Name: Isolation, Cultivation, and Transient Transfection of Primary Human T Cells to Generate Chimeric Antigen Receptor (CAR) T Cells (Video) | JoVE Journal
Uploaded: 2026-03-27T13:30:00.000+00:00
Duration: 8 min 33 s
Description: Learn to isolate, cultivate, and transiently transfect primary human T cells for CAR T cell generation using mRNA electroporation. This protocol enables rapid screening of CAR constructs for immunotherapy research.

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5 minutes

This work was supported by the Austrian Science Fund (FWF Project ESP 465-B). We acknowledge the Medical University Core Facilities, Flow Cytometry Unit. We thank all voluntary healthy donors for their participation and sample donation, and the nursing team for their support.

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</ol>

The authors declare no conflict of interest.

Isolation and cultivation of primary human T cells provide the foundation for in vitro and in vivo studies. It is well-established that upon activation and in vitro expansion, T cells differentiate into an effector-like phenotype, and that the choice of cytokine supplements strongly impacts those differentiation fates9. CAR T cells cultured with IL-7 and IL-15 demonstrated improved persistence and better anti-tumor activity in mouse models compared to T cells expanded with IL-229. Therefore, the choice of both activation reagents and cytokine supplements is important to yield T cells with the desired phenotype. We compared two activation reagents (CD3/28 beads and TransAct), that differentially influence T cell expansion kinetics, yield, and the CD4/CD8 ratio, over an expansion period of three weeks. In general, T cells expanded after CD3/28 bead activation showed a higher percentage of CD8+ and lower frequencies of CD4+ cells than after TransAct activation. As CD8&#8314; T cells preferentially differentiate towards an effector-like TEMRA phenotype, reagent selection is a key variable that should match the goals of the experiment. In addition to phenotypic surface markers, the upregulation of activation markers yields information about the T cell state and effector functions. Both CD25 and CD69 are sensitive activation markers and are typically upregulated at early time-points after activation. They are commonly used to assess activation of T cells, but also other cell types, such as natural killer (NK) cells30,31,32. In contrast, the activation markers CD137 and CD154 showed lower expression in expanded T cells, highlighting their potential to assess specific upregulation in CAR T cells upon co-culture with target cells. Notably, when applying the activation marker panel to assess CAR T cell activity upon co-culture with target cells (i.e., tumor cells), a careful choice of activation markers is required to avoid high background expression of those markers on T cells even in the absence of target cells.
When working with primary T cells, one should be aware that their expansion and differentiation profiles are highly donor dependent. However, storage of the initial sample at 4 &#176;C prior to T cell isolation and a short handling time overall improve the yield and the quality of isolated cells. Cell viability is influenced by the duration of freezing and thawing steps, since the freezing medium contains cryoprotectants such as dimethyl sulfoxide (DMSO). While DMSO is crucial for preserving cellular structures upon freezing, it is toxic to cells at room temperature. Therefore, fast handling and dilution of DMSO after thawing are required. When electroporating T cells, the cells are most sensitive after the electric pulse, which aims to make the cell wall permeable for mRNA. Handling the cells with care by avoiding additional shear stress and working with pre-warmed solutions may greatly increase cell recovery and viability. It can be beneficial to add a positive control mRNA, such as a CAR of known expression or a reporter protein such as GFP, when testing a new CAR construct. It is further recommended to include at least MOCK transfected cells (i.e., cells electroporated without mRNA) as negative control. For functional assays, mRNA encoding for a CAR directed against a different target may be used to assess CAR specific activation.
Since expression kinetics may depend on the mRNA sequence, assessing the kinetics individually is a valuable tool for choosing the ideal time point for the conduction of downstream assays. This transfection method is primarily intended as a rapid, easy way to screen multiple CAR constructs in parallel. Due to the short timeframe during which the CAR is expressed on the T cell surface, long-term assays cannot be conducted. However, from preliminary transfection experiments, one can determine the best CAR design to proceed to viral transduction, if desired.
Finally, antibodies or their conjugated fluorophores may be exchanged to fit the experimental setup. However, it is crucial to consider the respective excitation and emission spectra of the fluorophores to interpret the results reliably. Moreover, single staining with one antibody at a time can be conducted in preliminary experiments to assure correct compensation of a multi-color panel. While we did not require spectral compensation for the proposed antibody panel, it might be necessary to set up a compensation matrix when the panel is changed.
The presented IVT and electroporation protocol offers a simple and flexible alternative to viral transduction for introducing CAR constructs into primary human T cells. Because it relies entirely on RNA-based delivery, it can be performed in laboratories without access to BSL-2 infrastructure, significantly lowering the barrier for CAR T cell research. Although viral transduction is very efficient and is predominantly used in the CAR field, it is accompanied by safety concerns related to genomic integration, a potential cause of secondary malignancies, as well as labor and cost-intensive production33,34. Moreover, the transient nature of CAR expression makes this approach particularly suitable for rapid, parallel screening of multiple CAR designs35, allowing researchers to compare construct performance in a high-throughput manner. The drawback of this method is the limited time of CAR expression, which typically peaks around 6-12 h after electroporation22,24,35,36. With transient transfection, long-term cultures, such as repeated stimulation assays, cannot be performed due to CAR downregulation. However, we suggest identifying the most promising CAR construct(s) with mRNA electroporation before considering moving to stable expression systems with a limited number of candidates.
Electroporation of mRNA into primary cells is a versatile technology to express proteins such as antigens in different cell types, including PBMCs, T cells, B cells, NK cells, and dendritic cells37. Notably, another possibility for transient CAR delivery are lipid nanoparticles (LNPs)36,38, which allow&#160;the delivery of CAR-encoding mRNA or DNA into T cells without viral vectors and enables integration-free CAR expression. In contrast, the Sleeping Beauty transposon system39,40,41 achieves stable integration without the requirements of viral components. The most suitable choice of T cell transfection or transduction depends on the intended application and considerations for scalability and manufacturing. mRNA-based CAR T cells have been successfully used in preclinical and clinical studies against a variety of different entities, including hematological and solid malignancies and autoimmune diseases42,43,44. Transiently expressed CARs offer advantages compared to stably expressed CARs, since the limited surface expression can prevent or reduce side effects25.
In summary, this workflow provides a straightforward, step-by-step protocol for the isolation, activation, cultivation, analysis, and transfection of primary T cells to generate CAR T cells. It is simple to implement, does not require BSL-2 laboratory facilities, and can be readily integrated into laboratories equipped with standard molecular biology and cell culture facilities.

