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1.
Sci Rep ; 14(1): 4330, 2024 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-38383752

RESUMO

The therapeutic use of mesenchymal stem cells (MSCs) becomes more and more important due to their potential for cell replacement procedures as well as due to their immunomodulatory properties. However, protocols for MSCs differentiation can be lengthy and may result in incomplete or asynchronous differentiation. To ensure homogeneous populations for therapeutic purposes, it is crucial to develop protocols for separation of the different cell types after differentiation. In this article we show that, when MSCs start to differentiate towards adipogenic or osteogenic progenies, their dielectrophoretic behavior changes. The values of cell electric parameters which can be obtained by dielectrophoretic measurements (membrane permittivity, conductivity, and cytoplasm conductivity) change before the morphological features of differentiation become microscopically visible. We further demonstrate, by simulation, that these electric modifications make possible to separate cells in their early stages of differentiation by using the dielectrophoretic separation technique. A label free method which allows obtaining cultures of homogenously differentiated cells is thus offered.


Assuntos
Adipogenia , Células-Tronco Mesenquimais , Diferenciação Celular , Osteogênese , Células Cultivadas
2.
Ann Biomed Eng ; 52(1): 103-123, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37651029

RESUMO

Escherichia coli bacterium is a rod-shaped organism composed of a complex double membrane structure. Knowledge of electric field driven ion transport through both membranes and the evolution of their induced permeabilization has important applications in biomedical engineering, delivery of genes and antibacterial agents. However, few studies have been conducted on Gram-negative bacteria in this regard considering the contribution of all ion types. To address this gap in knowledge, we have developed a deterministic and stochastic Brownian dynamics model to simulate in 3D space the motion of ions through pores formed in the plasma membranes of E. coli cells during electroporation. The diffusion coefficient, mobility, and translation time of Ca2+, Mg2+, Na+, K+, and Cl- ions within the pore region are estimated from the numerical model. Calculations of pore's conductance have been validated with experiments conducted at Gustave Roussy. From the simulations, it was found that the main driving force of ionic uptake during the pulse is the one due to the externally applied electric field. The results from this work provide a better understanding of ion transport during electroporation, aiding in the design of electrical pulses for maximizing ion throughput, primarily for application in cancer treatment.


Assuntos
Eletroporação , Escherichia coli , Transporte de Íons , Transporte Biológico , Eletroporação/métodos , Íons
3.
Eur J Cancer ; 186: 62-68, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37030078

RESUMO

OBJECTIVE: To report efficacy and safety of percutaneous electrochemotherapy (ECT) in patients with radiotherapy-resistant metastatic epidural spinal cord compression (MESCC). MATERIAL/ METHODS: This retrospective study analyzed all consecutive patients treated with bleomycin-based ECT between February-2020 and September-2022 in a single tertiary referral cancer center. Changes in pain were evaluated with the Numerical Rating Score (NRS), in neurological deficit with the Neurological Deficit Scale, and changes in epidural spinal cord compression were evaluated with the epidural spinal cord compression scale (ESCCS) using an MRI. RESULTS: Forty consecutive solid tumour patients with previously radiated MESCC and no effective systemic treatment options were eligible. With a median follow-up of 5.1 months [1-19.1], toxicities were temporary acute radicular pain (25%), prolonged radicular hypoesthesia (10%), and paraplegia (7.5%). At 1 month, pain was significantly improved over baseline (median NRS: 1.0 [0-8] versus 7.0 [1.0-10], P < .001) and neurological benefits were considered as marked (28%), moderate (28%), stable (38%), or worse (8%). Three-month follow-up (21 patients) confirmed improved over baseline (median NRS: 2.0 [0-8] versus 6.0 [1.0-10], P < .001) and neurological benefits were considered as marked (38%), moderate (19%), stable (33.5%), and worse (9.5%). One-month post-treatment MRI (35 patients) demonstrated complete response in 46% of patients by ESCCS, partial response in 31%, stable disease in 23%, and no patients with progressive disease. Three-month post-treatment MRI (21 patients) demonstrated complete response in 28.5%, partial response in 38%, stable disease in 24%, and progressive disease in 9.5%. CONCLUSIONS: This study provides the first evidence that ECT can rescue radiotherapy-resistant MESCC.


