scholarly journals Growing neuronal islands on multi-electrode arrays using an Accurate Positioning-μCP device

2015 ◽  
Author(s):  
Robert Samhaber ◽  
Manuel Schottdorf ◽  
Ahmed El Hady ◽  
Kai Broeking ◽  
Andreas Daus ◽  
...  
Keyword(s):  
Cell Culture ◽  
Neuronal Networks ◽  
Standard Cell ◽  
Electrode Arrays ◽  
Unit Recording ◽  
Contact Printing ◽  
Novel Approach ◽  
Custom Made ◽  
Typical Cell

Multi-electrode arrays (MEAs) allow non-invasive multi-unit recording in-vitro from cultured neuronal networks. For sufficient neuronal growth and adhesion on such MEAs, substrate preparation is required. Plating of dissociated neurons on a uniformly prepared MEA's surface results in the formation of spatially extended random networks with substantial inter-sample variability. Such cultures are not optimally suited to study the relationship between defined structure and dynamics in neuronal networks. To overcome these shortcomings, neurons can be cultured with pre-defined topology by spatially structured surface modification. Spatially structuring a MEA surface accurately and reproducibly with the equipment of a typical cell-culture laboratory is challenging. In this paper, we present a novel approach utilizing micro-contact printing (μCP) combined with a custom-made device to accurately position patterns on MEAs with high precision. We call this technique AP-μCP (accurate positioning micro-contact printing). Other approaches presented in the literature using μCP for patterning either relied on facilities or techniques not readily available in a standard cell culture laboratory, or they did not specify means of precise pattern positioning. Here we present a relatively simple device for reproducible and precise patterning in a standard cell-culture laboratory setting. The patterned neuronal islands on MEAs provide a basis for high throughput electrophysiology to study the dynamics of single neurons and neuronal networks.

LTCC Based Multi-Electrode Arrays for In-Vitro Cell Culture

2015 ◽  
Vol 2015 (CICMT) ◽  
pp. 000269-000274
Author(s):  
Heike Bartsch ◽  
Dirk Stöpel ◽  
Marcel Himmerlich ◽  
Martin Baca ◽  
Philipp Stadie ◽  
...  
Keyword(s):  
Cell Culture ◽  
Laser Scanning ◽  
Neuronal Cell ◽  
Laser Scanning Microscopy ◽  
Gold Electrodes ◽  
Neural Cells ◽  
Electrode Arrays ◽  
Green Tape ◽  
High Level

Neurobiological concepts based on state-of-the art technology have so far lacked the complexity of actual high-level neurobiological systems. Two key advances are needed to improve our understanding of such systems: in vitro 3D-neuronal cell culture and 3D MEA systems for measuring such 3D-cultures. These requirements call for smart multilayer and packaging technology. The material Green Tape TM from DuPont Nemours is chosen for the presented works, because its compatibility and those of available metallisation with cell cultures is already proven. An LTCC multilayer circuit with gold electrodes is the base of the 3D MEA. The layout of the 3D MEA is designed to fit the MEA2100-System for in vitro recording from Multi Channel Systems and enable thus a comparable data processing to established 2D MEAs Slots. The surface topography of the thick film electrodes and the surface state is investigated with laser scanning microscopy, SEM, XPS and measurements of the wetting angle of contact. The impedance of the screen printed electrodes is discussed taking these data into account. Their impedance amounts to 24 kΩ and are falls thus below the impedance of commercially available electroplated gold electrodes of 30 kΩ. First promising results have been achieved using 3D MEAs for 2D culture of human pluripotent stem cell derived neural cells.

scholarly journals L-type voltage-gated calcium channel regulation of in vitro human cortical neuronal networks

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
William Plumbly ◽  
Nick Brandon ◽  
Tarek Z. Deeb ◽  
Jeremy Hall ◽  
Adrian J. Harwood
Keyword(s):  
Calcium Channel ◽  
Neuronal Networks ◽  
Gene Mutations ◽  
Synaptic Function ◽  
Neuronal Firing ◽  
Network Activity ◽  
Electrode Arrays ◽  
Voltage Gated ◽  
Voltage Gated Calcium Channel

