Modulation of mechanosensitive genes during embryonic aortic arch development.

BACKGROUND: Early embryonic aortic arches (AA) are a dynamic vascular structures that are in the process of shaping into the great arteries of cardiovascular system. Previously, a time-lapsed mechanosensitive gene expression map was established for AA subject to altered mechanical loads in the avian embryo. To validate this map, we investigated effects on vascular microstructure and material properties following the perturbation of key genes using an in-house microvascular gene knockdown system. RESULTS: All siRNA vectors show a decrease in the expression intensity of desired genes with no significant differences between vectors. In TGFß3 knockdowns, we found a reduction in expression intensities of TGFß3 (≤76%) and its downstream targets such as ELN (≤99.6%), Fbn1 (≤60%), COL1 (≤52%) and COL3 (≤86%) and an increase of diameter in the left AA (23%). MMP2 knockdown also reduced expression levels in MMP2 (≤30%) and a 6-fold increase in its downstream target COL3 with a decrease in stiffness of the AA wall and an increase in the diameter of the AA (55%). These in vivo measurements were confirmed using immunohistochemistry, western blotting and a computational growth model of the vascular extracellular matrix (ECM). CONCLUSIONS: Localized spatial genetic modification of the aortic arch region governs the vascular phenotype and ECM composition of the embryo and can be integrated with mechanically-induced congenital heart disease models.

Treating scars after burns with pulsed electric fields in the rat model.

Reducing scar size after severe burn injuries is an important and challenging medical, technology and social problem. We have developed a battery-powered pulsed electric field (PEF) device and surface needle electrode applicator to deliver pulsed electric fields to the healing dorsal burn wound in rats. PEF was used to treat residual burn wounds caused by metal contact in rats starting 10 days after the injury for 4 months every 11 or 22 days for 4 months using varying time applied voltages at 250-350V range, 400mA current, 40 pulses, 70 µs duration each, delivered at pulse repetition frequency 10 Hz at 5 locations inside the wound. We found 40-45% reduction in the scar size in comparison with untreated controls in both upper and lower dorsal locations on rats' backs two months after the last PEF application. We have not detected significant histopathological differences in the center of the scars besides the thickness of the newly generated epidermis, which was thicker in the PEF treated group.We showed that minimally invasively applied pulsed electric fields through needle electrodes are effective method and device for treating residual burn wounds in the rat model, reducing the size of the resulting scars, without any adverse reaction.

A high-titer scalable Chinese hamster ovary transient expression platform for production of biotherapeutics.

Transient gene expression (TGE) in Chinese hamster ovary (CHO) cells offers a route to accelerate biologics development by delivering material weeks to months earlier than what is possible with conventional cell line development. However, low productivity, inconsistent product quality profiles, and scalability challenges have prevented its broader adoption. In this study, we develop a scalable CHO-based TGE system achieving 1.9 g/L of monoclonal antibody in an unmodified host. We integrated continuous flow-electroporation and alternate tangential flow (ATF) perfusion to enable an end-to-end closed system from N-1 perfusion to fed-batch 50-L bioreactor production. Optimization of both the ATF operation for three-in-one application-cell growth, buffer exchange, and cell mass concentration-and the flow-electroporation process, led to a platform for producing biotherapeutics using transiently transfected cells. We demonstrate scalability up to 50-L bioreactor, maintaining a titer over 1 g/L. We also show comparable quality between both transiently and stably produced material, and consistency across batches. The results confirm that purity, charge variants and N-glycan profiles are similar. Our study demonstrates the potential of CHO-based TGE platforms to accelerate biologics process development timelines and contributes evidence supporting its feasibility for manufacturing early clinical material, aiming to strengthen endorsement for TGE's wider implementation.

Irreversible electroporation of localised prostate cancer downregulates immune suppression and induces systemic anti-tumour T-cell activation - IRE-IMMUNO study.

