Efficient electroporation in primary cells with PEDOT:PSS electrodes

Abstract

Precise and efficient delivery of macromolecules into cells enhances basic biology research and therapeutic applications in cell therapies, drug delivery, and personalized medicine. While pulsed electric field electroporation effectively permeabilizes cell membranes to deliver payloads without the need for toxic chemical or viral transduction agents, conventional bulk electroporation devices face major challenges with cell viability and heterogeneity due to variations in fields generated across cells and electrochemistry at the electrode-electrolyte interface. Here, we introduce the use of microfabricated electrodes based on the conducting polymer poly(3,4-ethylenedioxythiophene) doped with polystyrene sulfonate (PEDOT:PSS), which substantially increases cell viability and transfection efficiency. As a proof of concept, we demonstrate the enhanced delivery of Cas9 protein, guide RNA, and plasmid DNA into cell lines and primary cells. This use of PEDOT:PSS enables rapid modification of difficult-to-transfect cell types to accelerate their study and use as therapeutic platforms.

Fig. 1.. Illustration of system for PEDOT:PSS enhanced electroporation.

(A) Simplified schematic of fabrication process wherein gold microelectrodes are photolithographically patterned onto a glass substrate. Multiple layers of PEDOT:PSS are deposited on the substrate and then etched to coat just the electrode digits. The electrodes are then enclosed in a microfluidic channel. (B) (i) Bare glass substrate, (ii) uncoated gold electrode, and (iii) PEDOT:PSS coating are visible on each 20-μm-wide and spaced electrode. (C) The entire device is placed in a fluorescence microscope with a CO₂ and temperature-controlled environment [Created in BioRender. Fraser, I. (2024) BioRender.com/h54i032], and then (D) electrical and fluidic contact is established.

Fig. 2.. Workflow and stimulation parameters.

(A) Timeline of the experiment wherein cells are transferred into electroporation buffer and injected into the device. pDEP is used to bring cells close to the surface, and nDEP is used to pattern cells between electrodes before the electroporation stimulation. Permeabilized cells are flushed out of the chip. Images of cells in the device at time points (I) and (II) are shown in Fig. 3A. (B) Schematic of tested signals wherein the pDEP and nDEP waveforms are unchanged and the amplitude and frequency of the electroporation pulses are varied. (C) Electrochemical impedance spectra. Gray area highlights the range between the gold and PEDOT:PSS cutoff frequencies. (D) Time domain (top) and frequency domain (bottom) of 100-Hz bipolar square wave pulse. (E) Time domain (top) and frequency domain (bottom) of 1000-Hz bipolar square wave pulse.

Fig. 3.. Time course of permeabilization and cell viability.

(A) Cells patterned between electrodes. Dead cells express only PI (i), and living cells with undisrupted cell membranes express only calcein-AM (ii). After stimulation, cells between electrodes express both PI and calcein (iii); however, unstimulated cells do not express PI (iv). (B) The proportion of cells expressing PI in the 5 min following stimulation. (C) The proportion of cells expressing calcein-AM in the 90 min following stimulation The results are shown as the average across three Au (yellow) and three PEDOT:PSS (blue) devices with error bars for SD. *P < 0.05 (two-tailed Student’s t test).

Fig. 4.. Optimization of operating parameters.

(A) Permeabilization is quantified as the mean proportion of cells expressing PI on Au electrodes, or (B) PEDOT:PSS electrodes 4 min following stimulation on three devices. (C) Cell viability is characterized by the proportion of cells expressing calcein-AM on Au and (D) PEDOT:PSS electrodes 90 min after stimulation across three devices. (E) Pairwise multiplication of permeabilization and viability leads to an efficiency metric for Au and (F) PEDOT:PSS electrodes. Data are presented as a heatmap depicting the average proportion of fluorescent cells imaged on three devices.

Fig. 5.. Validation in difficult-to-transfect cell types.

(A) Schematic of RNP electroporation experiment in U937 and THP-1 cell lines. (B) Knockout efficiency following RNP transfection tracking the MFI of electroporated cells over 96 hours. (C) Relative transfection efficiency of mEmerald plasmid in THP-1 cells against Thermofisher Neon Transfection System 72 hours after stimulation. (D) Images of primary BMDMs 96 hours following mEmerald plasmid transfection. (E) Population distribution of fluorescence intensity in BMDMs 96 hours following mEmerald plasmid transfection. ***P < 0.001 (two-tailed Student’s t test).