Abstract The combination of in vitro multi-electrode arrays (MEAs) and the neuronal differentiation of stem cells offers the capability to study human neuronal networks from patient or engineered human cell lines. Here, we use MEA-based assays to probe synaptic function and network interactions of hiPSC-derived neurons. Neuronal network behaviour first emerges at approximately 30 days of culture and is driven by glutamate neurotransmission. Over a further 30 days, inhibitory GABAergic signalling shapes network behaviour into a synchronous regular pattern of burst firing activity and low activity periods. Gene mutations in L-type voltage gated calcium channel subunit genes are strongly implicated as genetic risk factors for the development of schizophrenia and bipolar disorder. We find that, although basal neuronal firing rate is unaffected, there is a dose-dependent effect of L-type voltage gated calcium channel inhibitors on synchronous firing patterns of our hiPSC-derived neural networks. This demonstrates that MEA assays have sufficient sensitivity to detect changes in patterns of neuronal interaction that may arise from hypo-function of psychiatric risk genes. Our study highlights the utility of in vitro MEA based platforms for the study of hiPSC neural network activity and their potential use in novel compound screening.

scholarly journals Organoids: An Emerging Tool To Study Aging Signature Across Human Tissues. Modeling Aging With Patient-derived Organoids

2021 ◽  
Author(s):  
Margalida Torrens-Mas ◽  
Catalina Perello-Reus ◽  
Cayetano Navas-Enamorado ◽  
Lesly Ibargüen ◽  
Andres Sanchez -Polo ◽  
...  
Keyword(s):  
Cell Culture ◽  
3D Cell Culture ◽  
Adverse Outcomes ◽  
Biological Aging ◽  
Preclinical Model ◽  
Biomarkers Of Aging ◽  
Novel Approach ◽  
New Biomarkers ◽  
Biology Of Aging

The biology of aging is focused on the identification of novel pathways that regulate the underlying processes of aging to develop interventions aimed at delaying the onset and progression of chronic diseases to extend lifespan. However, the research on the aging field has been conducted mainly in animal models, yeast, Caenorhabditis elegans and cell culture. Thus, it is unclear to what extent this knowledge is transferable to humans since they might not reflect the complexity of aging in people. Organoid culture is an in vitro 3D cell-culture technology that reproduces the physiological and cellular composition of the tissues and/or organs. This technology is being used in the cancer field to predict the response of a patient-derived tumor to a certain drug or treatment serving as patient stratification and drug-guidance approaches. Modeling aging with patient-derived organoids has a tremendous potential as a preclinical model tool to discover new biomarkers of aging, to predict adverse outcomes during aging and to design personalized approaches for prevention and treatment of aging-related diseases and geriatric syndromes. This could represent a novel approach to study chronological and/or biological aging paving the way to personalized interventions targeting the biology of aging.

scholarly journals L-type voltage-gated calcium channel regulation of in vitro human cortical neuronal networks

2018 ◽  
Author(s):  
William Plumbly ◽  
Nicholas J. Brandon ◽  
Tarek Z. Deeb ◽  
Jeremy Hall ◽  
Adrian J. Harwood
Keyword(s):  
Calcium Channel ◽  
Neuronal Networks ◽  
Gene Mutations ◽  
Synaptic Function ◽  
Neuronal Firing ◽  
Network Activity ◽  
Electrode Arrays ◽  
Voltage Gated ◽  
Voltage Gated Calcium Channel

The combination of in vitro multi-electrode arrays (MEAs) and the neuronal differentiation of stem cells offers the capability to study human neuronal networks from patient or engineered human cell lines. Here, we use MEA-based assays to probe synaptic function and network interactions of hiPSC-derived neurons. Neuronal network behaviour first emerges at approximately 30 days of culture and is driven by glutamate neurotransmission. Over a further 30 days, inhibitory GABergic signalling shapes network behaviour into a synchronous regular pattern of burst firing activity and low activity periods. Gene mutations in L-type voltage gated calcium channel subunit genes are strongly implicated as genetic risk factors for the development of schizophrenia and bipolar disorder. We find that, although basal neuronal firing rate is unaffected, there is a dose-dependent effect of L-type voltage gated calcium channel inhibitors on synchronous firing patterns of our hiPSC-derived neural networks. This demonstrates that MEA assays have sufficient sensitivity to detect changes in patterns of neuronal interaction that may arise from hypo-function of psychiatric risk genes. Our study highlights the utility of in vitro MEA based platforms for the study of hiPSC neural network activity and their potential use in novel compound screening.

