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.
- MeSH
- akutní myeloidní leukemie * terapie imunologie genetika MeSH
- allogeneické buňky imunologie MeSH
- antigeny CD19 * imunologie genetika MeSH
- B-buněčný lymfom terapie imunologie genetika MeSH
- chimerické antigenní receptory * genetika imunologie MeSH
- elektroporace MeSH
- imunoterapie adoptivní * metody MeSH
- lidé MeSH
- podkladové buňky MeSH
- T-lymfocyty imunologie metabolismus MeSH
- transpozibilní elementy DNA * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The application of pulsed electric fields (PEFs) is becoming a promising tool for application in biotechnology, and the food industry. However, real-time monitoring of the efficiency of PEF treatment conditions is challenging, especially at the industrial scale and in continuous production conditions. To overcome this challenge, we have developed a straightforward setup capable of real-time detection of yeast biological autoluminescence (BAL) during pulsing. Saccharomyces cerevisiae culture was exposed to 8 pulses of 100 μs width with electric field strength magnitude 2-7 kV cm-1. To assess the sensitivity of our method in detecting yeast electroporation, we conducted a comparison with established methods including impedance measurements, propidium iodide uptake, cell growth assay, and fluorescence microscopy. Our results demonstrate that yeast electroporation can be instantaneously monitored during pulsing, making it highly suitable for industrial applications. Furthermore, the simplicity of our setup facilitates its integration into continuous liquid flow systems. Additionally, we have established quantitative indicators based on a thorough statistical analysis of the data that can be implemented through a dedicated machine interface, providing efficiency indicators for analysis.
- MeSH
- elektroporace * metody MeSH
- Saccharomyces cerevisiae * růst a vývoj MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Electroporation is an effective technique for genetic manipulation of cells, both in vitro and in vivo. In utero electroporation (IUE) is a special case, which represents a fine application of this technique to genetically modify specific tissues of embryos during prenatal development. Commercially available electroporators are expensive and not fully customizable. We have designed and produced an inexpensive, open-design, and customizable electroporator optimized for safe IUE. We introduce NeuroPorator. METHOD: We used off-the-shelf electrical parts, a single-board microcontroller, and a cheap data logger to build an open-design electroporator. We included a safety circuit to limit the applied electrical current to protect the embryos. We added full documentation, design files, and assembly instructions. RESULT: NeuroPorator output is on par with commercially available devices. Furthermore, the adjustable current limiter protects both the embryos and the uterus from overcurrent damage. A built-in data acquisition module provides real-time visualization and recordings of the actual voltage/current pulses applied to each embryo. Function of NeuroPorator has been demonstrated by inducing focal cortical dysplasia in mice. SIGNIFICANCE AND CONCLUSION: The simple and fully open design enables quick and cheap construction of the device and facilitates further customization. The features of NeuroPorator can accelerate the IUE technique implementation in any laboratory and speed up its learning curve.
- MeSH
- design vybavení MeSH
- elektroporace * metody přístrojové vybavení MeSH
- embryo savčí MeSH
- myši MeSH
- technika přenosu genů * přístrojové vybavení MeSH
- těhotenství MeSH
- uterus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Radiofrekvenční energii bylo možné do současné doby považovat za zlatý standard katetrizační léčby fibrilace síní. Nicméně obecně s aplikací termické energie byla spojena řada limitací, jako je poškození kolaterálních struktur, časová náročnost a hlavně poměrně častá recidiva síňové arytmie, a to jak fibrilace síní, tak síňových tachykardií. Proto bylo potřeba hledat novou metodu s lepším bezpečnostním a výkonnostním profilem. Slibné výsledky přináší nově používaná ablace pomocí pulzního pole na principu ireverzibilní elektroporace, která na rozdíl od doposud použitých metod s termickým účinkem a tvorbou koagulační nekrózy způsobí převážně dezintegraci buněčné membrány. V tomto článku rozvádíme princip elektroporace, patofyziologické podklady i technické specifikace vývoje ablačních systémů využívajících pulzního pole. Dále jsou prezentovány výsledky prvních klinických studií ablační léčby s použitím metody pulzního pole u všech forem fibrilace síní a v neposlední řadě i první zkušenosti z praxe.
