Improving mRNA transfection outcomes in T-cells with high-throughput cytometry

This research investigates how to optimize mRNA transfection in human T-cells utilizing the jetMESSENGER^® transfection reagent. By using advanced bioanalytical instruments like the Incucyte^® Live-Cell Analysis System and the iQue^® High-Throughput Screening platform, Sartorius detected settings that improve T-cell viability, transfection efficiency, and eGFP expression alike.

Their results show the effective cytotoxic activity of transfected T-cells against A549 cancer cells, underscoring the potential of mRNA-based transfection for medical use cases, particularly in cancer immunotherapy. This study underscores precise T-cell engineering’s critical role in reaching elevated functionality and therapeutic efficacy levels.

Introduction

T-lymphocytes (T-cells) have been identified as critical components of the adaptive immune system, with a critical role in controlling immune responses, the secretion of cytokines, and detecting and getting rid of infected or malignant cells.¹

Considerable advancements in understanding T-cells have made way for the development of state-of-the-art therapeutic approaches that have demonstrated outstanding success in multiple domains, such as cancer immunotherapies, autoimmunities, and infectious diseases.^1,2

Among the different approaches utilized to employ T-cells for therapeutic effect, chimeric antigen receptor (CAR) T-cells are considered to be outstanding, with significant FDA approvals such as Kymriah and Yescarta for treating B-cell malignancies.^3,4 In spite of these therapies’ success, the efficacious manipulation of T-cells for study and medicinal purposes remains a key problem.

Conventional approaches for modifying T-cells include viral vectors, which are more costly and have a more limited cargo capacity in comparison to other approaches, while generating considerable risks, such as insertional mutagenesis and immunogenicity.⁵

Different approaches like electroporation depend on disrupting membranes, typically leading to low transfection efficiency and excessive cellular damage. Contrasting this, non-viral transfection reagents like lipid-based nanoparticles enable a safer, more cost-effective, and more effective option for T-cells and other difficult-to-transfect cellular varieties .^5,6

Alongside the intracellular delivery approach, the variety of genetic material should also be considered carefully. Recently, mRNA has arisen as a crucial part of genome-editing strategies owing to its capacity to engender precise and efficient improvements without integrating into the host genome.⁷

mRNA transfection is especially beneficial for T-cells, permitting the rapid and efficient production of necessary functional proteins. This strategy has demonstrated significant potential both in medicinal use cases and in T-cell biology research.

Modifying gene-delivery approaches for T-cells is critical for improving their potential in medicine. With this requirement, the principal goal of this research was to modify the transfection of human T-cells with mRNA utilizing the jetMESSENGER^® transfection reagent.

In particular, Sartorius wanted to identify the settings needed for optimizing cell proliferation, transfection efficiency, and cytotoxic activity against cancer cells. T-cells were prepared in specialized 4Cell® Nutri-T GMP media alongside high-quality cytokines from Sartorius to enable both their recovery and expansion.

For kinetic visualization and quantitative tests guiding the optimization process, Sartorius used the Incucyte^® LiveCell Analysis System and the iQue^® High-Throughput Screening (HTS) cytometer.

Figure 1. T-cell transfection workflow. A workflow diagram highlighting the key steps involved in eGFP-mRNA transfection of T-cells using jetMESSENGER^®, including timepoints for cell monitoring, sampling, and analysis. Image Credit: Sartorius

Materials and methods

The T-cell transfection workflow schematic (Figure 1) highlights various steps in the transfection process and underscores critical points in the timeline for tracking and analyzing cells.

Cell culture and T-cell activation

Human primary T-cells obtained from healthy donors were thawed after being frozen according to the vendor’s guidelines utilizing 4Cell^® Nutri-T GMP media supplemented with FBS for more support while thawing (Table 1). The cells were suspended again to a density of 1.5 × 10⁶ cells/mL and supplemented with either IL-2 or IL-21.

After adding cytokines, the T-cells were set aside to rest for 4 hours in a 37 °C, 5 % CO₂^-humidified incubator. To activate and proliferate the cells, T-Cell TransAct^™ was added, and the cells were then incubated for 44 hours more under the aforementioned settings.