52 USE OF TRANSWELL CELL CULTURE AND 3-DIMENSIONAL PRINTING TECHNOLOGY TO DEVELOP AN IN VITRO BOVINE OVIDUCT

2016 ◽  
Vol 28 (2) ◽  
pp. 156 ◽  
Author(s):  
M. A. M. M. Ferraz ◽  
H. H. W. Henning ◽  
K. M. A. Van Dorenmalen ◽  
P. L. A. M. Vos ◽  
T. A. E. Stout ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Growth ◽  
Liquid Culture ◽  
Primary Cilia ◽  
Negative Impact ◽  
Perfusion Culture ◽  
Vitro Fertilization ◽  
Custom Made

Oviduct epithelial cells (OECs) generate the microenvironment for mammalian fertilization. When cultured in vitro OECs rapidly lose their differentiated cell properties (e.g. secretory activity and cilia), while suspended cells have a limited lifespan. These limitations, likely due to the lack of folded tubular geometry of the oviduct, prompted us to combine transwell cell culture and 3-D printing technologies to mimic the in vivo OEC niche in order to better study the unique role of the oviduct and its microenvironment during the processes of fertilization and early embryonic development. U-shape inserts were 3-D printed using a multi-arm acrylate-based resin (PIC100) on an Envisiontec Perfactory P3 stereolithographer. Post-printing treatments of custom-made tubular transwell inserts were first tested in order to determine any possible negative impact of the plastics on cell growth. Inserts were either untreated, post-cured with 1000 flashes/side (Otoflash, 66 W), post-cured and Soxhlet-extracted overnight in isopropanol, or post-cured and Soxhlet-extracted over the weekend in water at 37°C. The post-cured and Soxhlet-extracted overnight in isopropanol inserts were selected as best pretreatment for culturing OECs. These inserts were mounted with track-etched PET membranes (12 µm thick, 0.4 µm pore diameter) to create a U-shape geometry that allows perfusion. Bovine OECs were obtained by squeezing the whole oviduct collected from slaughterhouse cows (on luteal phase) and cultured as monolayers for 7 days (n = 2 cows). These de-differentiated OECs were seeded on the membranes, grown to confluence (7 days), and cultured (1) at an air-liquid interface for 6 and 14 days (air-liquid culture) or (2) under perfusion (6 mL h–1) for 6 days (perfusion culture). OECs were also cultured on coverslips as monolayers (2-D culture) for 6 and 14 days. After this period, the OECs were fixed and immune labelled to determine their polarized state. Polarization of OECs (laminin and primary cilia detection) was observed on Day 6 for perfusion culture, on Day 14 for air-liquid culture, and was not detected in 2-D culture. The presence of secondary cilia (acetylated α-tubulin) was observed in 6% of the cells cultured under perfusion at Day 6; secondary cilia was not present in air-liquid or 2-D cultures during the period analysed. In conclusion, post-curing and Soxhlet extraction of leachable compounds is crucial to avoid toxic effects on cell growth. The U-shape custom-designed inserts are able to create a tube-like surface in which bovine oviducal cells can be cultured to confluency and thereafter repolarize (presence of primary cilia and detection of laminin); this polarization occurs faster when the U-shape culture is under perfusion. Further studies will examine the ability of the cells to differentiate further (development of secondary cilia and secretory ability) and support in vitro fertilization. To this end, 3-D designs will be tested to determine their use for live cell imaging and for collecting secreted fluids.

scholarly journals A Simple Aspect Ratio Dependent Method of Patterning Microwells for Selective Cell Attachment

2018 ◽  
Author(s):  
Erik A. Zavrel ◽  
Michael L. Shuler ◽  
Xiling Shen
Keyword(s):  
Cell Culture ◽  
Cell Attachment ◽  
In Vitro Models ◽  
Contact Printing ◽  
Deep Well ◽  
Biomimetic Approach ◽  
Authentic Environment ◽  
And Control

