Exploring The Benefits Of Electroporation In NK Cell Transfection

A conversation with Tanishka Kumar

As cell therapy developers push toward safer, faster, and more scalable solutions for introducing genetic material into cells, electroporation paints a compelling alternative to the status quo, transduction.

By using controlled electrical pulses to create temporary pores in cell membranes, this technique offers a non-viral route to precise payload delivery without the complexities and risks of viral vectors.

The implications and benefits are worth exploring. Electroporation promises flexibility across diverse cell types, reduces regulatory hurdles tied to viral systems, and opens doors for therapies that demand rapid turnaround. Yet, the shift isn’t without challenges: efficiency, cell viability, and manufacturing integration remain critical questions for developers.

At the heart of her master’s degree thesis at the Centre for Cell Manufacturing at the University of Galway, Tanishka Kumar explored optimizing this technique for natural killer (NK) cell therapy transfection. She offered to share what she found with us and answer some questions.

How do NK cells differ from T cells in terms of genetic modification?

NK cells are inherently more resistant and challenging to genetically modify than T cells. Whereas T cells can be efficiently engineered using viral vectors such as lentiviruses with higher transduction rates and stable integration, NK cells exhibit lower susceptibility to viral transduction and are prone to stress-induced functional exhaustion during genetic manipulation.^1,2 T cells recognize target cells via T cell receptors (TCRs), which require gene editing to avoid graft-versus-host disease (GvHD) in allogeneic settings.

In contrast, NK cells use HLA recognition mechanisms, which inherently reduce GvHD risk, making them better fits for off-the-shelf therapies but harder to genetically engineer.^3,4,5 These fundamental biological differences make non-viral delivery methods such as electroporation attractive for NK cells, even if technically more challenging. If we are to focus on safety, I think working toward improving electroporation is a better bet, though it has its downsides.

How does electroporation compare to the current state-of-art, viral transduction?

Viral transduction typically achieves more stable, high-efficiency delivery, but is less favorable for NK cells due to their stress sensitivity and low transduction rates. Electroporation suits GMP-compliant closed systems and allows rapid protocol adjustments but generally results in lower transfection efficiencies (here, ~10% GFP+ NK cells achieved) and transient expression compared to viral methods.^6,7 Even though electroporation offers safer, scalable, and feasible manufacturing routes for NK cells if optimized well, it still has a long way to go.

Can you talk about some of the key technical parameters, e.g., electric field and electrode setup, buffer chemistry, and nucleic acid concentration? How did you optimize them for NK cell electroporation?

In the study, the foundational protocol of Ingegnere et al. (2019)⁶ was adopted as a benchmark along with parallel studies,^8,9,10 while also adapting clinical manufacturing requirements using the CliniMACS closed electroporation system. The main parameters that were involved in the optimization of electroporation included voltage, pulse duration, pulse number, type of waveform, cell type, electrode gap, temperature, nucleic acid and cell concentrations, and electroporation buffer.

• Electric field strength and electrode setup: This was calculated as voltage divided by cuvette electrode gap (4 mm). Effective reversible permeabilization occurs typically at 0.25–3.0 kV/cm as per published research. I tried to optimize a two-pulse approach as performed in various published NK cell electroporation papers: a high-voltage short pulse (~1750–2125 V/cm, 150–300 µs) to induce pore formation followed by a lower-voltage, longer pulse (375–875 V/cm, up to 50,000 µs) to assist DNA entry. A huge limitation was that the voltages above 1,000 V performed using different electroporation systems like Neon NxT couldn’t be applied using CliniMACS Electroporator due to hardware constraints. In addition, the published research on electroporation performed by different systems was classified. The 4 mm cuvettes present nonuniform fields toward edges and risk arcing, limiting efficiency. Compared to microfluidic systems with low-volume, micro-gap chambers (Neon NxT, MaxCyte), they produce less homogeneous fields and greater stress.^11,12 The electric field strength applied inside the cuvette/electric pipette was different than CliniMACS but couldn’t be quantified exactly due to information being a copyright or trade secret. So, electric field strength calculations were estimative and this necessitated careful trial and error to avoid irreversible cell damage. Further individual cell and electrical environment studies of CliniMACS electroporator are required to fully optimize electroporation in a controlled manner.
• Buffer chemistry: Due to funding constraints, we used BTXpress proprietary low-conductivity buffer formulated to reduce heating and minimize arcing. Here the isolated DNA was dissolved in sterile distilled water to avoid ionic interference. Literature and thesis findings highlight that buffers minimizing NaCl and including mannitol osmoprotectants provide better transfection and viability.^13,6 During electroporation, arcing was observed and a stringy white precipitate formation showed stress. Therefore, if I were to continue optimization, I would start with buffer as well.
• Nucleic acid concentration: Plasmid DNA was kept constant at 120 μg/mL, which was shown to yield efficient delivery without excessive toxicity in the optimization Neon NXT published research paper. Cell density was targeted around 4x10^7 cells/mL but due to time constraints each sample electroporated contained 1x10^7 cells/mL.

