Enhanced Cell Viability and Migration of Primary Bovine Annular Fibrosus Fibroblast-like Cells Induced by Microsecond Pulsed Electric Field Exposure.

This study is the first to report the enhancement of cell migration and proliferation induced by in vitro microsecond pulsed electric field (µsPEF) exposure of primary bovine annulus fibrosus (AF) fibroblast-like cells. AF primary cells isolated from fresh bovine intervertebral disks (IVDs) are exposed to 10 and 100 µsPEFs with different numbers of pulses and applied electric field strengths. The results indicate that 10 µs-duration pulses induce reversible electroporation, while 100 µs pulses induce irreversible electroporation of the cells. Additionally, µsPEF exposure increased AF cell proliferation up to 150% while increasing the average migration speed by 0.08 µm/min over 24 h. The findings suggest that the effects of PEF exposure on cells are multifactorial-depending on the duration, intensity, and number of pulses used in the stimulation. This highlights the importance of optimizing the µsPEF parameters for specific cell types and applications. For instance, if the goal is to induce cell death for cancer treatment, then high numbers of pulses can be used to maximize the lethal effects. On the other hand, if the goal is to enhance cell proliferation, a combination of the number of pulses and the applied electric field strength can be tuned to achieve the desired outcome. The information gleaned from this study can be applied in the future to in vitro cell culture expansion and tissue regeneration.

Threshold Microsecond Pulsed Electric Field Exposures for Change in Spinach Quality.

Pulsed electric fields (PEFs) are often used to pretreat foods to enhance subsequent processes, such as drying, where maintaining food product quality is important for consumer satisfaction. This study aims to establish a threshold PEF exposure to determine the doses at which electroporation is viable for use on spinach leaves, wherein integrity is maintained postexposure. Three numbers of consecutive pulses (1, 5, 50) and two pulse durations (10 and 100 µs) have been examined herein at a constant pulse repetition of 10 Hz and 1.4 kV/cm field strength. The data indicate that pore formation in itself is not a cause for loss of spinach leaf food quality, i.e., significant changes in color and water content. Rather, cell death, or the rupture of the cell membrane from a high-intensity treatment, is necessary to significantly alter the exterior integrity of the plant tissue. PEF exposures thus can be used on leafy greens up until the point of inactivation before consumers would see any alterations, making reversible electroporation a viable treatment for consumer-intended products. These results open up future opportunities to use emerging technologies based on PEF exposures and provide useful information in setting parameters to avoid food quality diminishment.

Electrical impedance decreases in annulus fibrosus cartilage exposed to microsecond pulsed electric fieldsex vivo.

Electropermeabilization of biomembranes often is measured by microscopic imaging of a membrane-impermeable fluorophore that penetrates the cells following pulsed electric field (PEF) exposure. PEF exposure subsequently changes physiological properties of tissue. One way to probe these changes in tissue is measuring electrical properties by way of electrochemical impedance spectroscopy (EIS). In this study, we analyse impedance and conductivity of bovine annulus fibrosus (AF) cartilage before and after exposures to PEF of 100µs duration. Two PEF parameters-electric field amplitude and number of pulses-are varied, and total specific dose of PEF is calculated. AF tissue conductivity increases with both amplitude and number of pulses, indicating electropermeabilization of the AF cells. A Live/Dead cell imaging assay validates the EIS measurements, indicating intratissue cell permeabilization byµsPEF exposure. These results support the extension of EIS to monitor extent of electropermeabilization of cells within cartilage tissue.

Histone deacetylase 4 and 5 translocation elicited by microsecond pulsed electric field exposure is mediated by kinase activity.

Electroporation-based technologies using microsecond pulsed electric field (µsPEF) exposures are established as laboratory and clinical tools that permeabilize cell membranes. We demonstrate a µsPEF bioeffect on nucleocytoplasmic import and export of enzymes that regulate genetic expression, histone deacetylases (HDAC) -4 and -5. Their µsPEF-induced nucleocytoplasmic transport depends on presence and absence of extracellular calcium ions (Ca2+) for both MCF7 and CHO-K1 cells. Exposure to 1, 10, 30 and 50 consecutive square wave pulses at 1 Hz and of 100 µs duration with 1.45 kV/cm magnitude leads to translocation of endogenous HDAC4 and HDAC5. We posit that by eliciting a rise in intracellular Ca2+ concentration, a signaling pathway involving kinases, such as Ca2+/CaM-dependent protein kinase II (CaMKII), is activated. This cascade causes nuclear export and import of HDAC4 and HDAC5. The potential of µsPEF exposures to control nucleocytoplasmic transport unlocks future opportunities in epigenetic modification.

