Cultured neuronal network
From Wikipedia, the free encyclopedia
A cultured neuronal network is a cell culture of neurons that is used as a model to study the central nervous system, especially the brain. Often, cultured neuronal networks are connected to an input/output device such as a multi-electrode array (MEA), thus allowing two-way communication between the researcher and the network. This model has proved to be an invaluable tool to scientists studying the underlying principles behind neuronal learning, memory, plasticity, connectivity, and information processing.[1]
Cultured neurons are often connected via computer to a real or simulated robotic component, creating a hybrot or animat, respectively. Researchers can then thoroughly study learning and plasticity in a realistic context, where the neuronal networks are able to interact with their environment and receive at least some artificial sensory feedback. One example of this can be seen in the Multielectrode Array Art (MEART) system developed by the Potter Research Group at the Georgia Institute of Technology in collaboration with SymbioticA, The Centre for Excellence in Biological Art, at the University of Western Australia.[2] Another example can be seen in the neurally controlled animat.
Advantages
The use of cultured neuronal networks as a model for their in vivo counterparts has been an indispensable resource for decades.[4] It allows researchers to investigate neuronal activity in a much more controlled environment than would be possible in a live organism. Through this mechanism researchers have gleaned important information about the mechanisms behind learning and memory.
A cultured neuronal network allows researchers to observe neuronal activity from several vantage points. Electrophysiological recording and stimulation can take place either across the network or locally via an MEA, and the network development can be visually observed using microscopy techniques.[4] Moreover, chemical analysis of the neurons and their environment is more easily accomplished than in an in vivo setting.[4][5]
Disadvantages
Cultured neuronal networks are by definition disembodied cultures of neurons. Thus by being outside their natural environment, the neurons are influenced in ways that are not biologically normal. Foremost among these abnormalities is the fact that the neurons are usually harvested as neural stem cells from a fetus and are therefore disrupted at a critical stage in network development.[6] When the neurons are suspended in solution and subsequently dispensed, the connections previously made are destroyed and new ones formed. Ultimately, the connectivity (and consequently the functionality) of the tissue is changed from what the original template suggested.
Another disadvantage lies in the fact that the cultured neurons lack a body and are thus severed from sensory input as well as the ability to express behavior – a crucial characteristic in learning and memory experiments. It is believed that such sensory deprivation has adverse effects on the development of these cultures and may result in abnormal patterns of behavior throughout the network.[6]
Cultured networks on traditional MEAs are flat, single-layer sheets of cells with connectivity only two dimensions. Most in vivo neuronal systems, to the contrary, are large three-dimensional structures with much greater interconnectivity. This remains one of the most striking differences between the model and the reality, and this fact probably plays a large role in skewing some of the conclusions derived from experiments based on this model.
more on wikipedia
In vivo neural electrical activity is the essence of nervous system function, controlling sensory modalities, emotion, memory, behavior, and basic survival functions. Therefore, to study neurons in the laboratory it is important that in vitro neuronal models also support such electrical activity to reflect fundamental brain functions – and most human neuronal cultures are currently grown in vitro using the classic culture media DMEM (Dulbecco's Modified Eagle Medium), Neurobasal, or a mixture of the two. In contrast, laboratory experiments employing electrophysiological techniques – such as patch clamping (which allows the study of single or multiple ion channels in cells), calcium imaging – on brain slices or in culture are performed in a medium of artificial cerebrospinal fluid (aCSF).
Recently, scientists at the Salk Institute for Biological Studies, Sanford Consortium for Regenerative Medicine, La Jolla, CA, employing induced pluripotent stem cells (iPSCs) to model human neurological diseases in vitro used electrophysiology techniques to test DMEM and Neurobasal to determine their influence on fundamental neuronal activity. Surprisingly, the scientists discovered that, even though these are classic culture media, they strongly altered many crucial neurophysiological properties. Before deciding to design a new medium, the researchers tested all commercially available basal media that might be used for neuronal tissue culture; none of them supported electrophysiological activity as well as aCSF. However, even with the addition of various supplements, aCSF was not sufficient to maintain cell cultures for more than a day or two. The scientists therefore embarked on the challenging process of designing a new medium more adapted to supporting neuronal function, eventually producing BrainPhys, a novel medium that improves the differentiation and electrophysiological activity of neurons, supports long-term in vitro culture, mimics physiological conditions of the living brain, and allows for assessment of electrophysiological activity.
Read more at: https://medicalxpress.com/news/2015-05-brain-bottle-culture-medium-neuronal.html#
ALSO: http://journal.frontiersin.org/article/10.3389/fnins.2011.00046/full
Micro-Electrode Array Preparation
Micro-Electrode array were purchased from (MultiChannel Systems, Reutlingen, Germany). The MEA’s were sterilized according to the manufacturer‘s instructions. MEAs were washed with 70% EtOH, air dried on a sterile Petri dish, with UV on, in a laminar flow hood. MEA were then coated with 1 mg/ml poly D lysine (PDL) and left to stand overnight in covered Petri dish. The following day, electrodes were washed 4× with 0.22 μM filtered autoclaved DDW. MEA plates were placed onto custom fabricated square plastic clip with the electrodes face up. Thin slices of double-sided tape are adhered to a custom-made white plastic o-ring. The o-ring is firmly attached to the MEA plate. The inner wall of the plastic o-ring is coated with Silicon sealant, a glass cylinder is inserted and a plastic cap. The square plastic frame is disengaged.