3-D culture has been shown to provide cells with a more physiologically authentic environment than traditional 2-D (planar) culture [1, 2]. 3-D cues allow cells to exhibit more realistic functions and behaviors, e.g., adhesion, spreading, migration, metabolic activity, and differentiation. Knowledge of changes in cell morphology, mechanics, and mobility in response to geometrical cues and topological stimuli is important for understanding normal and pathological cell development [3]. Microfabrication provides unique in vitro approaches to recapitulating in vivo conditions due to the ability to precisely control the cellular microenvironment [4, 5]. Microwell arrays have emerged as robust alternatives to traditional 2D cell culture substrates as they are relatively simple and compatible with existing laboratory techniques and instrumentation [6, 7]. In particular, microwells have been adopted as a biomimetic approach to modeling the unique micro-architecture of the epithelial lining of the gastrointestinal (GI) tract [8–10]. The inner (lumen-facing) surface of the intestine has a convoluted topography consisting of finger-like projections (villi) with deep well-like invaginations (crypts) between them. The dimensions of villi and crypts are on the order of hundreds of microns (100–700 μm in height and 50–250 μm in diameter) [11]. While microwells have proven important in the development of physiologically realistic in vitro models of human intestine, existing methods of ensuring their surface is suitable for cell culture are lacking. Sometimes it is desirable to selectively seed cells within microwells and confine or restrict them to the microwells in which they are seeded. Existing methods of patterning microwells for cell attachment either lack selectivity, meaning cells can adhere and migrate anywhere on the microwell array, i.e., inside microwells or outside of them, or necessitate sophisticated techniques such as micro-contact printing, which requires precise alignment and control to selectively pattern the bottoms of microwells for cell attachment [12, 13].

Explorative Data Analysis of In-Vitro Neuronal Network Behavior Based on an Unsupervised Learning Approach

2010 ◽  
pp. 241-253
Author(s):  
A. Maffezzoli ◽  
E. Wanke
Keyword(s):  
Neuronal Networks ◽  
Analytical Techniques ◽  
Learning Approach ◽  
Electrode Arrays ◽  
Activity Data ◽  
New Information ◽  
Data Dimensionality Reduction ◽  
Long Time ◽  
Pros And Cons

In the present chapter authors want to expose new insights in the field of Computational Neuroscience at regard to the study of neuronal networks grown in vitro. Such kind of analyses can exploit the availability of a huge amount of data thanks to the use of Multi Electrode Arrays (MEA), a multi-channel technology which allows capturing the activity of several different neuronal cells for long time recordings. Given the possibility of simultaneous targeting of various sites, neuroscientists are so applying such recent technology for various researches. The chapter begins by giving a brief presentation of MEA technology and of the data produced in output, punctuating some of the pros and cons of MEA recordings. Then we present an overview of the analytical techniques applied in order to extrapolate the hidden information from available data. Then we shall explain the approach we developed and applied on MEAs prepared in our cell culture laboratory, consisting of statistical methods capturing the main features of the spiking, in particular bursting, activity of various neuron, and performing data dimensionality reduction and clustering, in order to classify neurons according to their spiking properties having showed correlated features. Finally the chapter wants to furnish to neuroscientists an overview about the quantitative analysis of in-vitro spiking activity data recorded via MEA technology and to give an example of explorative analysis applied on MEA data. Such study is based on methods from Statistics and Machine Learning or Computer Science but at the same time strictly related to neurophysiological interpretations of the putative pharmacological manipulation of synaptic connections and mode of firing, with the final aim to extract new information and knowledge about neuronal networks behavior and organization.

Synaptic Connectivity in Cultured Hypothalamic Neuronal Networks

1997 ◽  
Vol 77 (6) ◽  
pp. 3218-3225 ◽  
Author(s):  
Thomas H. Müller ◽  
D. Swandulla ◽  
H. U. Zeilhofer
Keyword(s):  
Neuronal Networks ◽  
Network Formation ◽  
Inhibitory Neurons ◽  
Synaptic Connectivity ◽  
Synaptic Connections ◽  
Cellular Mechanisms ◽  
Patch Clamp Technique ◽  
Novel Approach

Müller, Thomas H., D. Swandulla, and H. U. Zeilhofer. Synaptic connectivity in cultured hypothalamic neuronal networks. J. Neurophysiol. 77: 3218–3225, 1997. We have developed a novel approach to analyze the synaptic connectivity of spontaneously active networks of hypothalamic neurons in culture. Synaptic connections were identified by recording simultaneously from pairs of neurons using the whole cell configuration of the patch-clamp technique and testing for evoked postsynaptic current responses to electrical stimulation of one of the neurons. Excitatory and inhibitory responses were distinguished on the basis of their voltage and time dependence. The distribution of latencies between presynaptic stimulation and postsynaptic response showed multiple peaks at regular intervals, suggesting that responses via both monosynaptic and polysynaptic paths were recorded. The probability that an excitatory event is transmitted to another excitatory neuron and results in an above-threshold stimulation was found to be only one in three to four. This low value indicates that in addition to evoked synaptic responses other sources of excitatory drive must contribute to the spontaneous activity observed in these networks. The various types of synaptic connections (excitatory and inhibitory, monosynaptic, and polysynaptic) were counted, and the observations analyzed using a probabilistic model of the network structure. This analysis provides estimates for the ratio of inhibitory to excitatory neurons in the network (1:1.5) and for the ratio of postsynaptic cells receiving input from a single GABAergic or glutamatergic neuron (3:1). The total number of inhibitory synaptic connections was twice that of excitatory connections. Cell pairs mutually connected by an excitatory and an inhibitory synapse occurred significantly more often than predicted by a random process. These results suggests that the formation of neuronal networks in vitro is controlled by cellular mechanisms that favor inhibitory connections in general and specifically enhance the formation of reciprocal connections between pairs of excitatory and inhibitory neurons. These mechanisms may contribute to network formation and function in vivo.

