance transfection efficiency and cell viability.

Physical Methods

Physical techniques, such as electroporation and microfluidic-based transfection, introduce CAR constructs without viral or chemical carriers. Electroporation applies an electrical pulse to create transient pores in the cell membrane, allowing nucleic acids to enter. This method is widely used for non-viral CAR T cell generation, particularly with mRNA-based approaches that enable transient CAR expression. While mRNA electroporation avoids genomic integration, repeated dosing is needed for sustained therapeutic effects (Stadtmauer et al., 2020, Science Translational Medicine).

Microfluidic-based transfection, an emerging technique, uses controlled fluid dynamics to introduce genetic material into cells. It minimizes cellular stress compared to electroporation and has shown high efficiency in preclinical studies. Nanoparticle-mediated delivery systems are also being explored to enhance CAR gene transfer while preserving T cell viability. These methods offer flexibility in CAR T cell engineering but require further optimization for clinical scalability.

Activation And Expansion

After isolation, T cells undergo activation and expansion to reach the required quantity and functionality. This begins with stimulating the T cell receptor (TCR) complex using artificial antigen-presenting surfaces, such as magnetic beads coated with anti-CD3 and anti-CD28 antibodies. Anti-CD3 initiates TCR signaling, while CD28 enhances proliferation and survival. The bead-to-cell ratio is carefully calibrated to avoid overstimulation, which can lead to activation-induced cell death.

Cytokines play a key role in promoting T cell growth. While interleukin-2 (IL-2) has traditionally been used, newer protocols incorporate IL-7 and IL-15, which favor central memory (Tcm) and stem cell-like memory (Tscm) subsets. These subsets exhibit greater persistence and durability compared to effector T cells, which are prone to exhaustion. IL-7 and IL-15 supplementation increases the proportion of long-lived CAR T cells, potentially improving treatment outcomes.

Expansion occurs in controlled bioreactor systems or gas-permeable culture bags that regulate oxygenation and nutrient exchange. Bioreactors provide precise control over pH, glucose levels, and lactate accumulation, impacting cell viability. Wave-motion bioreactors are favored for large-scale manufacturing due to their ability to support high-density cultures. Maintaining appropriate cell density is crucial, as overcrowding can lead to nutrient depletion and metabolic stress, affecting the final CAR T cell product.

Quality Control Steps

Ensuring the safety, potency, and consistency of CAR T cell products requires stringent quality control. Flow cytometry is a key technique for assessing T cell subsets, verifying CAR expression, and detecting contaminants. Multiparametric flow cytometry panels distinguish between naïve, memory, and effector T cells, providing insight into therapeutic composition. Clinical-grade products aim for over 80% CAR expression to ensure robust anti-tumor activity.

Functional assays evaluate the cytotoxic capacity of CAR T cells. Cytokine release assays measure IFN-γ, TNF-α, and IL-2 secretion following antigen stimulation. Killing assays, often performed in co-culture with tumor cells, assess the ability of CAR T cells to recognize and eliminate malignant cells. Real-time impedance-based platforms, such as the xCELLigence system, provide quantitative data on tumor cell lysis over time.

Genomic integrity and safety testing are critical due to genetic modification. PCR-based assays and vector copy number (VCN) analysis quantify transgene integration and detect unintended mutations. Regulatory guidelines recommend maintaining a VCN below five copies per genome to minimize insertional mutagenesis risk. Whole-genome sequencing and karyotyping may also be used to detect chromosomal abnormalities that could affect stability or introduce oncogenic risks.

Cryopreservation Steps

Cryopreservation maintains CAR T cells for future use while preserving viability and functionality. After quality control, cells are prepared for storage using a controlled freezing protocol. Cryoprotectants like dimethyl sulfoxide (DMSO) prevent ice crystal formation, which can damage cell membranes. The typical DMSO concentration is 5–10%, often combined with human serum albumin or stabilizing agents to improve post-thaw recovery.

A gradual cooling rate of approximately -1°C per minute prevents intracellular ice formation. Programmable freezers ensure uniform cooling, reducing thermal shock. Once frozen, CAR T cells are stored in vapor-phase liquid nitrogen at temperatures below -150°C, halting metabolic activity. Stable storage conditions are essential, as temperature fluctuations can compromise cell integrity.

Before infusion, thawing is carefully managed to minimize damage. Rapid warming in a 37°C water bath followed by immediate dilution removes residual DMSO. Post-thaw viability assessments confirm that cells retain their proliferative capacity and cytotoxic function. Optimized cryopreservation protocols maintain viability above 80%, ensuring therapeutic potential remains intact.