scholarly journals In situ electroporation of mammalian cells through SiO2 thin film capacitive microelectrodes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
M. Maschietto ◽  
M. Dal Maschio ◽  
S. Girardi ◽  
S. Vassanelli
Keyword(s):  
Thin Film ◽  
Mammalian Cells ◽  
Genetic Manipulation ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Sio2 Thin Film ◽  
Low Amplitude ◽  
In Situ Electroporation

AbstractElectroporation is a widely used non-viral technique for the delivery of molecules, including nucleic acids, into cells. Recently, electronic microsystems that miniaturize the electroporation machinery have been developed as a new tool for genetic manipulation of cells in vitro, by integrating metal microelectrodes in the culture substrate and enabling electroporation in-situ. We report that non-faradic SiO2 thin film-insulated microelectrodes can be used for reliable and spatially selective in-situ electroporation of mammalian cells. CHO-K1 and SH-SY5Y cell lines and primary neuronal cultures were electroporated by application of short and low amplitude voltage transients leading to cell electroporation by capacitive currents. We demonstrate reliable delivery of DNA plasmids and exogenous gene expression, accompanied by high spatial selectivity and cell viability, even with differentiated neurons. Finally, we show that SiO2 thin film-insulated microelectrodes support a double and serial transfection of the targeted cells.

scholarly journals Spatio-temporally targeted in-situ electroporation of mammalian cells through SiO2 thin-film capacitive microelectrodes

2020 ◽  
Author(s):  
Marta Maschietto ◽  
Marco Dal Maschio ◽  
Stefano Girardi ◽  
Stefano Vassanelli
Keyword(s):  
Thin Film ◽  
Mammalian Cells ◽  
Genetic Manipulation ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Sio2 Thin Film ◽  
Low Amplitude ◽  
In Situ Electroporation

Abstract Electroporation is a widely used non-viral technique for the delivery of molecules, including nucleic acids, into cells. Recently, electronic microsystems that miniaturize the electroporation machinery have been developed as a new tool for genetic manipulation of cells in vitro, by integrating metal microelectrodes in the culture substrate and enabling electroporation in-situ. We report that non-faradic SiO2 thin film-insulated microelectrodes can be used for reliable and spatially selective in-situ electroporation of mammalian cells. CHO-K1 and SH-SY5Y cell lines and primary neuronal cultures were electroporated by application of short and low amplitude voltage transients leading to cell electroporation by capacitive currents. We demonstrate reliable delivery of DNA plasmids and exogenous gene expression, accompanied by high spatial selectivity and cell viability. Finally, we show that SiO2 thin film-insulated microelectrodes support a double and serial transfection of the targeted cells.

Differential addressing of 5-HT1A and 5-HT1B receptors in epithelial cells and neurons

1999 ◽  
Vol 112 (6) ◽  
pp. 967-976
Author(s):  
A. Ghavami ◽  
K.L. Stark ◽  
M. Jareb ◽  
S. Ramboz ◽  
L. Segu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Neuronal Cultures ◽  
Striatal Neurons ◽  
Primary Neuronal Cultures ◽  
Axon Terminals ◽  
In Vivo Experiments ◽  
In Vitro Systems ◽  
The Central Nervous System

The 5-HT1A and 5-HT1B serotonin receptors are expressed in a variety of neurons in the central nervous system. While the 5-HT1A receptor is found on somas and dendrites, the 5-HT1B receptor has been suggested to be localized predominantly on axon terminals. To study the intracellular addressing of these receptors, we have used in vitro systems including Madin-Darby canine kidney (MDCK II) epithelial cells and primary neuronal cultures. Furthermore, we have extended these studies to examine addressing in vivo in transgenic mice. In epithelial cells, 5-HT1A receptors are found on both apical and basolateral membranes while 5-HT1B receptors are found exclusively in intracellular vesicles. In hippocampal neuronal cultures, 5-HT1A receptors are expressed on somatodendritic membranes but are absent from axons. In contrast, 5-HT1B receptors are found on both dendritic and axonal membranes, including growth cones where they accumulate. Using 5-HT1A and 5-HT1B knockout mice and the binary tTA/tetO system, we generated mice expressing these receptors in striatal neurons. These in vivo experiments demonstrate that, in striatal medium spiny neurons, the 5-HT1A receptor is restricted to the somatodendritic level, while 5-HT1B receptors are shipped exclusively toward axon terminals. Therefore, in all systems we have examined, there is a differential sorting of the 5-HT1A and 5-HT1B receptors. Furthermore, we conclude that our in vivo transgenic system is the only model that reconstitutes proper sorting of these receptors.

scholarly journals A Method for Combining RNAscope In Situ Hybridization with Immunohistochemistry in Thick Free-Floating Brain Sections and Primary Neuronal Cultures

PLoS ONE ◽  
2015 ◽  
Vol 10 (3) ◽  
pp. e0120120 ◽  
Author(s):  
Tessa M. Grabinski ◽  
Andrew Kneynsberg ◽  
Fredric P. Manfredsson ◽  
Nicholas M. Kanaan
Keyword(s):  
In Situ Hybridization ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures

Protein self-assembly following in situ expression in artificial and mammalian cells

2017 ◽  
Vol 9 (5) ◽  
pp. 444-450 ◽  
Author(s):  
Urszula M. Migas ◽  
Michelle K. Quinn ◽  
Jennifer J. McManus
Keyword(s):  
Self Assembly ◽  
Mammalian Cells

The importance of in vitro measurements in explaining the mechanisms underlying protein self-assembly in physiologically relevant conditions has been demonstrated in solution and in artificial and mammalian cells.

scholarly journals Quantifying Amyloid Beta (Aβ)–Mediated Changes in Neuronal Morphology in Primary Cultures

2012 ◽  
Vol 17 (6) ◽  
pp. 835-842 ◽  
Author(s):  
Lan Nguyen ◽  
Sarah Wright ◽  
Mike Lee ◽  
Zhao Ren ◽  
John-Michael Sauer ◽  
...  
Keyword(s):  
Amyloid Beta ◽  
Cell Injury ◽  
Primary Cultures ◽  
Neuronal Morphology ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Blocking Antibody ◽  
Cortical Cultures ◽  
Hippocampal Cultures

Alzheimer’s disease (AD) is a devastating neurodegenerative disease affecting millions of people. The amyloid hypothesis suggests that the pathogenesis of AD is related to the accumulation of amyloid beta (Aβ) in the brain. Herein, the authors quantify Aβ-mediated changes in neuronal morphology in primary cultures using the Cellomics neuronal profiling version 3.5 (NPv3.5) BioApplication. We observed that Aβ caused a 33% decrease in neurite length in primary human cortical cultures after 24 h of treatment compared with control-treated cultures. We also determined that quantifying changes of neuronal morphology was a more sensitive indicator of nonlethal cell injury than traditional cytotoxicity assays. Aβ-mediated neuronal deficits observed in human cortical cultures were also observed in primary rat hippocampal cultures, where we demonstrated that the integrin-blocking antibody, 17E6, completely abrogated Aβ-mediated cytotoxicity. Finally, we showed that Aβ challenge to 21 days in vitro rat hippocampal cultures reduced synapsin staining to 14% of control-treated cultures. These results are consistent with the finding that loss of presynaptic integrity is one of the initial deficits observed in AD. The implementation of phenotypic screens to identify compounds that block Aβ-mediated cytotoxicity in primary neuronal cultures may lead to the development of novel strategies to prevent AD.

scholarly journals Non-proliferative neurogenesis in human periodontal ligament stem cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Carlos Bueno ◽  
Marta Martínez-Morga ◽  
Salvador Martínez
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Periodontal Ligament ◽  
Cell Biology ◽  
Basic Knowledge ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Periodontal Ligament Stem Cells ◽  
Sequence Of Events

AbstractUnderstanding the sequence of events from undifferentiated stem cells to neuron is not only important for the basic knowledge of stem cell biology, but also for therapeutic applications. In this study we examined the sequence of biological events during neural differentiation of human periodontal ligament stem cells (hPDLSCs). Here, we show that hPDLSCs-derived neural-like cells display a sequence of morphologic development highly similar to those reported before in primary neuronal cultures derived from rodent brains. We observed that cell proliferation is not present through neurogenesis from hPDLSCs. Futhermore, we may have discovered micronuclei movement and transient cell nuclei lobulation coincident to in vitro neurogenesis. Morphological analysis also reveals that neurogenic niches in the adult mouse brain contain cells with nuclear shapes highly similar to those observed during in vitro neurogenesis from hPDLSCs. Our results provide additional evidence that it is possible to differentiate hPDLSCs to neuron-like cells and suggest the possibility that the sequence of events from stem cell to neuron does not necessarily requires cell division from stem cell.

scholarly journals Tetracycline-regulated expression enables purification and functional analysis of recombinant connexin channels from mammalian cells

2004 ◽  
Vol 383 (1) ◽  
pp. 111-119 ◽  
Author(s):  
Irina V. KOREEN ◽  
Wafaa A. ELSAYED ◽  
Yu J. LIU ◽  
Andrew L. HARRIS
Keyword(s):  
Mammalian Cells ◽  
Cultured Cells ◽  
Expression System ◽  
Physiological Processes ◽  
Messenger Molecules ◽  
One Step ◽  
Cellular Control

Intercellular coupling mediated by gap junction channels composed of connexin protein underlies numerous physiological processes, such as cellular differentiation, tissue synchronization and metabolic homoeostasis. The distinct molecular permeability of junctional channels composed of different connexin isoforms allows cellular control of coupling via regulation of isoform expression. However, the permeability properties of most connexin isoforms have not been well characterized due to the difficulty of manipulating and measuring the diffusible concentrations of cytoplasmic messenger molecules and metabolites, and to a lack of control over channel isoform composition, in vivo. Here we present a method to express and purify active connexin hemichannels of a single isoform or a consistent ratio of two isoforms from cultured cells using the Tet-On inducible expression system and one-step anti-haemagglutinin immunoaffinity purification. The procedure yields 10–20 μg of pure connexin protein from 2.5×108 HeLa cells. The purified channels are shown to be useful for in vitro permeability analysis using well established techniques. This method has substantial advantages over existing methods for heterologous connexin expression, such as the ease of co-expression of two isoforms at a constant ratio, consistently high expression levels over many passages, and the ability to study channel properties in situ as well as in purified form. Furthermore, the generic cloning site of the new pBI-GT vector and the commercial availability of anti-haemagglutinin (clone HA-7)–agarose make this affinity tagging and purification procedure easily applicable to other proteins.