Primary human T cells represent a physiologically relevant and versatile model for studying T cell biology and for the development and evaluation of cell therapies, including CAR engineering. Unlike immortalized cell lines, T cells derived from healthy donors closely reflect the functional diversity, activation states, phenotypic diversity, and signaling dynamics of T cells in vivo, providing a more accurate representation of immune responses and therapeutic performance1,2. Moreover, the most commonly used Jurkat T cell line, which is derived from a lymphoblastic leukemia, was reported to exhibit genomic instability upon prolonged culture, a common phenomenon with cell lines.3 While T cell lines, including reporter cell lines4, can be valuable tools for early screening and enable a fast read-out, the use of primary T cells from patients more closely reflects the final application. However, the limited availability and suboptimal quality of patient-derived T cells often restrict their applicability, particularly during early stages of preclinical product development5,6,7. Using cells from healthy donors further minimizes variability introduced by disease-related immune alterations and previous treatments and allows reproducible assessment of T cell function, activation, and transgene expression across multiple donors.
Given that primary T cells are available, protocols for isolation, activation and expansion of primary T cells are critical prerequisites for their genetic manipulation and functional assays. Common activation strategies employ either magnetic beads coated with antibodies, polymeric reagents containing T cell agonists, or soluble antibody formulations. Usually, activation reagents mimic physiological T cell receptor (TCR) activation through the CD3 complex and co-stimulatory signaling through CD28, which resembles a stronger activation than solely via CD38. Activation with CD3/28 beads allows for precise control of activation strength and bead-to-cell ratio. After an initial stimulation phase, beads can either be removed or kept in the culture for continuous activation. T cells typically expand rapidly for three to four weeks before cell growth slows down. In contrast, TransAct contains a colloidal polymeric matrix, and excess reagent can simply be removed by centrifugation and exchange of the supernatant. Therefore, it provides an alternative that enables homogeneous activation in culture and resembles T cell activation with a lower stimulus compared to CD3/28 beads. However, proliferation is induced for approximately two weeks, and re-stimulation is required for longer expansion periods. After initial T cell activation with an activation reagent, T cells are cultured and expanded in vitro for one to four weeks before they can be used for experiments. Notably, the type of activation reagent, the choice of cytokines9,10,11, and the expansion period influence the absolute T cell numbers, phenotype and activation state. Typically, optimal conditions such as reagent concentration, culture density, cytokine supplementation, and expansion time should be initially tested and empirically determined to achieve robust proliferation without causing activation-induced cell death or T cell exhaustion. Here, we give a side-by-side comparison of two different activation protocols and their effect on the differentiation and activation profile of primary human T cells to support the decisions on the choice of activation reagent and timing of experiments.
Additionally, we suggest a flow cytometric panel useful for analyzing phenotypic composition and activation status of T cells, supporting experimental planning. For genetic modification of primary T cells, transient transfection using electroporation of messenger ribonucleic acid (mRNA) represents a safe, efficient, and flexible alternative to viral transduction12. While lentiviral vectors enable stable and long-term expression of transgenes, they require complex biosafety measures, involve integration into the host genome, and can lead to variable expression depending on the integration site. In addition, the production of lentiviral supernatants, followed by T cell transduction and selection of transduced cells is time-consuming. In contrast, mRNA electroporation allows rapid and transient expression of target genes without genomic integration or viral components, thereby reducing regulatory burden and enabling fast and iterative testing of constructs. This is particularly advantageous for early development stages, in which multiple CAR candidates are screened and evaluated. In recent publications, transfection with mRNA is explored as a therapeutic platform13,14,15. We suggest that transient transfection with mRNA16 is an approach that is easy to implement, especially for laboratories without the possibility to conduct lentiviral work in a biosafety level 2 (BSL-2) environment. A side-by-side comparison of different activation regiments prior lentiviral transduction is available elsewhere17.
This protocol describes the isolation, activation, cultivation, expansion, and transient transfection of primary human T cells using either anti-CD3/28 beads or TransAct for activation and mRNA electroporation for transient CAR expression. The outlined workflow enables reproducible generation of functional CAR T cells suitable for downstream applications such as cytotoxicity assays, phenotypic characterization, or in vitro modeling of adoptive T cell therapies.