Incucyte^® Cytolight Red A549 adenocarcinoma cells that stably expressed a cytoplasmic red fluorescent protein were preserved in F-12K media (Table 1) and utilized as target cells in the immune cell killing assay. The media formulations and materials utilized in the workflow are showcased in Table 1 and Table 2.

T-cell proliferation studies comparing IL-2 and IL-21

Research was carried out utilizing the protocol demonstrated in the T-cell transfection workflow shown in Figure 1.

On day 0 of the workflow, T-cells were washed with phosphate-buffered saline (PBS) (300 x g, 5 mins) and suspended once again in 4Cell^® Nutri-T GMP media at a density of 3 x 10⁶ cells/mL. The cells were equilibrated to serum-free settings for 30 minutes in a 37 °C, 5 % CO₂ humidified incubator prior to being seeded into a 96-well plate (187,500 cells/ well).

After a 30-minute cell-settling period at room temperature, the plate was put in the Incucyte^® LiveCell Analysis System (Incucyte^® system) to track proliferation. After 3.5 hours, the plate was taken away briefly to include fresh media carrying either IL-2 or IL-21 and TransAct^™ to the wells. Monitoring proliferation continued in the Incucyte^® system until full cell confluence was achieved.

High-definition (HD) phase contrast images were obtained at 3-hour intervals via a 20x objective (4 images/well) and examined with the integrated Incucyte^® Cell-By-Cell Analysis Software Module or the Incucyte^® AI Confluence Analysis software (offered via the Incucyte^® Base Analysis Software).

Table 1. Media formulations used for cell culture. Source: Sartorius

Table 2. Materials required for transfection and assays. Source: Sartorius

T-cell transfection

Transfection was carried out on day 0 (Figure 1). T-cells were washed and then equilibrated in media as outlined in the proliferation research. Over the 30 minutes during equilibration, mRNA was complexed with the jetMESSENGER^® transfection reagent.

Rising concentrations of CleanCap^® eGFP mRNA (Table 2) were blended with jetMESSENGER^® at a ratio of 1 µg mRNA to 3 µL transfection reagent and incubated for 15 minutes at room temperature. The mRNA-jetMESSENGER^® complex was added to a 96-well plate, followed by equilibrated T-cells (187,500 cells/well).

The plate was lightly rocked to enable proper mixing and then left to settle at room temperature for 30 minutes prior to being placed in the Incucyte^® system for both proliferation and monitoring transfection.

After 3.5 hours, the plate was removed for a short period of time to add fresh media with IL-21 and TransAct^™ to the wells. Where necessary, a Sartorius Opti-Green background suppressor was added to the wells to remove green autofluorescence from the media.

Proliferation and transfection tracking continued in the Incucyte^® system until full well coverage was achieved. HD phase contrast and green fluorescence images were obtained and examined as earlier outlined.

High-throughput cytometry assessments

To aid further examination, wells from transfection tests were sampled on both day 1 and day 2 for analysis via the iQue^® HTS platform utilizing the integrated iQue Forecyt^® software.

Cells were washed and stained according to the manufacturer’s guidelines to examine viability, immunophenotyping, T-cell activation, and transfection efficiency. The reagents utilized for these tests are available in Table 2 and include the iQue^® Human T Cell Activation Kit and the iQue^® Cell Membrane Integrity Dye (B/Red) Kit.

Immune cell killing assay

To examine the tumor-killing ability of transfected T-cells, an immune cell-killing assay was carried out with the Incucyte^® SX5 system. Cytolight Red A549 target cells were seeded at 5K cells/well into a 96-well plate in 4Cell^® Nutri-T GMP medium topped up with FBS.

Cells were permitted to adhere for 3 hours in a 37 °C, 5 % CO₂ humidified incubator. Throughout this period, T-cells transfected for 24 hours were pooled and centrifuged (300 x g, 5 mins), and the resultant pellet was suspended again in fresh media with IL-21. Following target cell attachment, the media was replaced with transfected T-cells at either 5:1, 10:1, or 20:1 effector-to-target (E:T) ratios.