scholarly journals Attenuation of Bluetongue Virus (BTV) in an in ovo Model Is Related to the Changes of Viral Genetic Diversity of Cell-Culture Passaged BTV

Viruses ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 481 ◽  
Author(s):  
Fabian Z. X. Lean ◽  
Matthew J. Neave ◽  
John R. White ◽  
Jean Payne ◽  
Teresa Eastwood ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Cell Culture ◽  
Bluetongue Virus ◽  
Genomic Analysis ◽  
Sequencing Analysis ◽  
Structural Protein ◽  
In Ovo ◽  
Novel Approach ◽  
Amino Acid Mutations

The embryonated chicken egg (ECE) is routinely used for the laboratory isolation and adaptation of Bluetongue virus (BTV) in vitro. However, its utility as an alternate animal model has not been fully explored. In this paper, we evaluated the pathogenesis of BTV in ovo using a pathogenic isolate of South African BTV serotype 3 (BTV-3) derived from the blood of an infected sheep. Endothelio- and neurotropism of BTV-3 were observed by immunohistochemistry of non-structural protein 1 (NS1), NS3, NS3/3a, and viral protein 7 (VP7) antigens. In comparing the pathogenicity of BTV from infectious sheep blood with cell-culture-passaged BTV, including virus propagated through a Culicoides-derived cell line (KC) or ECE, we found virus attenuation in ECE following cell-culture passage. Genomic analysis of the consensus sequences of segments (Seg)-2, -5, -6, -7, -8, -9, and -10 identified several nucleotide and amino-acid mutations among the cell-culture-propagated BTV-3. Deep sequencing analysis revealed changes in BTV-3 genetic diversity in various genome segments, notably a reduction of Seg-7 diversity following passage in cell culture. Using this novel approach to investigate BTV pathogenicity in ovo, our findings support the notion that pathogenic BTV becomes attenuated in cell culture and that this change is associated with virus quasispecies evolution.

scholarly journals Next-Generation Diamond Electrodes for Neurochemical Sensing: Challenges and Opportunities

Micromachines ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 128
Author(s):  
Erin Purcell ◽  
Michael Becker ◽  
Yue Guo ◽  
Seth Hara ◽  
Kip Ludwig ◽  
...  
Keyword(s):  
Surface Characteristics ◽  
Electrode Arrays ◽  
Diamond Electrodes ◽  
Boron Doped Diamond ◽  
Spatiotemporal Resolution ◽  
Bonding Structure ◽  
Boron Doped ◽  
Novel Approach

Carbon-based electrodes combined with fast-scan cyclic voltammetry (FSCV) enable neurochemical sensing with high spatiotemporal resolution and sensitivity. While their attractive electrochemical and conductive properties have established a long history of use in the detection of neurotransmitters both in vitro and in vivo, carbon fiber microelectrodes (CFMEs) also have limitations in their fabrication, flexibility, and chronic stability. Diamond is a form of carbon with a more rigid bonding structure (sp3-hybridized) which can become conductive when boron-doped. Boron-doped diamond (BDD) is characterized by an extremely wide potential window, low background current, and good biocompatibility. Additionally, methods for processing and patterning diamond allow for high-throughput batch fabrication and customization of electrode arrays with unique architectures. While tradeoffs in sensitivity can undermine the advantages of BDD as a neurochemical sensor, there are numerous untapped opportunities to further improve performance, including anodic pretreatment, or optimization of the FSCV waveform, instrumentation, sp2/sp3 character, doping, surface characteristics, and signal processing. Here, we review the state-of-the-art in diamond electrodes for neurochemical sensing and discuss potential opportunities for future advancements of the technology. We highlight our team’s progress with the development of an all-diamond fiber ultramicroelectrode as a novel approach to advance the performance and applications of diamond-based neurochemical sensors.

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