Assuntos
Eletroquimioterapia , Segunda Neoplasia Primária , Compressão da Medula Espinal , Neoplasias da Coluna Vertebral , Humanos , Compressão da Medula Espinal/diagnóstico por imagem , Compressão da Medula Espinal/etiologia , Compressão da Medula Espinal/radioterapia , Estudos Retrospectivos , Neoplasias da Coluna Vertebral/secundário , Descompressão Cirúrgica , Dor
4.
Front Vet Sci ; 9: 868989, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35968026

RESUMO

Electrochemotherapy (ECT) consists in the application of electric pulses to increase chemotherapeutic drug intake (bleomycin, cisplatin, or calcium) into the tumor cells. It has become a very valuable treatment option in veterinary oncology. It is an effective and safe treatment modality, which is not only beneficial as a palliative treatment, but also for a curative approach. Performing the treatment adequately will ensure the best results possible, in the minimum number of sessions, and reduce complications. Usually, only one session is enough to achieve excellent results, but the treatment can be repeated. Several sessions can be necessary in the case of incompletely treated or very extended lesions, as well as in the occurrence of new lesions. ECT is effective for superficial or oral tumors of any histology that are accessible to the electrodes. Intravenous bleomycin is the preferred drug and route of administration, leaving other ways of administration and drugs for selected cases. The guidelines presented here are destined to veterinarians who want to develop their understanding of the basis of ECT and wish to perform it adequately and effectively. In this paper, we also discuss common problems and how to solve them, and we include practical tips to improve the treatment results based on common questions and mistakes of beginner users.

5.
Bioelectrochemistry ; 147: 108218, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35933972

RESUMO

Electropulsation has become a powerful technological platform for electromanipulation of cells and tissues for various medical and biotechnological applications, but the molecular changes that underlay the very first initiation step of this process have not been experimentally observed. Here, we endowed a wide-field Coherent anti-Stokes Raman Scattering platform with an ad-hoc electromagnetic exposure device and we demonstrated, using artificial lipid vesicles (i.e. liposomes), that electropulsation is initiated by the increase of interstitial water content in liposome membranes. A pulse-dependent accumulation of the interstitial water molecules is observed in the membranes and a plausible mechanism supported by a computational electrochemical model is presented and discussed.


Assuntos
Lipossomos , Análise Espectral Raman , Eletricidade , Análise Espectral Raman/métodos , Água
6.
Int J Radiat Biol ; 98(1): 109-121, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34714724

RESUMO

BACKGROUND: Despite the numerous literature results about biological effects of electromagnetic field (EMF) exposure, the interaction mechanisms of these fields with organisms are still a matter of debate. Extremely low frequency (ELF) MFs can modulate redox homeostasis and we showed that 24 h exposure to 50 Hz-1 mT has a pro-oxidant effect and effects on the epigenome of SH-SY5Y cells, decreasing miR-34b/c expression through the hypermethylation of their promoter. METHODS: Here, we investigated the role of the electromagnetic deposited energy density (ED) during exposures lasting 24 h to 1 mT amplitude MFs at a frequency of 50 Hz in inducing the above mentioned effects. To this end, we delivered ultrashort electric pulses, in the range of microsecond and nanosecond duration, with the same ED of the previously performed magnetic exposure to SH-SY5Y cells. Furthermore, we explored the effect of higher deposited energy densities. Analysis of i) gene and microRNA expression, ii) cell morphology, iii) reactive oxygen species (ROS) generation, and iv) apoptosis were carried out. RESULTS: We observed significant changes in egr-1 and c-fos expression at very low deposited ED levels, but no change of the ROS production, miR-34b/c expression, nor the appearance of indicators of apoptosis. We thus sought investigating changes in egr-1 and c-fos expression caused by ultrashort electric pulses at increasing deposited ED levels. The pulses with the higher deposited ED caused cell electroporation and even other morphological changes such as cell fusion. The changes in egr-1 and c-fos expression were more intense, but, again, no change of the ROS production, miR-34b/c expression, nor apoptosis induction was observed. CONCLUSIONS: These results, showing that extremely low levels of electric stimulation (never investigated until now) can cause transcriptional changes, also reveal the safety of the electroporating pulses used in biomedical applications and open up the possibility to further therapeutic applications of this technology.