OBJECTIVES: To prospectively compare systemic anti-tumour immune responses induced by irreversible electroporation (IRE) and robot-assisted radical prostatectomy (RARP) in patients with localised intermediate-risk prostate cancer (PCa). PATIENTS AND METHODS: Between February 2021 and June 2022, before and after treatment (at 5, 14 and 30 days) peripheral blood samples of 30 patients with localised PCa were prospectively collected. Patient inclusion criteria were: International Society of Urological Pathologists Grade 2-3, clinical cancer stage ≤T2c, prostate-specific antigen level <20 ng/mL). Patients were treated with IRE (n = 20) or RARP (n = 10). Frequency and activation status of lymphocytic and myeloid immune cell subsets were determined using flow cytometry. PCa-specific T-cell responses to prostatic acid phosphatase (PSAP) and cancer testis antigen (New York oesophageal squamous cell carcinoma 1 [NY-ESO-1]) were determined by interferon-γ enzyme-linked immunospot assay (ELISpot). Repeated-measures analysis of variance and two-sided Student's t-tests were used to compare immune responses over time and between treatment cohorts. RESULTS: Patient and tumour characteristics were similar between the cohorts except for age (median 68 years [IRE] and 62 years [RARP], P = 0.01). IRE induced depletion of systemic regulatory T cells (P = 0.0001) and a simultaneous increase in activated cytotoxic T-lymphocyte antigen 4 (CTLA-4)+ cluster of differentiation (CD)4+ (P < 0.001) and CD8+ (P = 0.032) T cells, consistent with reduction of systemic immune suppression allowing for effector T-cell activation, peaking 14 days after IRE. Effects were positively correlated with tumour volume/ablation size. Accordingly, IRE induced expansion of PSAP and/or NY-ESO-1 specific T-cell responses in four of the eight immune competent patients. Temporarily increased activated myeloid derived suppressor cell frequencies (P = 0.047) were consistent with transient immunosuppression after RARP. CONCLUSIONS: Irreversible electroporation induces a PCa-specific systemic immune response in patients with localised PCa, aiding conversion of the tumour microenvironment into a more immune permissive state. Therapeutic efficacy might be further enhanced by combination with CTLA-4 checkpoint inhibition, potentially opening up a new synergistic treatment paradigm for high-risk localised or (oligo)metastatic disease.

Dendritic cell vaccination combined with irreversible electroporation for treating pancreatic cancer-a narrative review.

Background and Objective: Pancreatic ductal adenocarcinoma (PDAC) is 3rd most lethal cancer in the USA leading to a median survival of six months and less than 5% 5-year overall survival (OS). As the only potentially curative treatment, surgical resection is not suitable for up to 90% of the patients with PDAC due to late diagnosis. Highly fibrotic PDAC with an immunosuppressive tumor microenvironment restricts cytotoxic T lymphocyte (CTL) infiltration and functions causing limited success with systemic therapies like dendritic cell (DC)-based immunotherapy. In this study, we investigated the potential benefits of irreversible electroporation (IRE) ablation therapy in combination with DC vaccine therapy against PDAC. Methods: We performed a literature search to identify studies focused on DC vaccine therapy and IRE ablation to boost therapeutic response against PDAC indexed in PubMed, Web of Science, and Scopus until February 20th, 2023. Key Content and Findings: IRE ablation destructs tumor structure while preserving extracellular matrix and blood vessels facilitating local inflammation. The studies demonstrated IRE ablation reduces tumor fibrosis and promotes CTL tumor infiltration to PDAC tumors in addition to boosting immune response in rodent models. The administration of the DC vaccine following IRE ablation synergistically enhances therapeutic response and extends OS rates compared to the use of DC vaccination or IRE alone. Moreover, the implementation of data-driven approaches further allows dynamic and longitudinal monitoring of therapeutic response and OS following IRE plus DC vaccine immunoablation. Conclusions: The combination of IRE ablation and DC vaccine immunotherapy is a potent strategy to enhance the therapeutic outcomes in patients with PDAC.

Cardiomyocytes Permeabilization and Electrotransfection by Unipolar and Bipolar Asymmetric Electric Field Pulses.