<table><tbody><tr><td>1.5 mL Microcentrifuge Tubes, High Performance</td><td>VWR</td><td>525-1164</td><td></td></tr><tr><td>12-well plates, flat bottom, TC-treated&amp;nbsp;</td><td>VWR</td><td>734-2324</td><td></td></tr><tr><td>24-well plates, flat bottom, TC-treated</td><td>VWR</td><td>734-2325</td><td></td></tr><tr><td>6-well plates, flat bottom, TC-treated&amp;nbsp;</td><td>VWR</td><td>734-2323</td><td></td></tr><tr><td>7-AAD staining solution</td><td>Miltenyi Biotec</td><td>130-111-568</td><td></td></tr><tr><td>Anti-Flag DYKDDDDK Antibody, APC, REAfinity</td><td>Miltenyi Biotec</td><td>130-119-584</td><td></td></tr><tr><td>BD FACSCanto II Clinical Flow Cytometry System</td><td>BD Biosciences</td><td>FACSCanto II</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Merck</td><td>A4503-10G</td><td>For gravity flow isolation</td></tr><tr><td>CD134 (OX40) Antibody, anti-human, REAfinity</td><td>Miltenyi Biotec</td><td>130-126-048</td><td>Conjugate: PE</td></tr><tr><td>CD137 (4-1BB) Antibody, anti-human, REAfinity</td><td>Miltenyi Biotec</td><td>130-110-764</td><td>Conjugate: APC</td></tr><tr><td>CD154 (CD40L) Antibody, anti-human, REAfinity</td><td>Miltenyi Biotec</td><td>130-114-142</td><td>Conjugate: PerCP-VIO 700</td></tr><tr><td>CD25 Antibody, anti-human, REAfinity</td><td>Miltenyi Biotec</td><td>130-114-541</td><td>Conjugate: PE-Vio 770</td></tr><tr><td>CD3 Antibody, anti-human, REAfinity</td><td>Miltenyi Biotec</td><td>130-132-352</td><td>Conjugate: PE-Vio 670</td></tr><tr><td>CD4 Antibody, anti-human, REAfinity</td><td>Miltenyi Biotec</td><td>130-113-223</td><td>Conjugate: APC-Vio 770</td></tr><tr><td>CD4 MicroBeads, human</td><td>Miltenyi Biotec</td><td>130-045-101</td><td>For gravity flow isolation</td></tr><tr><td>CD45 Antibody, anti-human, REAfinity</td><td>Miltenyi Biotec</td><td>130-110-775</td><td>Conjugate: VioBlue</td></tr><tr><td>CD45RA Antibody, anti-human, REAfinity</td><td>Miltenyi Biotec</td><td>130-132-270</td><td>Conjugate: Vio Bright V600</td></tr><tr><td>CD62L Antibody, anti-human, REAfinity</td><td>Miltenyi Biotec</td><td>130-127-499</td><td>Conjugate: Vio Bright R720</td></tr><tr><td>CD69 Antibody, anti-human, REAfinity</td><td>Miltenyi Biotec</td><td>130-112-800</td><td>Conjugate: VioGreen</td></tr><tr><td>CD8 Antibody, anti-human, REAfinity</td><td>Miltenyi Biotec</td><td>130-110-677</td><td>Conjugate: FITC</td></tr><tr><td>CD8 MicroBeads, human</td><td>Miltenyi Biotec</td><td>130-045-201</td><td>For gravity flow isolation</td></tr><tr><td>Cellstar 96 Well Suspension Culture Plate, U-bottom</td><td>Greiner Bio-one</td><td>650 185</td><td></td></tr><tr><td>Centrifuge 5810&amp;nbsp;R</td><td>Eppendorf</td><td>5811000015</td><td></td></tr><tr><td>CO2 incubator ICO150</td><td>Memmert</td><td>ICO150</td><td></td></tr><tr><td>CoolNat Ice Machine</td><td>Ziegra Eismaschinen GmbH</td><td>ZBE 30-10</td><td></td></tr><tr><td>Corning CoolCell Freezer Container</td><td>Corning</td><td>CLS432000</td><td></td></tr><tr><td>Counting chamber, B&amp;uuml;rker-T&amp;uuml;rk pattern, BLAUBRAND</td><td>Brand</td><td>719520</td><td></td></tr><tr><td>CryoPure tubes, 2 mL, QuickSeal screw cap</td><td>Sarstedt</td><td>72,380</td><td></td></tr><tr><td>CytoFLEX LX Flow Cytometer</td><td>Beckmann Coulter</td><td>C11185</td><td></td></tr><tr><td>Deoxynucleotide (dNTP) Solution Mix</td><td>New England Biolabs</td><td>N0447S</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO) Cell culture grade</td><td>AppliChem</td><td>A3672</td><td></td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>Dynabeads Human T-Activator CD3/CD28</td><td>Gibco</td><td>11131D</td><td></td></tr><tr><td>Electroporation Cuvettes with 4 mm gap size, yellow cap</td><td>VWR</td><td>732-1137</td><td></td></tr><tr><td>Eppendorf Research plus pipette, 0.1&amp;ndash;2.5 &amp;micro;L</td><td>Eppendorf</td><td>3123000012</td><td></td></tr><tr><td>Eppendorf Research plus pipette, 100&amp;ndash;1000 &amp;micro;L</td><td>Eppendorf</td><td>3123000063</td><td></td></tr><tr><td>Eppendorf Research plus pipette, 2&amp;ndash;20 &amp;micro;L</td><td>Eppendorf</td><td>3123000039</td><td></td></tr><tr><td>Eppendorf Research plus pipette, 20&amp;ndash;200 &amp;micro;L</td><td>Eppendorf</td><td>3123000055</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Merck</td><td>E6758-100G</td><td>For gravity flow isolation</td></tr><tr><td>Falcon 