Assuntos
MicroRNAs , Neuroblastoma , Linhagem Celular , Campos Eletromagnéticos/efeitos adversos , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Neuroblastoma/metabolismo , Espécies Reativas de Oxigênio/metabolismo
7.
Pharmaceutics ; 13(6)2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34201070

RESUMO

Sonoporation is the process of cell membrane permeabilization, due to exposure to ultrasounds. There is a lack of consensus concerning the mechanisms of sonoporation: Understanding the mechanisms of sonoporation refines the choice of the ultrasonic parameters to be applied on the cells. Cells' classical exposure systems to ultrasounds have several drawbacks, like the immersion of the cells in large volumes of liquid, the nonhomogeneous acoustic pressure in the large sample, and thus, the necessity for magnetic stirring to somehow homogenize the exposure of the cells. This article reports the development and characterization of a novel system allowing the exposure to ultrasounds of very small volumes and their observation under the microscope. The observation under a microscope imposes the exposure of cells and Giant Unilamellar Vesicles under an oblique incidence, as well as the very unusual presence of rigid walls limiting the sonicated volume. The advantages of this new setup are not only the use of a very small volume of cells culture medium/microbubbles (MB), but the presence of flat walls near the sonicated region that results in a more homogeneous ultrasonic pressure field, and thus, the control of the focal distance and the real exposure time. The setup presented here comprises the ability to survey the geometrical and dynamical aspects of the exposure of cells and MB to ultrasounds, if an ultrafast camera is used. Indeed, the setup thus fulfills all the requirements to apply ultrasounds conveniently, for accurate mechanistic experiments under an inverted fluorescence microscope, and it could have interesting applications in photoacoustic research.

8.
Macromol Biosci ; 21(5): e2000391, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33645917

RESUMO

The work here reported analyzes the effect of increased efficiency of brain-derived neurotrophic factor (BDNF) production by electroporated Schwann cells (SCs) on the axonal extension in a coculture system on a biomaterial platform that can be of interest for the treatment of injuries of the nervous system, both central and peripheral. Rat SCs are electrotransfected with a plasmid coding for the BDNF protein in order to achieve an increased expression and release of this protein into the culture medium of the cells, performing the best balance between the level of transfection and the number of living cells. Gene-transfected SCs show an about 100-fold increase in the release of BDNF into the culture medium, compared to nonelectroporated SCs. Cocultivation of electroporated SCs with rat dorsal root ganglia (DRG) is performed on highly aligned substrates of polylactic acid (PLA) microfibers coated with the electroconductive polymer polypyrrol (PPy). The coculture of DRG with electrotransfected SCs increase both the axonal extension and the axonal sprouting from DRG neurons compared to the coculture of DRG with nonelectroporated SCs. Therefore, the use of PLA-PPy highly aligned microfiber substrates preseeded with electrotransfected SCs with an increased BDNF secretion is capable of both guiding and accelerating axonal growth.


Assuntos
Axônios , Fator Neurotrófico Derivado do Encéfalo/genética , Poliésteres/química , Polímeros/química , Pirróis/química , Células de Schwann/fisiologia , Transfecção/métodos , Animais , Materiais Biocompatíveis , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células Cultivadas , Técnicas de Cocultura , Meios de Cultura , Eletroporação , Gânglios Espinais/citologia , Neurônios/citologia , Plasmídeos , Ratos
9.
Int J Biometeorol ; 65(1): 59-67, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32335726

RESUMO

Mechanisms of how electromagnetic (EM) field acts on biological systems are governed by the same physics regardless of the origin of the EM field (technological, atmospheric...), given that EM parameters are the same. We draw from a large body of literature of bioeffects of a man-made electromagnetic field. In this paper, we performed a focused review on selected possible mechanisms of how atmospheric electromagnetic phenomena can act at the molecular and cellular level. We first briefly review the range of frequencies and field strengths for both electric and magnetic fields in the atmosphere. Then, we focused on a concise description of the current knowledge on weak electric and magnetic field bioeffects with possible molecular mechanisms at the basis of possible EM field bioeffects combined with modeling strategies to estimate reliable outcomes and speculate about the biological effects linked to lightning or pyroelectricity. Indeed, we bring pyroelectricity as a natural source of voltage gradients previously unexplored. While very different from lightning, it can result in similar bioeffects based on similar mechanisms, which can lead to close speculations on the importance of these atmospheric electric fields in the evolution.