Cell encapsulation in gelatin bioink impairs 3D bioprinting resolution.

Recent advances in three-dimensional (3D) bioprinting technologies have enabled precise patterning of cellular components along with biomimetic constructs for tissue engineering and regenerative medicine. The viscoelasticity of bioinks regulate printability and the smallest feature size in 3D bioprinted constructs. The impact of cellular components is typically neglected when choosing 3D bioprinting parameters. In this short communication, we quantified the effect of cell densities on the printability of hydrogel bioinks. Unexpectedly, our results show that encapsulated cells reduced the steady shear viscosity of gelatin-based bioinks by approximately 50% and the minimum force for onset of flow by approximately 30%. These results may justify the lower spatial resolution in 3D bioprinted cell-laden hydrogels.

Tracking Lysosome Migration within Chinese Hamster Ovary (CHO) Cells Following Exposure to Nanosecond Pulsed Electric Fields.

Above a threshold electric field strength, 600 ns-duration pulsed electric field (nsPEF) exposure substantially porates and permeabilizes cellular plasma membranes in aqueous solution to many small ions. Repetitive exposures increase permeabilization to calcium ions (Ca2+) in a dosage-dependent manner. Such exposure conditions can create relatively long-lived pores that reseal after passive lateral diffusion of lipids should have closed the pores. One explanation for eventual pore resealing is active membrane repair, and an ubiquitous repair mechanism in mammalian cells is lysosome exocytosis. A previous study shows that intracellular lysosome movement halts upon a 16.2 kV/cm, 600-ns PEF exposure of a single train of 20 pulses at 5 Hz. In that study, lysosome stagnation qualitatively correlates with the presence of Ca2+ in the extracellular solution and with microtubule collapse. The present study tests the hypothesis that limitation of nsPEF-induced Ca2+ influx and colloid osmotic cell swelling permits unabated lysosome translocation in exposed cells. The results indicate that the efforts used herein to preclude Ca2+ influx and colloid osmotic swelling following nsPEF exposure did not prevent attenuation of lysosome translocation. Intracellular lysosome movement is inhibited by nsPEF exposure(s) in the presence of PEG 300-containing solution or by 20 pulses of nsPEF in the presence of extracellular calcium. The only cases with no significant decreases in lysosome movement are the sham and exposure to a single nsPEF in Ca2+-free solution.

nsPEF-induced PIP2 depletion, PLC activity and actin cytoskeletal cortex remodeling are responsible for post-exposure cellular swelling and blebbing.

Cell swelling and blebbing has been commonly observed following nanosecond pulsed electric field (nsPEF) exposure. The hypothesized origin of these effects is nanoporation of the plasma membrane (PM) followed by transmembrane diffusion of extracellular fluid and disassembly of cortical actin structures. This investigation will provide evidence that shows passive movement of fluid into the cell through nanopores and increase of intracellular osmotic pressure are not solely responsible for this observed phenomena. We demonstrate that phosphatidylinositol-4,5-bisphosphate (PIP2) depletion and hydrolysis are critical steps in the chain reaction leading to cellular blebbing and swelling. PIP2 is heavily involved in osmoregulation by modulation of ion channels and also serves as an intracellular membrane anchor to cortical actin and phospholipase C (PLC). Given the rather critical role that PIP2 depletion appears to play in the response of cells to nsPEF exposure, it remains unclear how its downstream effects and, specifically, ion channel regulation may contribute to cellular swelling, blebbing, and unknown mechanisms of the lasting "permeabilization" of the PM.

Permeabilization of the nuclear envelope following nanosecond pulsed electric field exposure.