15 mL PP Centrifuge Tube, Conical Bottom</td><td>Corning</td><td>352196</td><td></td></tr><tr><td>Falcon 5 mL Round Bottom Polystyrene Test Tube</td><td>Corning</td><td>352008</td><td></td></tr><tr><td>Falcon 50 mL PP Centrifuge Tube, Conical Bottom</td><td>Corning</td><td>325098</td><td></td></tr><tr><td>Fetal Bovine Serum, Value FBS</td><td>Gibco</td><td>A52567-01</td><td></td></tr><tr><td>Ficoll PM 400</td><td>Sigma-Aldrich</td><td>F4375-10G</td><td>For density gradient isolation</td></tr><tr><td>Fisherbrand&amp;nbsp;Sterile Graduated Transfer Pipets</td><td>Fisher Scientific</td><td>13469108</td><td></td></tr><tr><td>Gene Pulser Xcell Total System</td><td>BioRad</td><td>1652660</td><td>with PC Module and CE Module</td></tr><tr><td>HiScribe T7 ARCA mRNA Kit (with tailing)</td><td>New England Biolabs</td><td>E2060S</td><td></td></tr><tr><td>Human IL-15, research grade</td><td>Miltenyi Biotec</td><td>130-093-955</td><td></td></tr><tr><td>Human IL-7, research grade</td><td>Miltenyi Biotec</td><td>130-095-367</td><td></td></tr><tr><td>Integra Biosciences Corp&amp;nbsp;PIPETBOY acu 2 pipet aid</td><td>Fisher Scientific</td><td>NC0085685</td><td></td></tr><tr><td>LE Agarose</td><td>Biozym</td><td>840004</td><td></td></tr><tr><td>LS Columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td>For gravity flow isolation</td></tr><tr><td>Microplate, 96 well, PS, V-bottom, clear, non-binding</td><td>Greiner Bio-one</td><td>651901</td><td></td></tr><tr><td>Monarch RNA Cleanup Kit (50 &amp;mu;g)</td><td>New England Biolabs</td><td>T2040S</td><td></td></tr><tr><td>Monarch Spin PCR &amp;amp; DNA Cleanup Kit</td><td>New England Biolabs</td><td>T1130S</td><td></td></tr><tr><td>MSC-Advantage Class II Biological Safety Cabinet</td><td>Thermo Scientific</td><td>51028226</td><td></td></tr><tr><td>Olympus IMT-2 Microscope</td><td>Spach Optics</td><td>OLYMPUS-IMT2</td><td></td></tr><tr><td>Opti-MEM I Reduced Serum Medium, no phenol red</td><td>Gibco</td><td>11058021</td><td></td></tr><tr><td>Pancoll human, Density: 1.077 g/mL</td><td>Pan Biotech</td><td>P04-60100</td><td>For density gradient isolation</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Q5 High-Fidelity DNA Polymerase</td><td>New England Biolabs</td><td>M0491S</td><td></td></tr><tr><td>QuadroMACS Separator</td><td>Miltenyi Biotec</td><td>130-090-976</td><td>For gravity flow isolation</td></tr><tr><td>RosetteSep Human T Cell Enrichment Cocktail</td><td>StemCell Technologies</td><td>15021</td><td>For density gradient isolation</td></tr><tr><td>Rotiphorese 50x TAE Buffer</td><td>Carl Roth</td><td>CL86.2</td><td></td></tr><tr><td>RPMI 1640 Medium, GlutaMAX Supplement</td><td>Gibco</td><td>61870-044</td><td></td></tr><tr><td>RPMI 1640 Medium, no phenol red</td><td>Gibco</td><td>11835030</td><td></td></tr><tr><td>SafeSeal SurPhob tips, 1000 &amp;micro;L, sterile</td><td>Biozym</td><td>VT0260</td><td></td></tr><tr><td>SafeSeal SurPhob tips, 20 &amp;micro;L, sterile</td><td>Biozym</td><td>VT0220</td><td></td></tr><tr><td>SafeSeal SurPhob tips, 200 &amp;micro;L, sterile</td><td>Biozym</td><td>VT0240</td><td></td></tr><tr><td>SafeSeal SurPhob tips,10 &amp;micro;L, sterile</td><td>Biozym</td><td>VT0200</td><td></td></tr><tr><td>Serological pipette, standard line, 10 mL</td><td>VWR</td><td>612-3700</td><td></td></tr><tr><td>Serological pipette, standard line, 2 mL</td><td>VWR</td><td>612-3704</td><td></td></tr><tr><td>Serological pipette, standard line, 25 mL</td><td>VWR</td><td>612-3698</td><td></td></tr><tr><td>Serological pipette, standard line, 5 mL</td><td>VWR</td><td>612-3702</td><td></td></tr><tr><td>Serological pipette, standard line, 50 mL</td><td>VWR</td><td>612-3696</td><td></td></tr><tr><td>Sterilin Polystyrene 30 mL Containers</td><td>Thermo Fisher</td><td>128A</td><td></td></tr><tr><td>T Cell TransAct, human</td><td>Miltenyi Biotec</td><td>130-111-160</td><td></td></tr><tr><td>UltraPure DNase/RNase-Free Distilled Water</td><td>Fisher Scientific</td><td>11538646</td><td></td></tr><tr><td>VWR, Cell Culture Flasks, TC-Treated, 182 cm&amp;sup2;</td><td>VWR</td><td>734-2315</td><td></td></tr><tr><td>VWR, Cell Culture Flasks, TC-Treated, 25 cm&amp;sup2;</td><td>VWR</td><td>734-2311</td><td></td></tr><tr><td>VWR, Cell Culture Flasks, TC-Treated, 75 cm&amp;sup2;</td><td>VWR</td><td>734-2313</td><td></td></tr></tbody></table>