Short electric field pulses represent a novel potential approach for achieving uniform electroporation within tissue containing elongated cells oriented in various directions, such as electroporation-based cardiac ablation procedures. In this study, we investigated how electroporation with nanosecond pulses with respect to different pulse shapes (unipolar, bipolar, and asymmetric) influences cardiomyocyte permeabilization and gene transfer. For this purpose, rat cardiomyocytes (H9c2) were used. The efficacy of the pulsed electric field protocols was assessed by flow cytometry and electrogene transfer by fluorescent and holotomographic microscopy. The response of the cells was assessed by the metabolic activity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [MTT] assay), F-actin distribution in cells by confocal microscopy, and muscle atrophy F-box (MAFbx) marker. We show nano- and microsecond pulse protocols, which are not cytotoxic for cardiac muscle cells and can be efficiently used for gene electrotransfection. Asymmetric nanosecond pulsed electric fields were similarly efficient in plasmid delivery as microsecond and millisecond protocols. However, the millisecond protocol induced a higher MAFbx expression in H9c2 cells.

Changes in Mitochondrial Membrane Potential in In Vitro Electroporation with Nano- and Microsecond Pulses.

With the introduction of nanosecond (ns) pulses, it was suggested that such pulses could be used to permeabilize intracellular membranes, including the mitochondrial membrane. The results presented thus far, however, are not conclusive. Interestingly, the effect of longer microsecond (µs) pulses on changes in mitochondria has never been investigated. We, therefore, investigated the changes in mitochondrial membrane permeability through changes in mitochondrial membrane potential (MMP) in CHO and H9c2 cells after electroporation with 4 ns, 200 ns, and 100 µs pulses. In the range of reversible electroporation, the decrease in MMP generally depended on the cell line. In CHO, ns pulses decreased MMP at lower electroporation intensities than µs. In H9c2, ns and µs were equally effective. In the range of irreversible electroporation, MMP decreased even further, regardless of pulse duration and cell type. The analysis at different time points showed that the changes in MMP within the first hour after pulse treatment are dynamic. Our results on the efficacy of ns pulses are consistent with published data, but with this study we show that µs pulses cause similar changes in MMP as ns pulses, demonstrating that electroporation affects MMP regardless of pulse duration. At the same time, however, differences in MMP changes were observed between different cell lines, indicating some dependence of MMP changes on cell type.

Good manufacturing practice-grade generation of CD19 and CD123-specific CAR-T cells using piggyBac transposon and allogeneic feeder cells in patients diagnosed with B-cell non-Hodgkin lymphoma and acute myeloid leukemia.

Background: The non-viral production of CAR-T cells through electroporation of transposon DNA plasmids is an alternative approach to lentiviral/retroviral methods. This method is particularly suitable for early-phase clinical trials involving novel types of CAR-T cells. The primary disadvantage of non-viral methods is the lower production efficiency compared to viral-based methods, which becomes a limiting factor for CAR-T production, especially in chemotherapy-pretreated lymphopenic patients. Methods: We describe a good manufacturing practice (GMP)-compliant protocol for producing CD19 and CD123-specific CAR-T cells based on the electroporation of transposon vectors. The lymphocytes were purified from the blood of patients undergoing chemotherapy for B-NHL or AML and were electroporated with piggyBac transposon encoding CAR19 or CAR123, respectively. Electroporated cells were then polyclonally activated by anti-CD3/CD28 antibodies and a combination of cytokines (IL-4, IL-7, IL-21). The expansion was carried out in the presence of irradiated allogeneic blood-derived mononuclear cells (i.e., the feeder) for up to 21 days. Results: Expansion in the presence of the feeder enhanced CAR-T production yield (4.5-fold in CAR19 and 9.3-fold in CAR123). Detailed flow-cytometric analysis revealed the persistence of early-memory CAR-T cells and a low vector-copy number after production in the presence of the feeder, with no negative impact on the cytotoxicity of feeder-produced CAR19 and CAR123 T cells. Furthermore, large-scale manufacturing of CAR19 carried out under GMP conditions using PBMCs obtained from B-NHL patients (starting number=200x10e6 cells) enabled the production of >50x10e6 CAR19 in 7 out of 8 cases in the presence of the feeder while only in 2 out of 8 cases without the feeder. Conclusions: The described approach enables GMP-compatible production of sufficient numbers of CAR19 and CAR123 T cells for clinical application and provides the basis for non-viral manufacturing of novel experimental CAR-T cells that can be tested in early-phase clinical trials. This manufacturing approach can complement and advance novel experimental immunotherapeutic strategies against human hematologic malignancies.

Dynamics of cell membrane lesions and adaptive conductance under the electrical stress.