Parameter optimization was iterative, empirical, and constrained by equipment limits. Balance between transfection efficiency and cell viability was a critical endpoint throughout.

Of those parameters, what technical issues did you encounter during electroporation optimization?

• Arcing: Electrical discharge events causing cuvette cap blowouts, visible sparks, and white protein precipitates during pulsing were observed. Arcing leads to protein denaturation and significant cell death. The CliniMACS software had to be forced to restart at the first trial of high voltages as seen in Ingegnere et. al. (2019)⁶ due to an error in the software. This made us led my supervisors, Maryam Sakhteh and Matthew Duggan, to rethink and reanalyze the different voltages and pulse duration to set and perform the trial and error. This effected the continuity of the electroporation process and reduced the number of trials and induced variations in the controlled electroporation environment.
• Viability vs. efficiency: Higher voltage or longer pulse durations increased DNA uptake but resulted in steep viability losses, showing an inverse relationship. For example, Protocol P1 in the first electroporation had the highest GFP (~3%) but lowest viability (~42%).
• Buffer conductivity: The BTXpress buffer’s conductivity seemed suboptimal, evidenced by arcing and cell death. Literature recommends low ionic strength buffers with mannitol; exclusion of chelating agents and use of pure water to re-suspend DNA improves outcomes.
• Equipment voltage limitations: The CliniMACS system was capped at 1000 V, restricting electric field strengths achievable compared to other platforms like Neon NxT.

You documented a workflow with an eye toward GMP compliance. Discuss the challenges in meeting rigorous quality control and data protocols.

While performing analytical assays to assess purity, sterility, efficiency, viability, and other in-process parameters, a challenge I faced was determining the appropriate quantitative and qualitative acceptance criteria to allow progression to the next stage. Establishing these thresholds required triangulating historical SOP data, industry literature, and reference protocols used by other companies. I faced another challenge regarding change control for R&D SOPs after obtaining negative results during early electroporation experiments. This required that I determine whether the deviation was due to experimental error or a gap in the procedure itself. I had to assess the data, consult with the team, and justify any proposed modifications while following proper documentation, version control, and approval workflows to maintain compliance and traceability.

What complications do you anticipate when scaling this for commercial production?

1. Donor variability: Biological variability between NK cell donors affects expansion capacity, phenotype, cytotoxicity, and transfection efficiency, complicating product standardization.³ This will also affect reproducibility of the product.
2. Cost-effectiveness: Securing consistent GMP-grade supply and qualification of buffers and consumables at scale should not be an issue once optimization is achieved. Balancing high cell dose requirements with manufacturing time, resource use, and costs, especially for allogeneic, off-the-shelf products is the main challenge that I am interested to learn via experience.
3. Scale-up of electric parameters: Maintaining uniform and reproducible electric field exposure to large cell volumes at scale while avoiding arcing or overheating is complex. Bulk automation to produce a commercial CGT product is something that I would like to research and develop in near future.
4. Process automation and closed-system integration: Achieving seamless integration of electroporation, expansion, sampling, and QC testing in fully automated, closed systems with minimal manual intervention is possible via optimization of systems like the CliniMACS prodigy system. I intend to be part of this R&D and have started a bit of skill development in bioinformatics to hopefully join an automation research team in near future.
5. Regulatory compliance: Finally, meeting stringent batch release criteria, environmental monitoring, and process validation at commercial scales is indeed complex. For a CGT product to be safe , successful, profitable, and effective against life-threatening diseases, I believe its manufacturing should be in a fully closed and automated process with the highest control on all parameters.

What does the whole analytical profile look like?

• Pre- and post-electroporation viability assessment via trypan blue exclusion
• Flow cytometry phenotyping gating on viable, single, CD3⁻CD56⁺ NK cells using viability dye DRAQ7
• Quantification of GFP-positive NK cells as a measure of gene delivery and transfection efficiency
• Purity assessment of NK cell population (&GT92%) to confirm minimal T cell contamination
• Quality control of plasmid DNA purity and concentration via Nanodrop spectrophotometry
• Use of fluorescence-minus-one controls and compensation beads for robust flow cytometry data accuracy
• Process documentation recording all batch and reagent information, SOP compliance, and deviation reports
• Data analysis with CytExpert and Excel for visualization and interpretation

These were all the analytical analyses performed during the thesis. It corresponds to a GMP-compatible quality control pipeline ensuring reproducible and analyzable cell product characterization. This list is not exhaustive, as several more assays are needed, including gel electrophoresis and sequencing to analyze plasmid, microbiological assays (mycoplasma tests), cytotoxicity, cytokine secretion, and migratory capacity to ensure that engineered NK cells retain their in vivo functional potential. For future work, integrating digitized, single-cell imaging (such as that reported by Golzio et al., 2002) could help directly observe the DNA’s electrophoretic movement, membrane poration, and binding/entry during pulsing in NK cells; which would provide a mechanistic link between pulse parameter, pore kinetics, and genetic delivery.¹⁴

Are there any long-term stability differences between transduced and electroporated cells?