isolation, cultivation, and transient transfection of primary human t cells to generate chimeric antigen receptor (car) t cells

Isolation, Cultivation, and Transient Transfection of Primary Human T Cells to Generate Chimeric Antigen Receptor (CAR) T Cells

To assess the efficacy and potency of chimeric antigen receptor (CAR) T cells, it is essential to isolate, activate, expand, characterize, and genetically engineer primary human T cells. Compared to common T cell lines, primary cells can provide a more realistic model system, since they are highly variable, capable of T cell-mediated killing, and able to differentiate. Here, we provide a step-by-step protocol for all essential steps for the cultivation of primary T cells, including their isolation from blood products, activation, in vitro expansion, and transient transfection. We describe differences in expansion kinetics and differentiation of primary T cells upon activation with two different reagents and provide an antibody panel for flow cytometric analysis of T cell phenotype and activation state. We offer detailed instructions for electroporation of human T cells with mRNA encoding a CAR and assessing CAR expression kinetics over time. This protocol provides recommendations on ideal timepoints for downstream potency and efficacy assays of CAR T cells and the influence of different amounts of mRNA on signal intensity. Given the transient nature of this method, it provides a rapid and easy-to-implement possibility to test and compare many CAR constructs in parallel.

Activation and expansion of primary human T cells 
Primary T cells were thawed and activated on day 0 with either CD3/28 beads or TransAct, expanded for 23 days, and counted every 2-3 days (Figure 2A). On counting days, cell densities were adjusted to 0.3-0.5 x 106 cells/mL. On days 0/2/5/9/14/20, T cells were used for flow cytometric analysis to assess the phenotype and expression of activation markers. Within the first week after activation, T cells proliferated and expanded rapidly with both activation reagents (Figure 2B), with a slightly lower fold-expansion for the following days for T cells activated by TransAct (Figure 2C). Notably, activation of T cells was also visible by microscopic analysis, with T cell clusters forming around the activation reagents (Figure 2D).
<img alt="T cell expansion timeline, experiment charts, microscopy images; CD3/28 beads vs. TransAct method." class="xfigimg" src="/files/ftp_upload/70162/70162fig2v3.jpg" data-altupdatedat="1784001604000" data-alt="Figure 2"> 
Figure 2: Activation and expansion of primary T cells using two different activation reagents. (A) Schematic representation of the experimental timeline. Cells were activated either with CD3/28 beads or TransAct on day 0 and cultured for 23 days. Blue dots depict days where the cells were counted, red dots depict days where the cells were analyzed by flow cytometry, and purple dots depict days where both analyses were performed. (B) and (C) Expansion of primary T cells during (B) the first week or (C) within 16 days of culture. Values depict the mean ± standard deviation of 4 (CD3/28 beads) or 3 (TransAct) different healthy T cell donors. (D) Representative microscopic images of activated T cells with visible cell clustering on days 1 and 2 after activation. Images were taken with an inverted microscope. The ruler corresponds to 200 µm. <a href="https://www.jove.com/files/ftp_upload/70162/70162fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Staining of T cell phenotype and activation markers 
For flow cytometric analysis, the following gating strategy was applied (Figure 3): A first gate (FSC-Height vs. SSC-Height) was applied to remove cell debris and beads, followed by gating for single cells (FSC-Width vs. SSC-Area). The percentage of CD3+ cells should be in the range of 98-100%. Further sub-gating yielded the percentage of CD4+ (helper) T cells and CD8+ (cytotoxic) T cells, which were further gated for different phenotypic T cell subsets using the markers CD45RA and CD62L. CD3+ T cells were additionally analyzed for the expression of activation markers (CD25/CD69/CD134/CD137/CD154).
<img alt="Flow cytometry gating diagram with scatter plots; CD3+ lymphocyte analysis, CD4/CD8 ratio evaluation." class="xfigimg" src="/files/ftp_upload/70162/70162fig3v3.jpg" data-altupdatedat="1784001604000" data-alt="Figure 3"> 