Assuntos
Eletricidade , Campos Eletromagnéticos , Campos Eletromagnéticos/efeitos adversos , Humanos
10.
Bioelectrochemistry ; 137: 107642, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32980738

RESUMO

The use of conductive nanoparticles (NPs) was previously proposed as a way to locally amplify the electric field (EF) intensity at the cell membrane to enhance cell electroporation. To achieve this, a close distance between the NPs and the cell membrane is mandatory. Here, a new method to improve the contact between NPs and cell surface using the effects of electric pulses (electrophoretic forces) is explored. The effects of two types of electric pulses are analyzed alone or combined in a two-pulse-train protocol on Chinese hamster DC-3F cells. Particularly we used 100 µs duration pulses, low intensity-millisecond pulses and combinations of both. Finally, we studied the use of surface coated NPs (PEGylated) for this application. Our results demonstrate that the delivery of an electric field prior to the electroporation pulses increases the accumulation of NPs around the cell membrane suggesting that NPs are pushed towards the cell surface through electrophoretic forces. This allowed reducing the need for long incubations between cells and NPs to observe an enhancement of electroporation mediated by conductive NPs. Thus low intensity-millisecond pulses can be used to increase the accumulation of either aggregated or individual (i.e. PEGylated) NPs supporting the electrophoretic nature of the observed effects.


Assuntos
Permeabilidade da Membrana Celular , Técnicas Eletroquímicas/métodos , Eletroforese/métodos , Ouro/química , Nanopartículas Metálicas/química , Animais , Bleomicina/farmacologia , Linhagem Celular , Cricetulus , Eletroporação , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/ultraestrutura , Microscopia Eletrônica de Transmissão
11.
Int J Biometeorol ; 65(1): 5-29, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33025117

RESUMO

There is an increasing interest to study the interactions between atmospheric electrical parameters and living organisms at multiple scales. So far, relatively few studies have been published that focus on possible biological effects of atmospheric electric and magnetic fields. To foster future work in this area of multidisciplinary research, here we present a glossary of relevant terms. Its main purpose is to facilitate the process of learning and communication among the different scientific disciplines working on this topic. While some definitions come from existing sources, other concepts have been re-defined to better reflect the existing and emerging scientific needs of this multidisciplinary and transdisciplinary area of research.


Assuntos
Biologia , Eletricidade
12.
Front Bioeng Biotechnol ; 8: 552261, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33072718

RESUMO

In the last years, microdosimetric numerical models of cells including intracellular compartments have been proposed, aiming to investigate the poration induced by the application of nanosecond pulsed electric fields (nsPEFs). A limitation of such models was the extremely approximate cell and organelle shapes, leading to an incorrect estimation of the electric field or transmembrane potential distribution in the studied domain. In order to obtain a reliable model of in vitro experiments and a one-to-one comparison between experimental and simulated results, here, a realistic model of 12 human mesenchymal stem cells was built starting from their optical microscopy images where different cell compartments were highlighted. The microdosimetric analysis of the cells group was quantified in terms of electric field and transmembrane potentials (TMPs) induced by an externally applied 10-ns trapezoidal pulse with rise and fall times of 2 ns, with amplitudes ranging from 2 to 30 MV/m. The obtained results showed that the plasma and endoplasmic reticulum (ER) membrane of each cell respond in a different way to the same electric field amplitude, depending on differences in shape, size, and position of the single cell with respect to the applied electric field direction. Therefore, also the threshold for an efficient electroporation is highly different from cell to cell. This difference was quantitatively estimated through the cumulative distribution function of the pore density for the plasma and ER membrane of each cell, representing the probability that a certain percentage of membrane has reached a specific value of pore density. By comparing the dose-response curves resulted from the simulations and those from the experimental study of De Menorval et al. (2016), we found a very good matching of results for plasma and ER membrane when 2% of the porated area is considered sufficient for permeabilizing the membrane. This result is worth of noting as it highlights the possibility to effectively predict the behavior of a cell (or of a population of cells) exposed to nsPEFs. Therefore, the microdosimetric realistic model described here could represent a valid tool in setting up more efficient and controlled electroporation protocols.