Catheter ablation of atrial fibrillation using radiofrequency energy has been the gold standard until these days. Nevertheless, there are limitations associated with the application of thermal energy such as damage to collateral structures, time-consuming nature, and, in particular, frequent recurrences of both atrial fibrillation and atrial tachycardias. Therefore, it was essential to find a brand new method with a better safety and efficiency profile. Promising results have been obtained with methods using pulsed field based on the principles of irreversible electroporation which, in comparison with the methods used so far that have a thermal effect and induce coagulation necrosis, predominantly cause disintegration of the cell membrane. This article deals with the principle of electroporation, pathophysiological background, and technical specification of the development of ablation systems using pulsed field energy. Also presented are the results of initial clinical trials with ablation treatment of all types of atrial fibrillation using the pulsed field method as well as the initial experience from the practice.
Radioterapie představuje vedle radikální prostatektomie základní léčebnou metodu lokalizovaného karcinomu prostaty. I přes významné pokroky v radioterapii stále dochází téměř u třetiny pacientů k lokální recidivě onemocnění. Současné možnosti kurativní terapie lokální recidivy karcinomu prostaty po radioterapii zahrnují radikální prostatektomii, brachyterapii, kryoablaci, fokusovaný ultrazvuk vysoké intenzity (high-intensity focused ultrasound, HIFU). Vzhledem ke změnám tkání po proběhlém ozáření jsou všechny tyto metody zatíženy významným rizikem klinicky závažné toxicity a selhání léčby. Proto je velká část pacientů s radiorekurentním karcinomem prostaty léčena pouze paliativní hormonální léčbou, případně sledována bez aktivní terapie. Jednu z perspektivních metod u této skupiny pacientů představuje ireverzibilní elektroporace (irreversible electroporation, IRE). Kazuistika prezentuje pacienta s lokální recidivou karcinomu prostaty ošetřeného metodou IRE na našem pracovišti.
Radiotherapy is beside surgery a standard therapeutic procedure in treatment of localized prostate cancer. However local failure occurs almost in one third of patients despite significant progress in radiotherapy. Current salvage treatment options include radical prostatectomy, brachytherapy, cryoablation, high-intensity focused ultrasound (HIFU). All these procedures can lead frequently to severe toxicity and treatment failure due to tissue changes after previous irradiation. Therefore many patients with radiorecurrent prostate cancer are treated with palliative hormonal therapy or observed. Irreversible electroporation (IRE) represents one of promising treatment methods in this setting. In our case report we present a patient with local failure after radiotherapy for prostate cancer treated by IRE.
- MeSH
- biopsie škodlivé účinky MeSH
- brachyterapie MeSH
- elektroporace * metody MeSH
- lidé středního věku MeSH
- lidé MeSH
- lokální recidiva nádoru MeSH
- magnetická rezonanční tomografie MeSH
- nádory prostaty * radioterapie terapie MeSH
- odmítnutí terapie pacientem MeSH
- PET/CT MeSH
- prostatický specifický antigen analýza MeSH
- výsledek terapie MeSH
- záchranná terapie klasifikace MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
- MeSH
- elektroporace * MeSH
- fibrilace síní * terapie MeSH
- katetrizační ablace metody MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- úvodníky MeSH
Aggregation of small neuronal protein α-synuclein (αSyn) in amyloid fibrils is considered to be one of the main causes of Parkinson's disease. Inhibition of this aggregation is a promising approach for disease treatment. Dozens of compounds able to inhibit αSyn fibrillization in solution were developed during the last decade. However, the applicability of most of them in the cellular environment was not established because of the absence of a suitable cell-based assay. In this work, we developed an assay for testing αSyn aggregation inhibitors in cells that is based on fluorescence resonance energy transfer (FRET) between labeled αSyn molecules in fibrils. The assay directly reports the amount of fibrillized αSyn and is more reliable than the assays based on cell viability. Moreover, we showed that cell viability decline does not always correlate with the amount of misfolded αSyn. The developed FRET-based assay does not interfere with the aggregation process and is suitable for high-throughput testing of αSyn aggregation inhibitors. Its application can sort out non-specific inhibitors and thus significantly facilitate the development of drugs for Parkinson`s disease.