Figure 3: Gating strategy for flow cytometric analysis. Representative dot plots of one T cell donor activated with CD3/28 beads on day 5 are shown (n = 4). Abbreviations; TEMRA = T effector memory cells re-expressing CD45RA; TSCM = stem cell-like memory T cells; TEM = effector memory T cells; TCM = central memory T cells. <a href="https://www.jove.com/files/ftp_upload/70162/70162fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
After activation with CD3/28 beads, the percentage of CD4+ T cells compared to CD8+ T cells decreased over time, with 74.3% (± 7.4%) of CD4+ T cells on day 5, which decreased to 14.6% (± 3.4%) on day 20 after activation. CD8+ T cells increased from 20.7% (± 6.4%) on day 5 to 82.4% (± 3.1%) on day 20 (Figure 4A). Similar trends were detected after activation with TransAct, with slightly lower percentages of CD8+ T cells on day 20 (72.5% ± 0.7%). Therefore, the slightly slower T cell expansion that was observed after activation with TransAct corresponded to a lower percentage of CD8+ and a higher percentage of CD4+ cells during the expansion period. The differences in T cell phenotypic composition were similar with both activation reagents (Figure 4B). However, the frequencies of T effector memory cells re-expressing CD45RA (TEMRA) were overall higher for CD8+ cells than for CD4+ cells. Concordantly, CD4+ T cells showed higher percentages of central memory T cells (TCM) and stem-cell-like memory T cells (TSCM) (Figure 4B).
CD134 was the only activation marker that was already upregulated after thawing of T cells, with 37.6% (± 4.1%) CD134+ T cells activated with CD3/28 beads and 36.1% (±4.4%) CD134+ T cells for TransAct-activated T cells (Figure 4C). In both activation strategies, CD25, CD69, and CD134 were highly upregulated by day 2. For CD3/28 beads, CD69 and CD134 expression reached their peak on day 2, while CD25 peaked on day 5. Similar results were seen for TransAct-activated T cells. CD134 and CD154 were upregulated only on day 14 for both CD3/28 beads and TransAct-activated cells. Overall, the observed trends were very similar between the two activation reagents. However, CD69 expression on day 14 was slightly higher in CD3/28-activated T cells than their TransAct counterparts (97.1% ± 0.9% versus 86.5% ± 5.1%). This corresponds to the prolonged high expansion rate seen with CD3/28 bead-activated cells. To sum up, the proposed activation marker panel includes markers that are upregulated both at early and later timepoints after activation, with varying sensitivity between the different activation markers, but overall, similar trends for both activation reagents.
<img alt="T-cell memory phenotype analysis; bar graph showing CD4/CD8 expression; frequency vs. time points." class="xfigimg" src="/files/ftp_upload/70162/70162fig4.jpg" data-altupdatedat="1784001604000" data-alt="Figure 4"> 
Figure 4: Analysis of T cell phenotype and activation marker expression with different activation reagents during a cultivation period of 20 days. (A) Percentages of CD4+/CD8+ T cells during cultivation after activation with CD3/28 beads (left) or TransAct (right). (B) T cell differentiation after activation with CD3/28 beads (two left panels) or TransAct (two right panels), each for either CD4+ or CD8+ cells. The phenotype was assessed using the markers CD45RA and CD62L. TEMRA: CD45RA+ CD62L-, TEM: CD45RA- CD62L-, TCM: CD45RA-CD62L+, TSCM: CD45RA+ CD62L+. (C) Expression of activation markers after activation with CD3/28 beads or TransAct and during the expansion period. Frequencies [%] of the parent population CD3+ T cells are shown (mean ± standard deviation of 4 [CD3/28 beads] or 3 [TransAct] different healthy T cell donors). Abbreviations; TEMRA = T effector memory cells re-expressing CD45RA; TSCM = stem cell-like memory T cells; TEM = effector memory T cells; TCM = central memory T cells. <a href="https://www.jove.com/files/ftp_upload/70162/70162fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Transient transfection of primary T cells and analysis of CAR expression 
Primary T cells were transfected with mRNA by electroporation on day 7 after activation with either CD3/28 beads or TransAct. As a template, mRNA encoding for a second-generation CAR targeting chondroitin sulfate proteoglycan 4 (CSPG4) with a CD28 co-stimulatory and a CD3ζ signaling domain was used. A DYKDDDDK-tag (flag-tag) was included to enable flow-cytometric detection. T cell viability and CAR expression were assessed 3, 6, 24, and 48 h after electroporation. Staining was performed as described in protocol section 6.