Permeabilization of cell membranes occurs upon exposure to a threshold absorbed dose (AD) of nanosecond pulsed electric fields (nsPEF). The ultimate, physiological bioeffect of this exposure depends on the type of cultured cell and environment, indicating that cell-specific pathways and structures are stimulated. Here we investigate 10 and 600 ns duration PEF effects on Chinese hamster ovary (CHO) cell nuclei, where our hypothesis is that pulse disruption of the nuclear envelope membrane leads to observed cell death and decreased viability 24 h post-exposure. To observe short-term responses to nsPEF exposure, CHO cells have been stably transfected with two fluorescently-labeled proteins known to be sequestered for cellular chromosomal function within the nucleus - histone-2b (H2B) and proliferating cell nuclear antigen (PCNA). H2B remains associated with chromatin after nsPEF exposure, whereas PCNA leaks out of nuclei permeabilized by a threshold AD of 10 and 600 ns PEF. A downturn in 24 h viability, measured by MTT assay, is observed at the number of pulses required to induce permeabilization of the nucleus.

Nanosecond pulsed electric fields modulate the expression of Fas/CD95 death receptor pathway regulators in U937 and Jurkat Cells.

In this publication, we demonstrate that exposure of Jurkat and U937 cells to nanosecond pulsed electrical fields (nsPEF) can modulate the extrinsic-mediated apoptotic pathway via the Fas/CD95 death receptor. An inherent difference in survival between these two cell lines in response to 10 ns exposures has been previously reported (Jurkat being more sensitive to nsPEF than U937), but the reason for this sensitivity difference remains unknown. We found that exposure of each cell line to 100, 10 ns pulses at 50 kV/cm caused a marked increase in expression of cFLIP (extrinsic apoptosis inhibitor) in U937 and FasL (extrinsic apoptosis activator) in Jurkat, respectively. Measurement of basal expression levels revealed an inherent difference between U937 cells, having a higher expression of cFLIP, and Jurkat cells, having a higher expression of FasL. From these data, we hypothesize that the sensitivity difference between the cells to nsPEF exposure may be directly related to expression of extrinsic apoptotic regulators. To validate this hypothesis, we used siRNA to knockdown cFLAR (coding for cFLIP protein) expression in U937, and FasL expression in Jurkat and challenged them to 100, 10 ns pulses at 150 kV/cm, a typical lethal dose. We observed that U937 survival was reduced nearly 60% in the knockdown population while Jurkat survival improved ~40%. These findings support the hypothesis that cell survival following 10 ns pulse exposures depends on extrinsic apoptotic regulators. Interestingly, pretreatment of U937 with a 100-pulse, 50 kV/cm exposure (to amplify cFLAR expression) significantly reduced the lethality of a 150 kV/cm, 100-pulse exposure applied 24 h later. From these data, we conclude that the observed survival differences between cells, exposed to 10 ns pulsed electric fields, is due to inherent cell biochemistry rather than the biophysics of the exposure itself. Understanding cell sensitivity to nsPEF may provide researchers/clinicians with a predicable way to control or avoid unintended cell death during nsPEF exposure.

Disruption of the actin cortex contributes to susceptibility of mammalian cells to nanosecond pulsed electric fields.

Nanosecond pulsed electric fields (nsPEFs) perturb membranes of cultured mammalian cells in a dose-dependent manner with different types of cells exhibiting characteristic survivability. Adherent cells appear more robust than non-adherent cells during whole-cell exposure. We hypothesize that cellular elasticity based upon the actin cytoskeleton is a contributing parameter, and the alteration of a cell's actin cortex will significantly affect viability upon nsPEF exposure. Chinese hamster ovary (CHO) cells that are (a) untreated, (b) treated with latrunculin A to inhibit actin polymerization, or (c) exposed to nsPEFs have been probed using atomic force microscopy (AFM) force-indentations. Exposure to 50 or 100 pulses of 10 ns duration and 150 kV/cm in a single dosage approximately lowers average CHO cell elastic modulus by half, whereas latrunculin lowers it more than 75%. Latrunculin pre-treatment disrupts the actin cortex enough that it negates cumulative damage by equally fractionated (i.e., two rounds of 50 pulses each, separated by 10 min) dosages of nsPEFs as seen in untreated and dimethyl sulfoxide (DMSO)-treated cells with propidium uptake, phosphatidylserine externalization, and 24 h viability according to MTT and CellTiter Glo assays. These results suggest a correlation among cell stiffness, cytoskeletal integrity, and susceptibility to recurrent exposures to nsPEFs, which emphasizes a mechanobiological underpinning of nsPEF bioeffects.