As a last step, fresh media supplemented with IL-21 and TransAct^™ was added to the wells prior to being placed in the Incucyte^® system for T-cell cytotoxic activity tests. Orange fluorescence and HD phase contrast images were gathered every hour (20x objective, 4 images/ well) and examined with the Incucyte^® Classic Confluence.

Analyzing orange fluorescence area/well and orange green fluorescence overlap area/well for quantitative assessments of target cell number and E:T interactions..

Results

Using live-cell analysis and HTS cytometry for T-cell characterization

Many cytokines are widely utilized for T-cell culture, with IL-2 and IL-21 being the most commonly used. IL-2 is known to enable both T-cell proliferation and activation. Meanwhile, IL-21 is linked to improving effector function.

To contrast T-cells cultured with IL-2 versus IL-21, cells were seeded into a 96-well plate and tracked for proliferation as shown in Figures 2A and 2B. Furthermore, wells were sampled at 24 hours for viability, phenotypic, and functional analysis utilizing the iQue^® HTS platform shown in Figure 2C.

The images and kinetic growth curves (Figures 2A and 2B) demonstrate how the proliferation dynamics of T-cells were similar for IL-2 and IL-21, with growth curves demonstrating small differences and overlapping exponential rates and plateaus. Data gathered utilizing the iQue^® HTS platform shown in Figure 2C supported this data, demonstrating similar percentages of viable cells for the two cytokines (86.3 ± 1.8% and 87.9 ± 0.7% for IL-2 and IL-21).

Immunophenotyping showed similar percentages of CD3 T-cells, alongside CD4 and CD8 subsets. For instance, the percentage of CD8 T-cells stood at 39.2 ± 0.5% for IL-2 and 38.1 ± 0.68% for IL-21. Among the CD8 subset, the percentage expressing the late activation marker CD25 was also similar (35.2 ± 1.0% and 39.0 ± 2.3% for IL-2 and IL-21).

Figure 2. Using Live-cell Analysis and HTS Cytometry for T-Cell Characterization in the presence of IL-2 or IL-21 Cells were monitored over 60 hours using live-cell analysis and HTS cytometry. A) HD phase-contrast images and Cell-by-Cell segmentation showing T-cells at 36 hours. B) Kinetic T-cell proliferation profile for both cytokines. C) Representative histograms and scatter plots showing T-cell characterization gating strategies at 24 hours. Data are presented as mean ± SEM with n = 1 donor (2-6 replicates, 4 images per well). Image Credit: Sartorius

References and further reading

1. Sun, L., et al. (2023). T cells in health and disease. Signal Transduction and Targeted Therapy, (online) 8(1). https://doi.org/10.1038/s41392-023-01471-y.
2. Waldman, A., Fritz, J. and Lenardo, M. (2020). A guide to cancer immunotherapy: from T cell basic science to clinical practice. Nature Reviews Immunology, 20(20), pp.1–18. https://doi.org/10.1038/s41577-020-0306-5.
3. U.S. Food & Drug Administrations. KYMRIAH (tisagenlecleucel). https://www.fda.gov/vaccinesblood-biologics/cellular-gene-therapy-products/ kymriah-tisagenlecleucel.
4. FDA (2025). US food and drug administration. (online) Fda.gov. Available at: https://www.fda.gov/.
5. Stewart, M., et al. (n.d.). Publisher: NPG; Journal: Nature: Nature; Article Type: Review. [online] https://doi.org/10.1038/nature19764.
6. Chong, Z.X., Yeap, S.K. and Ho, W.Y. (2021). Transfection types, methods and strategies: a technical review. PeerJ, (online) 9. https://doi.org/10.7717/peerj.11165.
7. Qin, S., et al. (2022). mRNA-based therapeutics: powerful and versatile tools to combat diseases. Signal Transduction and Targeted Therapy, (online) 7(1), pp.1–35. https://doi.org/10.1038/s41392-022-01007-w.

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