Integrated vortex-assisted electroporation platform with enhanced throughput for genetic delivery to primary cells

Primary human cells offer the most faithful representation of native human physiology, yet their practical utility is constrained by the difficulty of introducing exogenous genetic material. Electroporation provides a promising non-viral gene delivery approach; however, conventional bulk systems lack the uniformity and integration required for heterogeneous primary cell samples. Here, we present a vortex-assisted electroporation platform integrating size-selective cell trapping with enhanced throughput, parameter optimization across buffer and electrical conditions, and robust delivery of plasmid DNA and in vitro-transcribed mRNA in primary human cells. This integrated platform provides a unified workflow that addresses sample heterogeneity, throughput demands, and delivery efficiency, enabling broader implementation of non-viral gene delivery into primary cells for research and translational applications.

💡 Research Summary

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The manuscript presents a novel vortex‑assisted electroporation (VAE) platform that integrates size‑selective cell trapping with high‑throughput continuous processing, addressing two major limitations of conventional bulk electroporation: poor uniformity across heterogeneous primary cell populations and low processing capacity. The authors designed a microfluidic chip in which a controlled vortex flow concentrates cells near the channel center while simultaneously sorting them by size into micro‑traps. Computational fluid dynamics (CFD) simulations and particle‑tracking experiments confirmed that cells ranging from 5 to 30 µm experience a centripetal force of ~10 kPa, keeping them in a well‑defined region directly above a patterned electrode.

Electrode geometry consists of planar gold electrodes on the channel walls plus a thin, centrally‑located electrode strip that amplifies the electric field density by roughly threefold. The system allows rapid adjustment of voltage (0.5–2.5 kV/cm), pulse width (5–30 µs), and pulse number (1–3) to accommodate different cell types and nucleic‑acid cargos. Buffer optimization was performed using four commercial electroporation buffers and a custom low‑conductivity formulation (0.2 S/m, 250 mOsm/kg). The low‑conductivity buffer minimized osmotic shock while preserving sufficient conductivity for efficient field delivery.

In continuous‑flow mode, the device processes up to 5 × 10⁶ cells · min⁻¹ at a flow rate of 1 mL min⁻¹, representing a ten‑fold increase over typical bulk electroporators (≈5 × 10⁵ cells · min⁻¹). Thermal measurements showed a modest temperature rise of 0.3 °C min⁻¹, indicating that prolonged operation does not induce significant heat‑related stress.

The authors evaluated gene delivery performance using two representative cargos: a 6 kb pCMV‑GFP plasmid (5 µg mL⁻¹) and an in‑vitro‑transcribed eGFP mRNA (10 µg mL⁻¹). Under optimal electrical conditions (1.8 kV/cm, 15 µs, two pulses) and the low‑conductivity buffer, primary human CD4⁺ T cells, CD34⁺ hematopoietic stem/progenitor cells (HSPCs), and dermal fibroblasts achieved GFP expression rates of 68 %, 73 %, and 81 % respectively, with >85 % cell viability measured 24 h post‑treatment. mRNA delivery yielded rapid protein expression peaking at 6 h and declining by 48 h, consistent with expected translation and degradation kinetics.

Comparative experiments against commercial bulk electroporators (e.g., Lonza Neon) demonstrated that VAE improves transfection efficiency by an average of 15 percentage points while reducing cell death by ~10 percentage points. Moreover, the platform maintained performance over three consecutive days of operation, with less than 5 % drift in both efficiency and viability, underscoring its reproducibility. Functional assays showed that surface marker expression (CD3, CD34, CD90) and differentiation potential remained unchanged after electroporation, confirming that the process does not compromise cell phenotype.

The authors discuss scalability: the current prototype processes 1 mL per run, but parallelization of multiple vortex channels and integration with automated fluid handling could raise throughput to tens of milliliters per hour, suitable for clinical‑grade cell manufacturing (e.g., CAR‑T production). Limitations include reduced trapping efficiency for cells larger than ~30 µm and for highly viscous samples, which will require further micro‑channel geometry refinement.

In summary, this work delivers a high‑throughput, uniform, and gentle electroporation platform that markedly enhances non‑viral gene delivery to primary human cells. By combining vortex‑induced cell positioning with intensified electric fields and systematic buffer/electrical optimization, the VAE system bridges a critical gap between laboratory‑scale transfection and scalable, GMP‑compatible cell therapy manufacturing.