Exceeding physiological limits of the cell membrane potential compromises structural integrity, enabling the passage of normally impermeant solutes and disrupting cell function. Electropermeabilization has been studied extensively at the cellular scale, but not at the individual membrane lesion level. We employed fast total internal reflection fluorescence (TIRF) imaging of Ca2+ entry transients to discern individual lesions in a hyperpolarized cell membrane and characterize their focality, thresholds, electrical conductance, and the lifecycle. A diffuse and momentary membrane permeabilization without a distinct pore formation was observed already at a -100 mV threshold. Polarizing down to -200 mV created focal pores with a low 50- to 300-pS conductance, which disappeared instantly once the hyperpolarization was removed. Charging to -240 mV created high-conductance (> 1 nS) pores which persisted for seconds even at zero membrane potential. With incremental hyperpolarization steps, persistent pores often emerged at locations different from those where the short-lived, low-conductance pores or diffuse permeabilization were previously observed. Attempts to polarize membrane beyond the threshold for the formation of persistent pores increased their conductance adaptively, preventing further potential build-up and "clamping" it at a certain limit (-270 ± 6 mV in HEK cells, -284 ± 5 mV in CHO cells, and -243 ± 9 mV in neurons). The data suggest a previously unknown role of electroporative lesions as a protective mechanism against a potentially fatal membrane overcharging and cell disintegration.

Well Plate-Based Localized Electroporation Workflow for Rapid Optimization of Intracellular Delivery.

Efficient and nontoxic delivery of foreign cargo into cells is a critical step in many biological studies and cell engineering workflows with applications in areas such as biomanufacturing and cell-based therapeutics. However, effective molecular delivery into cells involves optimizing several experimental parameters. In the case of electroporation-based intracellular delivery, there is a need to optimize parameters like pulse voltage, duration, buffer type, and cargo concentration for each unique application. Here, we present the protocol for fabricating and utilizing a high-throughput multi-well localized electroporation device (LEPD) assisted by deep learning-based image analysis to enable rapid optimization of experimental parameters for efficient and nontoxic molecular delivery into cells. The LEPD and the optimization workflow presented herein are relevant to both adherent and suspended cell types and different molecular cargo (DNA, RNA, and proteins). The workflow enables multiplexed combinatorial experiments and can be adapted to cell engineering applications requiring in vitro delivery. Key features • A high-throughput multi-well localized electroporation device (LEPD) that can be optimized for both adherent and suspended cell types. • Allows for multiplexed experiments combined with tailored pulse voltage, duration, buffer type, and cargo concentration. • Compatible with various molecular cargoes, including DNA, RNA, and proteins, enhancing its versatility for cell engineering applications. • Integration with deep learning-based image analysis enables rapid optimization of experimental parameters.

Analyses of Neurite and Spine Formation by In Utero Electroporation in Mice.

Dendrite morphology and dendritic spines are key features of the neuronal networks in the brain. Abnormalities in these features have been observed in patients with psychiatric disorders and mouse models of these diseases. In utero electroporation is an easy and efficient gene transfer system for developing mouse embryos in the uterus. By combining with the Cre-loxP system, the morphology of individual neurons can be clearly and sparsely visualized. Here, we describe how this labeling system can be applied to visualize and evaluate the dendrites and dendritic spines of cortical neurons.

In Vitro and In Vivo Analysis of Neuronal Polarity.

Neuronal development is characterized by the unidirectional flow of signal from the axon to the dendrites via synapses. Neuronal polarization is a critical step during development that allows the specification of the different neuronal processes as a single axon and multiple dendrites both structurally and functionally, allowing the unidirectional flow of information. Along with extrinsic and intrinsic signaling, a whole network of molecular complexes involved in positive and negative feedback loops play a major role in this critical distinction of neuronal processes. As a result, neuronal morphology is drastically altered during establishment of polarity. In this chapter, we discuss how we can analyze the morphological alterations of neurons in vitro in culture to assess the development and polarity status of the neuron. We also discuss how these studies can be conducted in vivo, where polarity studies pose a greater challenge with promising results for addressing multiple pathological conditions. Our experimental model is limited to rodent hippocampal/cortical neurons in culture and cortical neurons in brain tissues, which are well-characterized model systems for understanding neuronal polarization.