Although the viability of both CD3/28 bead- and TransAct-activated T cells was low 3 h after electroporation, overall, the viability of transfected CAR T cells ranged from 51-85% and did not differ significantly between the two activation strategies (Figure 5A). The CAR was detectable already at this early point after electroporation. Although the percentage of CAR+ cells gradually decreased over time, the highest frequency of CAR+ cells was detectable within 24 h after electroporation (Figure 5B, left panel). Notably, the signal intensity was stable within the first 6 h after electroporation and gradually decreased afterwards (Figure 5B, right panel). CAR expression was almost absent after 48 h, demonstrating the transient nature of cell manipulation and the importance of choosing an early time window for downstream assays. Representative dot plots for CAR-expression of one donor are shown in Figure 5C. For both activation reagents, the CD4/CD8 ratio did not differ between CAR and MOCK-transfected cells (Figure 5D). Consistent with our previous results, T cells activated with CD3/28 beads showed a slightly higher frequency of CD8+ T cells than cells activated with TransAct. For the phenotypic analysis, one representative donor is shown due to large donor-dependent variations (Figure 5E). While both the activation reagent and CD4/CD8 state influence the T cell phenotype, CAR expression does not cause a change in phenotype compared to MOCK-transfected cells.
To assess the influence of mRNA concentration on expression levels, primary T cells were transfected with mRNA encoding for a mutant version of GFP (muGFP) using 0.1, 0.3, 1, 3 or 6 µg of mRNA. The GFP signal was measured by flow cytometry 24 h after electroporation. As expected, already as little amounts of mRNA as 0.1 µg led to a detectable GFP signal, with a gradual increase of signal intensity with increasing mRNA amounts. In our experiments, 1 µg of mRNA was already saturating, and larger amounts (3 and 6 µg of mRNA) did not lead to an increased GFP signal (Figure 5F). This highlights the possibility of using varying amounts of mRNA to titrate expression levels, which can be beneficial for testing the influence of different CAR- or even target antigen expression levels28.
<img alt="CAR T cell viability and frequency post-electroporation; flow cytometry data analysis, mRNA titration." class="xfigimg" src="/files/ftp_upload/70162/70162fig5v3.jpg" data-altupdatedat="1784001604000" data-alt="Figure 5"> 
Figure 5: Expression kinetics of CARs and phenotype analysis of transfected CAR T cells. (A) Viability of CAR T cells after electroporation with CAR-encoding mRNA. 2 µL of 7-AAD in 100 µL of staining buffer was used to assess viability after 10 min incubation. n = 2-3 individual donors (B) Staining for CAR expression with an anti-DYKDDDDK antibody (conjugated with APC, 1:50 final dilution). After gating on viable cells, either the percentage of CAR-positive cells in relation to MOCK-transfected controls (left panel) or the geometric median fluorescence intensity (GeoMean, right panel) of the whole population was analyzed. Shown are averages of n = 3 individual donors ± S.D. (C) Representative dot plots of one donor from (B) demonstrating the kinetics of CAR expression over time. (D) Analysis of CD4+/CD8+ T cells and comparison of MOCK-transfected with CAR-transfected T cells and two different activation strategies 24 h after electroporation. Shown are averages of n = 3 individual donors ± S.D. (E) Phenotypic analysis of MOCK-transfected and CAR-transfected T cells shown in (B) 24 h after electroporation. Shown is one representative donor of n = 3. (F) Analysis of muGFP expression 24 h after electroporation with different amounts of mRNA. Gating for live cells was performed before the GeoMean for GFP was quantified. Shown are averages of n = 3 donors ± S.D. Abbreviations; CAR = chimeric antigen receptor; mRNA = messenger ribonucleic acid; 7-AAD = 7-aminoactinomycin; RT = room temperature; muGFP = mutant green fluorescent protein; S.D. = standard deviation. <a href="https://www.jove.com/files/ftp_upload/70162/70162fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