In situ study of the formation of NiSi2 nanoplates and Ni nanocrystals embedded in a Si(001) wafer and in a deposited Ni-doped SiO2 thin film

2021 ◽  
pp. 160345
Author(s):  
Daniel da Silva Costa ◽  
Guinther Kellermann ◽  
Aldo F. Craievich ◽  
Lisandro J. Giovanetti ◽  
Cristián Huck-Iriart ◽  
...  
Keyword(s):  
Thin Film ◽  
In Situ Study ◽  
Sio2 Thin Film

scholarly journals Control of Cardiac Function in vivo with a Light-Regulated Drug

2018 ◽  
Author(s):  
Fabio Riefolo ◽  
Carlo Matera ◽  
Aida Garrido-Charles ◽  
Alexandre M. J. Gomila ◽  
Luca Agnetta ◽  
...  
Keyword(s):  
Cardiac Function ◽  
Mammalian Cells ◽  
Acetylcholine Receptors ◽  
Genetic Manipulation ◽  
Muscarinic Acetylcholine Receptors ◽  
Therapeutic Interventions ◽  
Muscarinic Acetylcholine ◽  
First Time

<p>Remote control of physiological functions with light offers the promise of unveiling their complex spatiotemporal dynamics in vivo, and enabling highly focalized therapeutic interventions with reduced systemic toxicity. Optogenetic methods have been implemented in the heart, but the need of genetic manipulation jeopardizes clinical applicability. This study aims at developing, testing and validating the first light-regulated drug with cardiac effects, in order to avoid the requirement of genetic manipulation offered by optogenetic methods. A M2 muscarinic acetylcholine receptors (mAChRs) light-regulated drug (PAI) was designed, synthesized and pharmacologically characterized. The design was based on the orthosteric mAChRs agonist Iperoxo, an allosteric M2 ligand, and a photoswitchable azobenzene linker. PAI can be reversibly photoisomerized between <i>cis</i> and <i>trans</i> configurations under ultraviolet (UV) and visible light, respectively, and it reversibly photoswitches the activity of M2 muscarinic acetylcholine receptors. We have evaluated <i>in vitro</i> photoresponses using a calcium imaging assay in genetically unmodified receptors overexpressed in mammalian cells. Furthermore, using this new chemical tool, we demonstrate for the first time photoregulation of cardiac function <i>in vivo</i> in wildtype frog tadpoles and in rats with a method that does not require genetic manipulation. Such a new approach may enable enhanced spatial and temporal selectivity for cardiovascular drugs.</p>

scholarly journals Isoflurane but not Fentanyl Causes Apoptosis in Immature Primary Neuronal Cells

2017 ◽  
Vol 11 (1) ◽  
pp. 39-47
Author(s):  
Monika Berns ◽  
Anna Christine Wolter ◽  
Christoph Bührer ◽  
Stefanie Endesfelder ◽  
Thoralf Kerner
Keyword(s):  
Cell Viability ◽  
Neuronal Cells ◽  
Neuronal Cultures ◽  
Primary Neurons ◽  
Primary Neuronal Cultures ◽  
Amino Butyric Acid ◽  
Wide Range ◽  
Primary Neuronal Cells ◽  
The Impact

Background: Anaesthetics are widely used in new-borns and preterm infants, although it is known that they may adversely affect the developing brain. Objective: We assessed the impact of the volatile anaesthetic, isoflurane, and the intravenous analgesic, fentanyl, on immature and mature embryonic neuronal cells. Methods: Primary neuronal cultures from embryonic rats (E18) cultured for 5 (immature) or 15 days (mature) in vitro (DIV), respectively, were exposed to isoflurane (1.5 Vol.%) or fentanyl (0.8 - 200 ng/ml) for 24 hours. Experiments were repeated in the presence of the γ-amino butyric acid-A (GABAA) receptor antagonists, bicuculline or picrotoxin (0.1 mmol/l), or the pancaspase inhibitor zVAD-fmk (20 nmol/l). Cell viability was assessed by methyltetrazolium (MTT) metabolism or lactate dehydrogenase (LDH) release. Results: Isoflurane reduced cell viability significantly in primary neuronal cells cultured for 5 DIV (Δ MTT -28 ±13%, Δ LDH +143 ±15%). Incubation with bicuculline, picrotoxin or zVAD-fmk protected the cells mostly from isoflurane toxicity. After 15 DIV, cell viability was not reduced by isoflurane. Viability of primary neurons cultured for 5 DIV did not change with fentanyl over the wide range of concentrations tested. Conclusion: Immature primary neurons may undergo apoptosis following exposure to isoflurane but are unaffected by fentanyl. Mature primary neurons were not affected by isoflurane exposure.

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