Preparation of Cortical Neuronal Cultures
Post-natal day 1 (P1) mice (WT and Atm−/−) were decapitated. The entire brains were excised into room temperate L-15 medium. Meninges were removed, left and right cortices placed in 8 ml 0.06% w/v trypsin solution inserted in its place. The cortex in trypsin was incubated at 37°C for 15 min, occasionally rotating. After removing trypsin, the tissue was washed once with MEM growth medium. Mechanical disaggregation of cells was performed through repeated pipetting followed by centrifugation for 4 min at 300 g. The medium was replaced with 4 ml fresh 37°C MEM growth medium and re-disaggregated. Filter using gross cell strainer (BD Falcon, 70 μm, BD Bioscience, ON, Canada). One million cells were plated in 3 ml MEM growth medium per MEA. Large networks of about 2 cm2 area and about 1 × 106 neurons were prepared.
ALSO: http://ieeexplore.ieee.org/document/939810/
Four basic aspects of the design and development of a cultured probe, coated with rat cortical or dorsal root ganglion neurons, are described. First, the importance of optimization of the cell-electrode contact is presented. It turns out that impedance spectroscopy, and detailed modeling of the electrode-cell interface, is a very helpful technique, which shows whether a cell is covering an electrode and how strong the sealing is. Second, the dielectrophoretic trapping method directs cells efficiently to desired spots on the substrate, and cells remain viable after the treatment. The number of cells trapped is dependent on the electric field parameters and the occurrence of a secondary force, a fluid flow (as a result of field-induced heating). It was found that the viability of trapped cortical cells was not influenced by the electric field. Third, cells must adhere to the surface of the substrate and form networks, which are locally confined, to one electrode site. For that, chemical modification of the substrate and electrode areas with various coatings, such as polyethyleneimine (PEI) and fluorocarbon monolayers promotes or inhibits adhesion of cells. Finally, it is shown how PEI patterning, by a stamping technique, successfully guides outgrowth of collaterals from a neonatal rat lumbar spinal cord explant, after six days in culture.
From Wikipedia, the free encyclopedia
A cultured neuronal network is a cell culture of neurons that is used as a model to study the central nervous system, especially the brain. Often, cultured neuronal networks are connected to an input/output device such as a multi-electrode array (MEA), thus allowing two-way communication between the researcher and the network. This model has proved to be an invaluable tool to scientists studying the underlying principles behind neuronal learning, memory, plasticity, connectivity, and information processing.[1]
Cultured neurons are often connected via computer to a real or simulated robotic component, creating a hybrot or animat, respectively. Researchers can then thoroughly study learning and plasticity in a realistic context, where the neuronal networks are able to interact with their environment and receive at least some artificial sensory feedback. One example of this can be seen in the Multielectrode Array Art (MEART) system developed by the Potter Research Group at the Georgia Institute of Technology in collaboration with SymbioticA, The Centre for Excellence in Biological Art, at the University of Western Australia.[2] Another example can be seen in the neurally controlled animat.
Advantages
The use of cultured neuronal networks as a model for their in vivo counterparts has been an indispensable resource for decades.[4] It allows researchers to investigate neuronal activity in a much more controlled environment than would be possible in a live organism. Through this mechanism researchers have gleaned important information about the mechanisms behind learning and memory.
A cultured neuronal network allows researchers to observe neuronal activity from several vantage points. Electrophysiological recording and stimulation can take place either across the network or locally via an MEA, and the network development can be visually observed using microscopy techniques.[4] Moreover, chemical analysis of the neurons and their environment is more easily accomplished than in an in vivo setting.[4][5]
Disadvantages
Cultured neuronal networks are by definition disembodied cultures of neurons. Thus by being outside their natural environment, the neurons are influenced in ways that are not biologically normal. Foremost among these abnormalities is the fact that the neurons are usually harvested as neural stem cells from a fetus and are therefore disrupted at a critical stage in network development.[6] When the neurons are suspended in solution and subsequently dispensed, the connections previously made are destroyed and new ones formed. Ultimately, the connectivity (and consequently the functionality) of the tissue is changed from what the original template suggested.