13.
Bioelectrochemistry ; 136: 107627, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32784102

RESUMO

This study presents electrical measurements (both conductivity during the pulses and impedance spectroscopy before and after) performed in liver tissue of mice during electroporation with classical electrochemotherapy conditions (8 pulses of 100 µs duration). A four-needle electrode arrangement inserted in the tissue was used for the measurements. The undesirable effects of the four-electrode geometry, notably concerning its sensitivity, were quantified and discussed showing how the electrode geometry chosen for the measurements can impact the results. Numerical modelling was applied to the information collected during the pulse, and to the impedance spectra acquired before and after the pulses sequence. Our results show that the numerical results were not consistent, suggesting that other collateral phenomena not considered in the model are at work during electroporation in vivo. We show how the modification in the volume of the intra and extra cellular media, likely caused by the vascular lock effect, could at least partially explain the recorded impedance evolution. In the present study we demonstrate the significant impact that physiological effects have on impedance changes following electroporation at the tissue scale and the potential need of introducing them into the numerical models. The code for the numerical model is publicly available at https://gitlab.inria.fr/poignard/4-electrode-system.


Assuntos
Eletroporação/métodos , Fígado/fisiologia , Modelos Biológicos , Animais , Espectroscopia Dielétrica , Impedância Elétrica , Camundongos
14.
Sci Rep ; 10(1): 10471, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591612

RESUMO

The permeabilization of the live cells membrane by the delivery of electric pulses has fundamental interest in medicine, in particular in tumors treatment by electrochemotherapy. Since underlying mechanisms are still not fully understood, we studied the impact of electric pulses on the biochemical composition of live cells thanks to label-free optical methods: confocal Raman microspectroscopy and terahertz microscopy. A dose effect was observed after cells exposure to different field intensities and a major impact on cell peptide/protein content was found. Raman measurements reveal that protein structure and/or environment are modified by the electric pulses while terahertz measurements suggest a leakage of proteins and other intracellular compounds. We show that Raman and terahertz modalities are a particularly attractive complement to fluorescence microscopy which is the reference optical technique in the case of electropermeabilization. Finally, we propose an analytical model for the influx and efflux of non-permeant molecules through transiently (electro)permeabilized cell membranes.


Assuntos
Membrana Celular/metabolismo , Eletroquimioterapia/psicologia , Eletroporação/métodos , Microscopia de Fluorescência/métodos , Animais , Linhagem Celular , Permeabilidade da Membrana Celular/fisiologia , Cães , Eletricidade , Eletroquimioterapia/métodos , Células Madin Darby de Rim Canino , Neoplasias/metabolismo , Proteínas/metabolismo
15.
Cancers (Basel) ; 12(1)2020 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-31963132