600 ns pulse electric field-induced phosphatidylinositol4,5-bisphosphate depletion.

The interaction between nsPEF-induced Ca(2+) release and nsPEF-induced phosphatidylinositol4,5-bisphosphate (PIP2) hydrolysis is not well understood. To better understand this interrelation we monitored intracellular calcium changes, in cells loaded with Calcium Green-1 AM, and generation of PIP2 hydrolysis byproducts (inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG)) in cells transfected with one of two fluorescent reporter genes: PLCδ-PH-EGFP or GFP-C1-PKCγ-C1a. The percentage fluorescence differences (ΔF %) after exposures were determined. Upon nsPEF impact, we found that in the absence of extracellular Ca(2+) the population of IP3 liberated during nsPEF exposure (ΔF 6%±3, n=22), is diminished compared to the response in the presence of calcium (ΔF 84%±15, n=20). The production of DAG in the absence of extracellular Ca(2+) (ΔF 29%±5, n=25), as well as in cells exposed to thapsigargin (ΔF 40%±12, n=15), was not statistically different from cells exposed in the presence of extracellular calcium (ΔF 22±6%, n=18). This finding suggests that the change in intracellular calcium concentration is not solely driving the observed response. Interestingly, the DAG produced in the absence of Ca(2+) is the strongest near the membrane regions facing the electrodes, whereas the presence of extracellular Ca(2+) leads to a whole cell response. The reported observations of Ca(2+) dynamics combined with IP3 and DAG production suggest that nsPEF may cause a direct effect on the phospholipids within the plasma membrane.

Activation of intracellular phosphoinositide signaling after a single 600 nanosecond electric pulse.

Exposure to nanosecond pulsed electrical fields (nsPEFs) results in a myriad of observable effects in mammalian cells. While these effects are often attributed to the direct permeabilization of both the plasma and organelle membranes, the underlying mechanism(s) are not well understood. We hypothesize that nsPEF-induced membrane disturbance will initiate complex intracellular lipid signaling pathways, which ultimately lead to the observed multifarious effects. In this article, we show activation of one of these pathways--phosphoinositide signaling cascade. Here we demonstrate that nsPEF initiates phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis or depletion from the plasma membrane, accumulation of inositol-1,4,5-trisphosphate (IP3) in the cytoplasm and increase of diacylglycerol (DAG) on the inner surface of the plasma membrane. All of these events are initiated by a single 16.2 kV/cm, 600 ns pulse exposure. To further this claim, we show that the nsPEF-induced activation mirrors the response of M1-acetylcholine Gq/11-coupled metabotropic receptor (hM1). This demonstration of PIP2 hydrolysis by nsPEF exposure is an important step toward understanding the mechanisms underlying this unique stimulus for activation of lipid signaling pathways and is critical for determining the potential for nsPEFs to modulate mammalian cell functions.

Nanosecond pulsed electric field thresholds for nanopore formation in neural cells.

The persistent influx of ions through nanopores created upon cellular exposure to nanosecond pulse electric fields (nsPEF) could be used to modulate neuronal function. One ion, calcium (Ca(2+)), is important to action potential firing and regulates many ion channels. However, uncontrolled hyper-excitability of neurons leads to Ca(2+) overload and neurodegeneration. Thus, to prevent unintended consequences of nsPEF-induced neural stimulation, knowledge of optimum exposure parameters is required. We determined the relationship between nsPEF exposure parameters (pulse width and amplitude) and nanopore formation in two cell types: rodent neuroblastoma (NG108) and mouse primary hippocampal neurons (PHN). We identified thresholds for nanoporation using Annexin V and FM1-43, to detect changes in membrane asymmetry, and through Ca(2+) influx using Calcium Green. The ED50 for a single 600 ns pulse, necessary to cause uptake of extracellular Ca(2+), was 1.76 kV/cm for NG108 and 0.84 kV/cm for PHN. At 16.2 kV/cm, the ED50 for pulse width was 95 ns for both cell lines. Cadmium, a nonspecific Ca(2+) channel blocker, failed to prevent Ca(2+) uptake suggesting that observed influx is likely due to nanoporation. These data demonstrate that moderate amplitude single nsPEF exposures result in rapid Ca(2+) influx that may be capable of controllably modulating neurological function.