The thesis did not experimentally address long-term stability differences due to time constraints, but literature shows that viral transduction of DNA leads to stable, integrated transgene expression with longer persistence in vivo as compared to RNA and electroporation.

Electroporation (especially plasmid DNA or mRNA-based) typically yields transient expression lasting days to a week, without genomic integration.¹⁵,¹⁶ Clinically, mRNA electroporation yields potent but temporary protein expression, suited for short-term modulation; stable gene delivery (e.g., CAR constructs) may require optimized DNA or viral methods. Functional NK properties post-electroporation are generally retained short-term, but sustained effects require further study and optimization of several parameters, including electroporation buffer. This influences clinical application design depending on transient vs. durable gene expression needs.

Starting material and donor quality weren’t part of your research, but based on donor-to-donor variations, how does that impact the manufacturing process?

Donor variability is a significant factor influencing NK cell manufacturing based on published research. Genotypic differences (KIR and HLA haplotypes) affect NK phenotype, expansion potential, cytokine responsiveness, and cytotoxicity.³ Variations impact cell culture growth, electroporation efficiency, and functional outcomes, which will lead to batch variability. Controlling donor variability requires donor screening, a pooling strategy, or process adaptations to ensure reproducible product quality important for regulatory approval.

References:

1. REZVANI, K., et al. 2017. Engineering Natural Killer Cells for Cancer Immunotherapy. Molecular Therapy, 25, 1769-1781.
2. STRELTSOVA, M. A., et al. I. 2017. Retroviral gene transfer into primary human NK cells activated by IL-2 and K562 feeder cells expressing membrane-bound IL-21. J Immunol Methods, 450, 90-94.
3. JONCKER, N. T., et al. 2009. NK cell responsiveness is tuned commensurate with the number of inhibitory receptors for self-MHC class I: the rheostat model. J Immunol, 182, 4572-80.
4. OH, I.-H., et al. 2004. IL-10 Is a Novel Ligand for Hematopoietic Stem Cell Self-Renewal. Blood, 104, 2164-2164.
5. LEE, D. A., et al. 2016. Haploidentical Natural Killer Cells Infused before Allogeneic Stem Cell Transplantation for Myeloid Malignancies: A Phase I Trial. Biology of Blood and Marrow Transplantation, 22, 1290-1298.
6. INGEGNERE, T., et al. 2019. Human CAR NK Cells: A New Non-viral Method Allowing High Efficient Transfection and Strong Tumor Cell Killing. Frontiers in Immunology, Volume 10 - 2019.
7. OBERSCHMIDT, et al. 2019. Development of Automated Separation, Expansion, and Quality Control Protocols for Clinical-Scale Manufacturing of Primary Human NK Cells and Alpharetroviral Chimeric Antigen Receptor Engineering. Hum Gene Ther Methods, 30, 102-120.
8. ALZUBI, J., et al. 2021. Automated generation of gene-edited CAR T cells at clinical scale. Mol Ther Methods Clin Dev, 20, 379-388.
9. SCHWARZE, L. I., et al. 2021. Automated production of CCR5-negative CD4(+)-T cells in a GMP-compatible, clinical scale for treatment of HIV-positive patients. Gene Ther, 28, 572-587.
10. DOUKA, S., et al. 2023. Lipid nanoparticle-mediated messenger RNA delivery for ex vivo engineering of natural killer cells. Journal of Controlled Release, 361, 455-469.
11. SAHU, P., et al. 2025. Round Well Inset for Uniform Electric Field Distribution in Electroporation Applications. Bioengineering (Basel), 12.
12. KENNEDY, S. M., et al. 2008. Quantification of electroporative uptake kinetics and electric field heterogeneity effects in cells. Biophys J, 94, 5018-27.
13. NG, M. M. 2019. Exclusive Compendium: Optimizing an Electroporation Protocol. Holliston, Massachusetts, USA: BTX, a division of Harvard Bioscience, Inc.
14. CALIN, V. L., et al. 2021. Digital holographic microscopy evaluation of dynamic cell response to electroporation. Biomed Opt Express, 12, 2519-2530.
15. CARLSTEN, M. & CHILDS, R. W. 2015. Genetic Manipulation of NK Cells for Cancer Immunotherapy: Techniques and Clinical Implications. Frontiers in Immunology, Volume 6 - 2015.
16.  VAN TENDELOO, et al. 2001. Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells. Blood, 98, 49-56.

About The Expert:

Tanishka Kumar is a bioprocess technician and quality analyst who recently received her M.Sc. from the University of Galway. Her graduate thesis explored optimizing NK cell electroporation. Connect with her on LinkedIn.