RESUMO

The effectiveness of electrochemotherapy (ECT) in local eradication of tumours in human and veterinary medicine has been proven. ECT consists of increasing the uptake of cytotoxic drugs by means of pulsed electric fields (PEFs) that transiently permeabilise the cell membrane. Still, this tumour treatment includes some drawbacks that are linked to the characteristics of the intense electric pulses (EPs) used. Meanwhile, the emerging field of cancer therapies that are based on the application of non-thermal plasmas (NTP) has recently garnered interest because of their potentialities as rich sources of reactive species. In this work, we investigated the potential capabilities of the combined application of indirect NTP treatment and microsecond PEFs (µsPEFs) to outperform in vitro cell electropermeabilisation, the basis of ECT. Thus, phosphate-buffered saline (PBS) was plasma-treated (pPBS) and used afterwards to explore the effects of its combination with µsPEFs. Analysis of two different cell lines (DC-3F Chinese hamster lung fibroblasts and malignant B16-F10 murine melanoma cells), by flow cytometry, revealed that this combination resulted in significant increases of the level of cell membrane electropermeabilisation, even at very low electric field amplitude. The B16-F10 cells were more sensitive to the combined treatment than DC-3F cells. Importantly, the percentage of permeabilised cells reached values similar to those of cells exposed to classical electroporation field amplitude (1100 V/cm) when the cells were treated with pPBS before and after being exposed only to very low PEF amplitude (600 V/cm). Although the level of permeabilisation of the cells that are treated by the pPBS and the PEFs at 600 V/cm is lower than the level reached after the exposure to µsPEFs alone at 1100 V/cm, the combined treatment opens the possibility to reduce the amplitude of the EPs used in ECT, potentially allowing for a novel ECT with reduced side-effects.

16.
IEEE Trans Biomed Eng ; 67(4): 1040-1049, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31329545

RESUMO

OBJECTIVE: The purpose of this work is to assess the ability of sine waves to perform electrochemotherapy (ECT) and to study the dependence of the frequency of the applied sine wave on the treatment efficacy. METHODS: A subcutaneous tumor model in mice was used, and the electric field was delivered in combination with bleomycin. Sinusoidal electric fields of different frequencies, amplitudes, and durations were compared to square waves. Computer simulations were additionally performed. RESULTS: The results confirmed the ability of a sinusoidal electric field to obtain successful ECT responses. A strong dependence on frequency was obtained. The efficacy of the treatment decreased when the frequency of the sine waves was increased. At low sinusoidal frequency, the efficacy of the treatment is very similar to that obtained with a square wave. The collateral effects such as skin burns and muscle contractions decreased for the highest frequency assayed. CONCLUSION: The use of sine wave burst represents a feasible option for the treatment of cancer by ECT. SIGNIFICANCE: These results could have important implications for the treatment of cancer in the clinical world where ECT is performed with dc square pulses.


Assuntos
Eletroquimioterapia , Neoplasias , Animais , Bleomicina/uso terapêutico , Simulação por Computador , Camundongos , Resultado do Tratamento
17.
Nanotechnology ; 30(49): 495101, 2019 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-31422958

RESUMO

Conducive nanoparticles (NPs) were proposed to locally amplify the external electric field (EF) intensity at the cell surface to improve cell electroporation. To better understand the physical mechanisms behind this improvement, different types of NPs and several incubation conditions were applied to adherent cells in the present study. The enhancement of electroporation was observed in the presence of conductive NPs but not when non-conductive NPs were used. Experimental data demonstrate the influence of the incubation conditions between cells and NPs, which impact on the number and quality (aggregated or isolated) of the NPs surrounding the cells. While NPs can increase the number of electroporated cells, they have a more pronounced impact on the level permeabilization of each individual cell. Our results reveal the potential of conductive NPs to enhance the efficiency of electroporation via the amplification of the local EF at the cell surface as shown by numerical simulations.


Assuntos
Condutividade Elétrica , Eletroporação/métodos , Células Epiteliais/metabolismo , Nanopartículas/química , Animais , Bleomicina/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cricetulus , Eletrodos , Campos Eletromagnéticos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Nanopartículas/metabolismo , Nanopartículas/ultraestrutura , Platina/química , Platina/farmacologia , Dióxido de Silício/química , Dióxido de Silício/farmacologia
18.
Bioelectrochemistry ; 129: 218-227, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31200252

RESUMO

In this study the evolution in the efficiency of electrochemotherapy (reversible electroporation) with pulse number was assessed in vitro. Experiments were performed using 100 µs pulses at different electric field intensities and the chemotherapeutic agent bleomycin. Additionally, electrical impedance spectroscopy measurements were used as a different method to study in real time the changes produced on cells with pulse number during trains of consecutive pulses. Our results show that the relation between pulse number and the observed outcome is complex and difficult to fully characterize. This relation can display a highly linear behaviour up to a certain number of pulses and/or field intensity applied. However, the relation between the number of pulses and the observed outcome always evolves to a saturation or at least a reduction in the electric field effects that is displayed when either electric field intensity or pulse number are increased. An exponential model was found to best describe this relation within the range of experimental conditions considered. Electrical impedance measurements confirmed the results and gave a more precise quantification of this dependence. The study highlights the importance that pulse number has in the electrochemotherapy protocols and establishes some limits in the use of this parameter.


