Advantages and limitations of various treatment chamber designs for reversible and irreversible electroporation in life sciences.

The fundamental mechanisms of pulsed electric fields on biological cells are not yet fully elucidated, though it is apparent that membrane electroporation plays a crucial role. Little is known about treatment-chamber-specific effects, and systematic studies are scarce. Thus, the present study evaluates the (dis-)advantages of various treatment chamber designs for liquid applications at differing scales. Three chambers, namely parallel plate microfluidic (V̇: 0.1 ml/min; titanium electrodes), co-linear meso (V̇: 5.0 ml/min; stainless steel electrodes), and co-linear macro (V̇: 83.3 ml/min; stainless steel electrodes) chambers, were studied. Electroporation effects on Escherichia coli in media with 0.1-10.0 mS/cm were evaluated by plate counts and flow cytometry at 8, 16, and 20 kV/cm. For the microfluidic chamber, predominantly irreversible electroporation (2.5 logs10 reductions) was seen at 0.1 mS/cm, while high irreversible electroporation (4.2 logs10 reductions) at 10.0 mS/cm was observed for the macro chamber. The meso chamber indicated a similar trend towards increased conductivity, even though only low inactivation levels were present. Variation in conductivity and electrode configuration or area likely induces effects resulting in distinct electroporation levels, as observed for the micro and macro chamber. Suitable application scenarios, depending on targeted electroporation effects, were suggested.

Capacitive coupling increases the accuracy of cell-specific tumour disruption by electric fields.

Irreversible electroporation holds great potential for cell-specific lysis due to the size-dependent susceptibility of cells to externally imposed electric fields. Previous attempts at selective cell lysis lead to significant overlap between affected populations and struggle with inconsistent biological outcome. We propose that charge transfer at the electrode-liquid interface is responsible by inducing multifactorial effects originating from both the electric field and electrochemical reactions. A promising remedy is the coating of electrodes with a high-k dielectric layer. The resulting capacitive coupling restores the selective potential of electric field mediated lysis in a microfluidic setup. Initial experiments show the consistent depletion of erythrocytes from whole blood while leaving leukocytes intact. The same is true for the reproducible and selective depletion of Jurkat and MCF-7 cells in a mixture with leukocytes. Unexpectedly, the observed order of lysis cannot be correlated with cell size. This implies that the cellular response to capacitive coupling features a selective characteristic that is different from conventional lysis configurations.

Controllable cell manipulation in a microfluidic pipette-tip design using capacitive coupling of electric fields.

Systems designed toward cell manipulation by electric fields are inherently challenged by energy dissipation along the electrode-electrolyte interface. A promising remedy is the introduction of high-k electrode passivation, enabling efficient capacitive coupling of electric fields into biological samples. We present the implementation of this strategy in a reusable pipette tip design featuring a 10 µl chamber volume for life science applications. Prototype validation and comparison to conductive gold-coated electrodes reveal a consistent and controllable biological effect that significantly increases the reproducibility of lysis events. The system provides precise descriptions of HEK-293 lysis dependency to variables such as field strength, frequency, and conductivity. Over 80% of cells were reversibly electroporated with minimal electrical lysis over a broad range of field settings. Successful transfection requires exponential decay pulses and showcases how modulating capacitive coupling can advance our understanding of fundamental mechanics in the field of electroporation.

High-k Dielectric Passivation: Novel Considerations Enabling Cell Specific Lysis Induced by Electric Fields.

A better understanding of the electrodynamic behavior of cells interacting with electric fields would allow for novel scientific insights and would lead to the next generation of cell manipulation, diagnostics, and treatment. Here, we introduce a promising electrode design by using metal oxide high-k dielectric passivation. The thermally generated dielectric passivation layer enables efficient electric field coupling to the fluid sample comprising cells while simultaneously decoupling the electrode ohmically from the electrolyte, allowing for better control and adjustability of electric field effects due to reduced electrochemical reactions at the electrode surface. This approach demonstrates cell-size specific lysis with electric fields in a microfluidic flow-through design resulting in 99.8% blood cell lysis at 6 s exposure without affecting the viability of Gram-positive and Gram-negative bacterial spike-ins. The advantages of this new approach can support next-generation investigations of electrodynamics in biological systems and their exploitation for cell manipulation in multiple fields of medicine, life science, and industry.

R5 clade C SHIV strains with tier 1 or 2 neutralization sensitivity: tools to dissect env evolution and to develop AIDS vaccines in primate models.

BACKGROUND: HIV-1 clade C (HIV-C) predominates worldwide, and anti-HIV-C vaccines are urgently needed. Neutralizing antibody (nAb) responses are considered important but have proved difficult to elicit. Although some current immunogens elicit antibodies that neutralize highly neutralization-sensitive (tier 1) HIV strains, most circulating HIVs exhibiting a less sensitive (tier 2) phenotype are not neutralized. Thus, both tier 1 and 2 viruses are needed for vaccine discovery in nonhuman primate models. METHODOLOGY/PRINCIPAL FINDINGS: We constructed a tier 1 simian-human immunodeficiency virus, SHIV-1157ipEL, by inserting an "early," recently transmitted HIV-C env into the SHIV-1157ipd3N4 backbone [1] encoding a "late" form of the same env, which had evolved in a SHIV-infected rhesus monkey (RM) with AIDS. SHIV-1157ipEL was rapidly passaged to yield SHIV-1157ipEL-p, which remained exclusively R5-tropic and had a tier 1 phenotype, in contrast to "late" SHIV-1157ipd3N4 (tier 2). After 5 weekly low-dose intrarectal exposures, SHIV-1157ipEL-p systemically infected 16 out of 17 RM with high peak viral RNA loads and depleted gut CD4+ T cells. SHIV-1157ipEL-p and SHIV-1157ipd3N4 env genes diverge mostly in V1/V2. Molecular modeling revealed a possible mechanism for the increased neutralization resistance of SHIV-1157ipd3N4 Env: V2 loops hindering access to the CD4 binding site, shown experimentally with nAb b12. Similar mutations have been linked to decreased neutralization sensitivity in HIV-C strains isolated from humans over time, indicating parallel HIV-C Env evolution in humans and RM. CONCLUSIONS/SIGNIFICANCE: SHIV-1157ipEL-p, the first tier 1 R5 clade C SHIV, and SHIV-1157ipd3N4, its tier 2 counterpart, represent biologically relevant tools for anti-HIV-C vaccine development in primates.