- MeSH
- alfa-synuklein analýza antagonisté a inhibitory metabolismus MeSH
- benzodioxoly farmakologie MeSH
- elektroporace metody MeSH
- HeLa buňky MeSH
- intracelulární tekutina chemie účinky léků metabolismus MeSH
- lidé MeSH
- proteinové agregáty účinky léků fyziologie MeSH
- pyrazoly farmakologie MeSH
- rezonanční přenos fluorescenční energie metody MeSH
- viabilita buněk účinky léků fyziologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
PURPOSE: To demonstrate the feasibility of irreversible electroporation (IRE) for treating biliary metal stent occlusion in an experimental liver model. METHODS AND MATERIALS: IRE was performed using an expandable tubular IRE-catheter placed in nitinol stents in the porcine liver. A 3-electrode IRE-catheter was connected to an IRE-generator and one hundred 100μs pulses of constant voltage (300, 650, 1000, and 1300 V) were applied. Stent occlusion was simulated by insertion of liver tissue both ex vivo (n = 94) and in vivo in 3 pigs (n = 14). Three scenarios of the relationship between the stent, electrodes, and inserted tissue (double contact, single contact, and stent mesh contact) were studied. Electric current was measured and resistance and power calculated. Pigs were sacrificed 72 h post-procedure. Harvested samples (14 experimental, 13 controls) underwent histopathological analysis. RESULTS: IRE application was feasible at 300 and 650 V for the single and double contact setup in both ex vivo and in vivo studies. Significant differences in calculated resistance between double contact and single contact settings were observed (ex-vivo p ˂ 0.0001, in-vivo p = 0.02; Mann-Whitney). A mild temperature increase of the surrounding liver parenchyma was noted with increasing voltage (0.9-5.9 °C for 300-1000 V). The extent of necrotic changes in experimental samples in vivo correlated with the measured electric current (r2 = 0.39, p = 0.01). No complications were observed during or after the in-vivo procedure. CONCLUSION: Endoluminal IRE using an expandable tubular catheter in simulated metal stent occlusion is feasible. The relationship of active catheter electrodes to stent ingrowth tissue can be estimated based on resistance values.
- MeSH
- ablace * MeSH
- elektroporace * MeSH
- katétry MeSH
- prasata MeSH
- stenty MeSH
- teoretické modely MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- fibrilace síní patofyziologie terapie MeSH
- ireverzibilní elektroporace (terapie) MeSH
- katetrizační ablace * metody škodlivé účinky MeSH
- lidé MeSH
- srdeční arytmie * MeSH
- výsledek terapie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
Česká republika (ČR) zaujímá ve výskytu nádorů ledvin první místo na světě. Mortalita na uvedené onemocnění v ČR v posledních letech mírně klesá. U nemetastazujících nádorů ledvin je řešení možné chirurgickou ablací (resekce či nefrektomie), tepelně ablačními metodami, sledováním či paliativní embolizací. Resekce ledviny je preferovaným řešením pro nádory kategorie cT1. Pro tumory kategorie cT2 je preferována minimálně invazivní nefrektomie. Indikace tepelně ablačních metod je možná na základě individuálního posouzení spíše u osob nevhodných k chirurgickému odstranění. U lokálně pokročilého (nemetastazujícího) tumoru ledviny je radikální nefrektomie metodou volby. Biopsie nádorového ložiska je indikována především u méně typických případů, kde znalost histologie může ovlivnit způsob řešení. Sledování ložiska u pacientů nevhodných k aktivnímu řešení poskytuje příznivé nádorově specifické přežití v krátkodobém a střednědobém horizontu.
Renal tumor incidence is the highest worldwide in Czech Republic. However, the mortality here is slowly declining in recent years. Possible management in non-metastatic renal tumors includes surgical removal (partial or total nephrectomy), thermal ablation methods, surveillance, or palliative embolization. Partial nephrectomy is a preferred method for cT1 renal tumors. For those of cT2 category a minimally invasive nephrectomy is preferred. Thermal ablation is indicated following individual assessment in patients not suitable for surgical removal. In locally advanced (non-metastatic) tumors a radical nephrectomy is a method of choice. Renal mass biopsy is indicated usually in less typical cases, where a knowledge of histology may affect management choice. Tumor surveillance in patients not suitable for active treatment provides favorable disease specific shortand mid-term survival.
- MeSH
- elektroporace metody MeSH
- karcinom z renálních buněk * chirurgie epidemiologie terapie MeSH
- ledviny chirurgie diagnostické zobrazování MeSH
- lidé MeSH
- mikrovlny terapeutické užití MeSH
- miniinvazivní chirurgické výkony MeSH
- nefrektomie * metody MeSH
- radiofrekvenční ablace metody MeSH
- teplá ischemie škodlivé účinky MeSH
- terapeutická embolizace metody MeSH
- trombektomie metody MeSH
- Check Tag
- lidé MeSH