Assuntos
Eletroquimioterapia/métodos , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/farmacologia , Bleomicina/administração & dosagem , Bleomicina/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Impedância Elétrica , Modelos Biológicos , Neoplasias/tratamento farmacológico
19.
Stem Cell Res Ther ; 9(1): 203, 2018 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-30053888

RESUMO

BACKGROUND: Differentiation of mesenchymal stem cells to osteoblasts is widely performed in research laboratories. Classical tests to prove this differentiation employ procedures such as cell fixation, cell lysis or cell scraping. Very few studies report gentle dissociation of mesenchymal stem cells undergoing an osteodifferentiation process. Here we used this technique to reveal the presence of several cell layers during osteogenesis and to study their different properties. METHODS: Through the sequential enzymatic detachment of the cells, we confirm the presence of several layers of differentiated cells and we compare them in terms of enzymatic sensitivity for dissociation, expression of cluster of differentiation, cytosolic calcium oscillations and osteogenic potential. Adipogenic and neurogenic differentiations were also performed in order to compare the cell layers. RESULTS: The cells undergoing differentiation formed one layer in the neurogenic differentiation, two layers in the adipogenic differentiation and at least four layers in the osteogenic differentiation. In the latter, the upper layers, maintained by a collagen I extracellular matrix, can be dissociated using collagenase I, while the remaining lowest layer, attached to the bottom of the dish, is sensitive only to trypsin-versene. The action of collagenase I is more efficient before the mineralization of the extracellular matrix. The collagenase-sensitive and trypsin-sensitive layers differ in their cluster of differentiation expression. The dissociation of the cells on day 15 reveals that cells could resume their growth (increase in cell number) and rapidly differentiate again in osteoblasts, in 2 weeks (instead of 4 weeks). Cells from the upper layers displayed a higher mineralization. CONCLUSIONS: MSCs undergoing osteogenic differentiation form several layers with distinct osteogenic properties. This could allow the investigators to use upper layers to rapidly produce differentiated osteoblasts and the lowest layer to continue growth and differentiation until an ulterior dissociation.


Assuntos
Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Diferenciação Celular , Humanos
20.
Biosens Bioelectron ; 117: 207-216, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-29906768

RESUMO

This paper reports the comparative analysis, by means of electric impedance spectroscopy measurements, of three different cell lines subjected to electroporative pulses. The multifrequency information is recorded simultaneously at 21 frequency values in the range between 5 kHz and 1.3 MHz using a multisine based measuring approach. The analysis of the pre-electroporation impedance spectra shows how the system is able to detect differences and similarities between the cell lines under analysis. Particularly, a good agreement is found between the average cell diameter and the characteristic frequency (the frequency corresponding to a maximum in the imaginary part of the impedance). The measurements performed during electroporation at three different electric field intensities show how the impedance spectra changes dynamically between the consecutive pulses of a train of 8,100 µs pulses delivered at 1 Hz repetition rate. There are clear differences between the changes in the impedance measured at low and high frequency. The multifrequency information has been fitted to an electrical equivalent model in order to understand the different contributions in the observed impedance changes (mainly separate between membrane permeabilization and the conductivity changes in the extracellular medium). Finally, a ratio of the low and high frequency impedance information is used to estimate the accumulated impedance decay and to compare it to the internalization of a fluorescent permeabilization reporter. The comparison between both techniques at the three electroporation electric field intensities assayed confirms the ability of impedance measurements to detect in a precise way the level of membrane permeabilization. Additionally, this study demonstrates how the real time information obtained thanks to impedance measurements can provide a more precise quantification of the membrane permeabilization extent.


Assuntos
Permeabilidade da Membrana Celular/fisiologia , Espectroscopia Dielétrica , Fenômenos Eletrofisiológicos , Linhagem Celular , Eletroporação
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