In Ovo Electroporation of Embryonic Chicken Spinal Cord in Combination with Ex Vivo Slice Culture for Live Imaging of Vertebrate Neuronal Differentiation.

To investigate the cell behavior underlying neuronal differentiation in a physiologically relevant context, differentiating neurons must be studied in their native tissue environment. Here, we describe an accessible protocol for fluorescent live imaging of differentiating neurons within ex vivo embryonic chicken spinal cord slice cultures, which facilitates long-term observation of individual cells within developing tissue.

Ex Vivo Live Imaging in Brain Slices for Analysis of Neurite Formation in Combination with In Utero Electroporation.

The use of time-lapse live imaging enables us to track the dynamic changes in neurites during their formation. Ex vivo live imaging with acute brain slices provides a more physiological environment than cultured cells. To accomplish this, a certain method of labeling is necessary to visualize and identify neurite morphology. To understand the dynamics of neurite structure at early stages of neurite formation, we describe in this chapter ex vivo live imaging using a confocal microscope at P0 in combination with in utero electroporation (IUE).

Significant hemolysis is present during irreversible electroporation of cardiomyocytes in vitro.

BACKGROUND: Pulsed-field ablation (PFA) of atrial fibrillation (AF) is a new method in clinical practice. Despite a favorable safety profile of PFA in AF ablation, rare cases of renal failure, probably due to hemolysis, have been recently reported. OBJECTIVE: The aim of this study was to determine the rate of hemolysis and cardiac cell death during in vitro PFA with different electric field intensities. METHODS: Blood samples from healthy volunteers and mouse HL-1 cardiomyocyte cell lines were subjected to in vitro irreversible electroporation (IRE) using 216 bipolar pulses, each lasting 2 µs with 5 µs intervals, repeated 20 times at a frequency of 1 Hz. These pulses varied in from 500 to 1500 V. Cell-free hemoglobin levels were assessed spectrophotometrically, and red blood cell microparticles (RBCµ) were evaluated using flow cytometry. Cardiomyocyte death was quantified using propidium iodide. RESULTS: PF energy (1000 V/cm, 1250 V/cm, and 1500 V/cm) was associated with a significant increase in cell-free hemoglobin (0.31 ± 0.16 g/l, 2.33 ± 0.90 g/l, and 5.7 ± 0.20 g/l, p< 0.05), and similar increase in the concentration of RBCµ. Significant rates of cardiomyocyte death were observed at electric field strengths of 750 V/cm, 1000 V/cm, 1250 V/cm and 1500 V/cm (26.5 ± 5.9%, 44.3 ± 6.2%, 55.5 ± 6.9% and 74.5 ± 17.8% of cardiomyocytes, p < 0.05). CONCLUSION: The most effective induction of cell death in vitro was observed at 1500 V/cm. This intensity was also associated with a significant degree of hemolysis.

Electroporation saves the day again: Pulsed-field ablation for phrenic nerve-sparing in right atrial tachycardia.

INTRODUCTION: Pulsed-field ablation (PFA) is a novel nonthermal energy that shows unique features that can be of use beyond pulmonary vein ablation, like tissue selectivity or proximity rather than contact dependency. METHODS AND RESULTS: We report three cases of right focal atrial tachycardias arising from the superior cavoatrial junction and the crista terminalis, in close relationship with the phrenic nerve, effectively ablated using a commercially available PFA catheter designed for pulmonary vein isolation without collateral damage. CONCLUSION: PFA can be useful for treating right atrial tachycardias involving sites near the phrenic nerve, avoiding the need for complex nerve-sparing strategies.

Genome editing using type I-E CRISPR-Cas3 in mice and rat zygotes.

The type I CRISPR system has recently emerged as a promising tool, especially for large-scale genomic modification, but its application to generate model animals by editing zygotes had not been established. In this study, we demonstrate genome editing in zygotes using the type I-E CRISPR-Cas3 system, which efficiently generates deletions of several thousand base pairs at targeted loci in mice with 40%-70% editing efficiency without off-target mutations. To overcome the difficulties associated with detecting the variable deletions, we used a newly long-read sequencing-based multiplex genotyping approach. Demonstrating remarkable versatility, our Cas3-based technique was successfully extended to rats as well as mice, even by zygote electroporation methods. Knockin for SNP exchange and genomic replacement with a donor plasmid were also achieved in mice. This pioneering work with the type I CRISPR zygote editing system offers increased flexibility and broader applications in genetic engineering across different species.

