Compact High-Voltage AC Generator with Pulse Transformer for High-Frequency Irreversible Electroporation (H-FIRE)

This paper is focused on a design of a high-voltage (HV) generator, which is proposed for a high-frequency irreversible electroporation (H-FIRE). The generator produces bursts of bipolar symmetrical pulses. Most HV sources used for cell electroporation are based on a controlled discharge of a capacitor into a resistive load. This solution is very simple, but it is associated with a certain risk of an uncontrolled discharge of the capacitor. We present a different type of the generator, where a DC-AC inverter with pulse transformer is used and where the mentioned risk is eliminated. Our generator is able to deliver bursts with variable length from 50 to 150 μs and a gap between bursts can be set from 0.5 to 1.5 s. Pulse frequency can be varied from 65 to 470 kHz and the output voltage is controlled in two ranges from 0 to 1.3 kV or from 0 to 2.5 kV. Results are presented with resistive load and with tissue impedance load.

Deep Learning and Computer Vision Strategies for Automated Gene Editing with a Single-Cell Electroporation Platform

Single-cell delivery platforms like microinjection and nanoprobe electroporation enable unparalleled control over cell manipulation tasks but are generally limited in throughput. Here, we present an automated single-cell electroporation system capable of automatically detecting cells with artificial intelligence (AI) software and delivering exogenous cargoes of different sizes with uniform dosage. We implemented a fully convolutional network (FCN) architecture to precisely locate the nuclei and cytosol of six cell types with various shapes and sizes, using phase contrast microscopy. Nuclear staining or reporter fluorescence was used along with phase contrast images of cells within the same field of view to facilitate the manual annotation process. Furthermore, we leveraged the near-human inference capabilities of the FCN network in detecting stained nuclei to automatically generate ground-truth labels of thousands of cells within seconds, and observed no statistically significant difference in performance compared to training with manual annotations. The average detection sensitivity and precision of the FCN network were 95±1.7% and 90±1.8%, respectively, outperforming a traditional image-processing algorithm (72±7.2% and 72±5.5%) used for comparison. To test the platform, we delivered fluorescent-labeled proteins into adhered cells and measured a delivery efficiency of 90%. As a demonstration, we used the automated single-cell electroporation platform to deliver Cas9–guide RNA (gRNA) complexes into an induced pluripotent stem cell (iPSC) line to knock out a green fluorescent protein–encoding gene in a population of ~200 cells. The results demonstrate that automated single-cell delivery is a useful cell manipulation tool for applications that demand throughput, control, and precision.

Simulation of single cell electroporation

This paper attempts to introduce the dynamics of electroporation into the single cell model. The main characteristics of the model were described. Results show the generation and rise of pores in a round cell with a radius of 50 μm exposed to 40 kV/m electric field for 1 ms were analysed, so that the transmembrane potential, the number of pores and the membrane conductance could be calculated. Finally, how the model can help explain the experiment is discussed.

535 Electroporation of B cells is correlated with cell size change during B cell expansion

BackgroundPrimary B cells are an important target for investigation and transfection of B cells is considered difficult. Electroporation is a very useful technology for transfection but its application on B cells has been unsatisfactory with low efficiency and low viability. The first reason is the small size of B cells compared to cell lines and the second reason is the low abundance of B cells in human PBMC. Since we had previous exprience with T cell electroporation, we sought to extend our knowledge on electroporation to B cells.MethodsHere we studied the B cell electroporation in PBMC samples and found that it is preferrable to electroporate the B cells in the PBMC mixture and B cells can be purified after electroporation if necessary. In this fashion, the total cell number in electroporation is boosted by other cell types in the PBMC and it helps B cell electroporation. Furthermore, we studied expanded B cells and found that they have a larger size than unstimulated B cells and the size increase is correlated to a decrease in electroporation voltage, consistent with the electroporation principle that larger cells need a lower voltage.ResultsWhen B cells are expanded, the electroporation efficiency is similar to common cell lines and it becomes easy to do gene expression or genomic modification.ConclusionsOur studies elucidated the mechanism of difference between unstimulated B cells and expanded B cells and could be useful in helping the research on B cells.