Another disadvantage lies in the fact that the cultured neurons lack a body and are thus severed from sensory input as well as the ability to express behavior – a crucial characteristic in learning and memory experiments. It is believed that such sensory deprivation has adverse effects on the development of these cultures and may result in abnormal patterns of behavior throughout the network.[6]
Cultured networks on traditional MEAs are flat, single-layer sheets of cells with connectivity only two dimensions. Most in vivo neuronal systems, to the contrary, are large three-dimensional structures with much greater interconnectivity. This remains one of the most striking differences between the model and the reality, and this fact probably plays a large role in skewing some of the conclusions derived from experiments based on this model.
more on wikipedia
In vivo neural electrical activity is the essence of nervous system function, controlling sensory modalities, emotion, memory, behavior, and basic survival functions. Therefore, to study neurons in the laboratory it is important that in vitro neuronal models also support such electrical activity to reflect fundamental brain functions – and most human neuronal cultures are currently grown in vitro using the classic culture media DMEM (Dulbecco's Modified Eagle Medium), Neurobasal, or a mixture of the two. In contrast, laboratory experiments employing electrophysiological techniques – such as patch clamping (which allows the study of single or multiple ion channels in cells), calcium imaging – on brain slices or in culture are performed in a medium of artificial cerebrospinal fluid (aCSF).
Recently, scientists at the Salk Institute for Biological Studies, Sanford Consortium for Regenerative Medicine, La Jolla, CA, employing induced pluripotent stem cells (iPSCs) to model human neurological diseases in vitro used electrophysiology techniques to test DMEM and Neurobasal to determine their influence on fundamental neuronal activity. Surprisingly, the scientists discovered that, even though these are classic culture media, they strongly altered many crucial neurophysiological properties. Before deciding to design a new medium, the researchers tested all commercially available basal media that might be used for neuronal tissue culture; none of them supported electrophysiological activity as well as aCSF. However, even with the addition of various supplements, aCSF was not sufficient to maintain cell cultures for more than a day or two. The scientists therefore embarked on the challenging process of designing a new medium more adapted to supporting neuronal function, eventually producing BrainPhys, a novel medium that improves the differentiation and electrophysiological activity of neurons, supports long-term in vitro culture, mimics physiological conditions of the living brain, and allows for assessment of electrophysiological activity.
Read more at: https://medicalxpress.com/news/2015-05-brain-bottle-culture-medium-neuronal.html#
ALSO: http://journal.frontiersin.org/article/10.3389/fnins.2011.00046/full
Micro-Electrode Array Preparation
Micro-Electrode array were purchased from (MultiChannel Systems, Reutlingen, Germany). The MEA’s were sterilized according to the manufacturer‘s instructions. MEAs were washed with 70% EtOH, air dried on a sterile Petri dish, with UV on, in a laminar flow hood. MEA were then coated with 1 mg/ml poly D lysine (PDL) and left to stand overnight in covered Petri dish. The following day, electrodes were washed 4× with 0.22 μM filtered autoclaved DDW. MEA plates were placed onto custom fabricated square plastic clip with the electrodes face up. Thin slices of double-sided tape are adhered to a custom-made white plastic o-ring. The o-ring is firmly attached to the MEA plate. The inner wall of the plastic o-ring is coated with Silicon sealant, a glass cylinder is inserted and a plastic cap. The square plastic frame is disengaged.
Preparation of Cortical Neuronal Cultures
Post-natal day 1 (P1) mice (WT and Atm−/−) were decapitated. The entire brains were excised into room temperate L-15 medium. Meninges were removed, left and right cortices placed in 8 ml 0.06% w/v trypsin solution inserted in its place. The cortex in trypsin was incubated at 37°C for 15 min, occasionally rotating. After removing trypsin, the tissue was washed once with MEM growth medium. Mechanical disaggregation of cells was performed through repeated pipetting followed by centrifugation for 4 min at 300 g. The medium was replaced with 4 ml fresh 37°C MEM growth medium and re-disaggregated. Filter using gross cell strainer (BD Falcon, 70 μm, BD Bioscience, ON, Canada). One million cells were plated in 3 ml MEM growth medium per MEA. Large networks of about 2 cm2 area and about 1 × 106 neurons were prepared.
ALSO: http://ieeexplore.ieee.org/document/939810/
Four basic aspects of the design and development of a cultured probe, coated with rat cortical or dorsal root ganglion neurons, are described. First, the importance of optimization of the cell-electrode contact is presented. It turns out that impedance spectroscopy, and detailed modeling of the electrode-cell interface, is a very helpful technique, which shows whether a cell is covering an electrode and how strong the sealing is. Second, the dielectrophoretic trapping method directs cells efficiently to desired spots on the substrate, and cells remain viable after the treatment. The number of cells trapped is dependent on the electric field parameters and the occurrence of a secondary force, a fluid flow (as a result of field-induced heating). It was found that the viability of trapped cortical cells was not influenced by the electric field. Third, cells must adhere to the surface of the substrate and form networks, which are locally confined, to one electrode site. For that, chemical modification of the substrate and electrode areas with various coatings, such as polyethyleneimine (PEI) and fluorocarbon monolayers promotes or inhibits adhesion of cells. Finally, it is shown how PEI patterning, by a stamping technique, successfully guides outgrowth of collaterals from a neonatal rat lumbar spinal cord explant, after six days in culture.
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