This protocol describes the essential steps for the cultivation of primary T cells, including their isolation, activation, expansion, and transient transfection by mRNA electroporation. Additionally, a workflow and antibody panel for flow cytometric analysis of phenotype, activation markers, viability, and chimeric antigen receptor (CAR) expression are proposed.

To assess the efficacy and potency of chimeric antigen receptor (CAR) T cells, it is essential to isolate, activate, expand, characterize, and genetically ...

My lab focuses on next-generation cell therapies with T-cells expressing chimeric antigen receptor, or short, CARs. T-cell lines only poorly represent a diversity of primary human T-cells. This protocol provides guidelines for isolating, engineering, and analyzing primary T-cells for the generation of CAR T-cells.To begin, obtain primary T-cells at the exponential growth phase and dilute them to a density of 0.3 times 10 to the power six cells per milliliter in full T-cell medium one day before the experiment. On the day of transfection, pre-warm all required media at 37 degrees Celsius. Calculate the required seeding volume to achieve a final density of 0.5 to 1.0 times 10 to the power of six cells per milliliter after electroporation.Add full T-cell medium in a 12-well plate to reach the required density and pre-warm the plate to 37 degrees Celsius until electroporation. Count the T-cells and calculate the required volume to use one to five times 10 to the power of six cells for each chimeric antigen receptor messenger ribonucleic acid delivery. Transfer the required volume of T-cell suspension into a conical centrifuge tube.Spin the tube at 300g at room temperature for five minutes and discard the supernatant. Perform the first washing step by adding 20 milliliters of RPMI medium with phenol red and without supplements on top of the cell pellet. Centrifuge at 300g at room temperature for five minutes, discard the supernatant, and repeat the washing twice:once with RPMI without phenol red and supplements and once with reduced serum medium.After the final wash, discard the supernatant thoroughly by pipetting off the remaining liquid without disturbing the pellet. Resuspend the cell pellet in 100 microliters of reduced serum medium per electroporation. During the final T-cell washing step, collect all required mRNA aliquots from the freezer and keep them on ice.Label the electroporation cuvettes and the plate with the pre-warmed media and set up the conditions on the electroporation device using a Gene Pulser Xcell Total System. Select the Square Wave Protocol and set the required parameters. Once the T-cell suspension is ready, combine 100 microliters of the suspension with three to five micrograms of mRNA per electroporation and mix gently without pipetting up and down.Now, transfer the mRNA and T-cell mixture to an electroporation cuvette. Ensure that the suspension fully covers the bottom of the cuvette without pipetting up and down. Next, quickly transfer the cuvette to the electroporation device and perform electroporation using the previously prepared settings.After electroporation, add 100 microliters of pre-warmed full T-cell medium from the well plate to the cuvette to recover the T-cells. Transfer the cells back to the plate. Rinse the cuvette with an additional 100 microliters of medium and transfer the rinse to the same well.Incubate the Transfected T-cells at 37 degrees Celsius with 5%carbon dioxide. Within 24 hours post-transfection, transfer 50, 000 to 100, 000 cells per staining reaction into a suitable polystyrene round bottom tube. Wash the cells by adding one milliliter of staining buffer consisting of phosphate buffer saline, or PBS, with 0.5%fetal bovine serum.Centrifuge at 300g at four degrees Celsius for five minutes. From this point onward, keep the cells on ice and work with cooled reagents. Prepare a master mix containing the required fluorophore conjugated antibodies required for staining.Dilute the antibodies as required in a final staining volume of 50 microliters. After centrifugation of the cells, discard the supernatant thoroughly by pipetting off residual liquid without disturbing the cell pellet. Resuspend each pellet in 50 microliters of the prepared antibody mix.Incubate the tubes at four degrees Celsius in the dark for at least 30 minutes. Wash the cells by adding one milliliter of staining buffer directly to each tube. Centrifuge at 300g at four degrees Celsius for five minutes and discard the supernatant thoroughly by pipetting off residual liquid.Resuspend the cell pellets in 100 microliters of staining buffer per tube. Finally, before starting the measurement, add two microliters of 7-Aminoactinomycin D solution to each tube and mix well. Incubate for 10 minutes at room temperature in the dark and proceed immediately to fluorescence measurement.The viability of transfected chimeric antigen receptor, or CAR, T-cells ranged from 51 to 85%and did not differ significantly between CD3/28 bead and TransAct-activated T-cells. The highest frequency of CAR-positive cells was detectable within 24 hours after electroporation. The signal intensity of CAR expression was stable within the first six hours after electroporation and gradually decreased thereafter.CAR expression was almost absent after 48 hours, demonstrating the transient nature of cell manipulation and the importance of choosing an early time window for downstream assays. The CD4 positive to CD8 positive ratio did not differ between CAR and MOCK transfected T-cells for both activation reagents. CAR expression did not alter the T-cell phenotype compared to MOCK transfected cells.Electroporation width as little as 0.1 micrograms of mRNA led to a detectable mutant green fluorescent protein signal after 24 hours. Signal intensity increased, with increasing mRNA amounts up to one microgram, while three in six micrograms did not further increase the signal. A key challenge is donor-dependent variability in primary T-cells.Therefore, sufficient biological replicates are essential to ensure reproducibility and reliable interpretation of experimental results. This protocol can be combined with CAR T-cell efficacy analysis, including cytotoxicity, or repeated stimulation assays, or the measurement of circulated cytokines for broader functional evaluation.

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Immunology and Infection

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