citations

                    A solution for highly efficient electroporation of primary cytotoxic T lymphocytes
                

Authors:

Alawar N, Schirra C, Hohmann M, Becherer U

In:

Source: BMC Biotechnol
Publication Date: (2024)
Issue: 24(1): 16

Research Area:

Cancer Research/Cell Biology

Immunotherapy / Hematology

Basic Research

Regenerative medicine

Drug Discovery

Cells used in publication:

Natural killer Cells (NK), human: Species: human; Tissue Origin: blood
CD4+, human: Species: human; Tissue Origin: blood

Platform:

Nucleofector® I/II/2b

4D-Nucleofector® X-Unit

Experiment

Electroporation: Electroporation was performed on day 5 mouse CTLs using the Nucleofector™ 2b Device (Lonza). About 6 × 10^6 cells were resuspended with 2 µg plasmid DNA in 100 µl electroporation buffer (Mouse T Cell Nucleofector Kit, VPA-1006; Lonza). The mixture was then transferred to electroporation cuvettes. Electroporation was immediately performed using the special pulse (X-001) for mouse CTLs. The transfected cells were then directly transferred to 3 ml prewarmed recovery media and kept in 12 well plates at 32 °C with 5% CO2. After 12 hours of transfection, cells were washed and cultured in AIM-V with 10% FCS at 37 °C and used for experiments 14–18 hours post-electroporation.

Electroporation was performed on day 5 human CTLs using the P3 Primary Cell 4D-Nucleofector X Kit (V4XP- 3024; Lonza). About 6 × 10^6 cells were resuspended with 1.5 µg plasmid DNA in 100 µl electroporation buffer. The mixture was then transferred to electroporation cuvettes and electroporation was immediately performed using the EO-115 program. Cells were then directly transferred to 3 ml prewarmed recovery media.

NK cells were electroporated 2 days after isolation. 5 × 10^6 NK cells were nucleofected with pmax-GFP Vector (2 µg) using the P3 Primary Cell 4D-Nucleofector
Kit (Lonza). Electroporation was performed using the 4D-Nucleofector (Lonza) with the EH-115 program as it appeared to be the most promising based on [21]. The
cells were then placed in AIM-V with 10% FCS or our RM for 12 hours at 32 °C. Cells were then used for flow cytometry analysis.

CD4+ cells were electroporated 3 days after isolation. Stimulation beads were removed, and 5 × 10^6 CD4+ T cells were nucleofected with pmax-GFP Vector (2 µg) using the P3 Primary Cell 4D-Nucleofector Kit (Lonza). Electroporation was performed using the 4D-Nucleofector (Lonza) with the EO-115 program. Cells were then placed in AIM-V with 10% FCS or our RM for 12 hours at 32 °C. Cells were then used for flow cytometry analysis.

Abstract

Background: Cytotoxic T lymphocytes (CTLs) are central players in the adaptive immune response. Their functional characterization and clinical research depend on efficient and reliable transfection. Although various methods have been utilized, electroporation remains the preferred technique for transient gene over-expression. However, the efficiency of electroporation is reduced for human and mouse primary CTLs. Lonza offers kits that effectively improve plasmid DNA transfection quality. Unfortunately, the removal of key components of the cell recovery medium considerably reduced the efficiency of their kit for CTLs. Our aim was to develop a new recovery medium to be used with Lonza's Nucleofector system that would significantly enhance transfection rates.

Results: We assessed the impact of different media in which the primary CTLs were placed to recover after electroporation on cell survival, transfection rate and their ability to form an immunological synapse and to perform exocytosis. We transfected the cells with pmax-GFP and large constructs encoding for either CD81-super ecliptic pHluorin or granzyme B-pHuji. The comparison of five different media for mouse and two for human CTLs demonstrated that our new recovery medium composed of Opti-MEM-GlutaMAX supplemented with HEPES, DMSO and sodium pyruvate gave the best result in cell survival (> 50%) and transfection rate (> 30 and 20% for mouse and human cells, respectively). More importantly, the functionality of CTLs was at least twice as high as with the original Lonza recovery medium. In addition, our RM significantly improved transfection efficacy of natural killer cells that are notoriously hard to electroporate.

Conclusion: Our results show that successful transfection depends not only on the electroporation medium and pulse sequence but also on the medium applied for cell recovery. In addition, we have reduced our reliance on proprietary products by designing an effective recovery medium for both mouse and human primary CTLs and other lymphocytes that can be easily implemented by any laboratory. We expect that this recovery medium will have a significant impact on both fundamental and applied research in immunology.

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