Preclinical evaluation of reversible pulsed electrical field: electrophysiological and histological assessment of myocardium.

Background: Pulsed field ablation, as a non-thermal ablation modality, has received increasing attention. The aim of this study is to explore whether a reversible pulsed electric field (RPEF) can temporarily inhibit electrical conduction and provide a novel method for precise ablation of arrhythmia. Methods: RPEF energy was delivered from an ablation catheter to the atrium of six dogs, followed by a series of electrogram and histology assessments. Results: RPEF ablation of ordinary myocardium resulted in an average reduction of 68.3% (range, 53.7%-83.8%) in electrogram amplitude, while 5 min later, the amplitude in eight electrograms returned to 77.9% (range, 72.4%-87.3%) of baseline. Similarly, the amplitude of the sinoatrial node electrograms reduced by an average of 73.0% (range, 60.2%-84.4%) after RPEF ablation, but recovered to 84.9% (range, 80.3%-88.5%) of baseline by 5 min. No necrotic change was detected in histopathology. Transient third-degree atrioventricular block occurred following the ablation of the maximum His potential sites with RPEF, the duration of which was voltage dependent. The histopathological results showed necrosis of the myocardium at the ablation sites but no injury to His bundle cells. Conclusions: RPEF can be applied to transiently block electrical conduction in myocardial tissues contributing to precise ablation.

Quantifying Muscle Regeneration: Activated Muscle Satellite Cells and New Regenerated Myofibers in Chronic and Acute Degeneration Models.

Regeneration is a remarkable characteristic of the skeletal muscle. Triggered by common lesions, regeneration is stimulated resulting in muscle fiber repair and restoration of muscle homeostasis in normal muscle. In genetic dystrophic muscle, the cycle of degeneration/regeneration is an endless loop that leads to impaired regeneration and substitution of muscle fibers by connective and adipose tissue, causing muscle weakness. Identification and characterization of muscle regeneration steps can help discover potential therapy targets for muscle diseases and aging. Muscle regeneration markers such as the number of satellite cells in the muscle, the proportion of activated satellite cells, and the quantity of regenerating muscle fiber can be quantified using immunolabeling.Here we are presenting a quantitative method to measure muscle regeneration that can be applied to different proposals. To demonstrate the protocol applicability, we used models for acute and chronic muscle injuries. As model of acute degeneration, a wild-type C57BL6 mice with muscle injury induced by electroporation was used, and the muscle was analyzed after 5 and 10 days post-injury. DMDmdx mouse muscle was used as a model of chronic degeneration. The methodologies presented here are among the gold standard methodologies for muscle regeneration analysis and can be easily applied to any type of muscle regeneration study.

Tuning the Locally Enhanced Electric Field Treatment (LEEFT) between Electrophysical and Electrochemical Mechanisms for Bacteria Inactivation.

Efficient drinking water disinfection methods are critical for public health. Locally enhanced electric field treatment (LEEFT) is an antimicrobial method that uses sharp structures, like metallic nanowires, to enhance the electric field at tips and cause bacteria inactivation. Electroporation is the originally designed mechanism of LEEFT. Although oxidation is typically undesired due to byproduct generation and electrode corrosion, it can enhance the overall disinfection efficiency. In this work, we conduct an operando investigation of LEEFT, in which we change the electrical parameters to tune the mechanisms between electrophysical electroporation and electrochemical oxidation. Pure electroporation (i.e., without detectable oxidation) could be achieved under a duty cycle of ≤0.1% and a pulse width of ≤2 µs. Applying 2 µs pulses at 7-8 kV/cm and 0.1% duty cycle results in 80-100% bacteria inactivation with pure electroporation. A higher chance of oxidation is found with a higher duty cycle and a longer pulse width, where the antimicrobial efficiency could also be enhanced. For water with a higher conductivity, a higher antimicrobial efficiency can be achieved under the same treatment conditions, and electrochemical reactions could be induced more easily. The findings shown in this work improve the fundamental understanding of LEEFT and help optimize the performance of LEEFT in real applications.