The authors thank Giorgio Carlini for the technical support in developing the confinement structure and dott. Ornella LoBrutto for the thorough revision of the manuscript. The research leading to these results has received funding from the European Union&#39;s 7th Framework Programme (ICT-FET FP7/2007-2013, FET Young Explorers scheme) under grant agreement 284772 BRAIN BOW (www.brainbowproject.eu).

The experimental protocol was approved by the European Animal Care Legislation (2010/63/EU), by the Italian Ministry of Health in accordance with the D.L. 116/1992 and by the guidelines of the University of Genova (Prot. N. 13130, May 2011). All efforts were made to reduce the number of animals for the project and to minimize their suffering.
1. Preparation of Materials and Supports
<ol>
	<li>Construct the mold to build the PDMS (Poly-Dimethyl-Siloxane) constraint by means of a CNC (Computer Numerical Control) milling machine. Such a mold is realized in polycarbonate with the central cylinder in polytetrafluoroethylene (PTFE). This material allows an easier extraction of the mask once the PDMS has hardened. 
	NOTE: The design of the mold has been performed by Computer-Aided-Design (CAD) and then delivered to the CNC milling machine.</li>
	<li>Prepare the PDMS elastomer. PDMS is an organic polymer. It is composed of two elements, a curing agent and a polymer. Mix them by a volume ratio of 1:10, one part of curing agent and nine parts of polymer. Put the two components in a Petri dish, shake and insert the mix in the vacuum chamber for 10 min&#160;to eliminate air bubbles. 3 g&#160;of PDMS are sufficient for one constraint.</li>
	<li>Construct the PDMS constraint. Insert the PDMS material into the molder and put it into the oven for 30 min&#160;at 120 &#176;C. The PDMS constraint has the shape of an ideal cylinder with an external and internal diameter of 22.0 and 3.0 mm, respectively and a height of 650 &#181;m.</li>
	<li>The day before the plating, couple the mask to the active area of the Micro-Electrode Arrays (MEAs) by means of a tweezer under a stereomicroscope and sterilize the PDMS mask in the oven at 120 &#176;C.</li>
	<li>Sterilize the glass microbeads (nominal diameter of 40&#177;2 &#181;m; certified mean diameter of 42.3&#177;1.1 &#181;m) in 70% ethanol for 2 hr&#160;in a conical vial and rotate every 30 min&#160;to expose all microbeads.&#160;</li>
	<li>Remove the ethanol solution from the vial and rinse the microbeads two times with sterilized water.</li>
	<li>Condition the central part of the MEA area delimited by the PDMS mask (diameter equals to 3.0 mm) with 24 &#181;l of mixed solution of Poly-D-Lysine and Laminin at 0.05 &#956;g/ml.</li>
	<li>Coat the microbeads with adhesion proteins, Laminin and Poly-D-Lysine at 0.05 &#181;g/ml and leave them overnight in the incubator at 37 &#176;C, to obtain a stable and long-lasting neuronal network.</li>
	<li>Remove the adhesion factors both from the glass surface of the MEA and from the glass microbeads. Aspirate approximately 95% of the coating solution with a pipette and apply a small volume- 24&#181;l- of sterile water on the MEA surface. Aspirate again more than 95% of the water and let the MEA dry under the laminar hood for 1 hr&#160;before plating the cells. 
	NOTE: In the case of microbeads instead, aspirate the coating solution and make a first wash with sterilized water and a second one with the basal medium and its supplement such as B27, in order to condition the glass surface where neurons will be plated. Do not let the microbeads dry. Leave them in suspension in the culture medium inside the vial.</li>
	<li>Distribute, by means of a pipette -200 &#181;l-, the suspension of treated microbeads (about 32,000) onto a multiwell plate with membrane insert (pore diameter 0.4 &#181;m) where they will self-assemble forming a uniform layer.</li>
	<li>Fill each well of the membrane with 0.5 ml of medium and put it in the incubator (T = 37.0 oC, CO2 = 5%) until the neurons are ready to be plated (3.2).</li>
</ol>
2. Dissection of Embryos and Dissociation of Tissue
<ol>
	<li>House adult female rats (200-250 g) at a constant temperature (22 &#177; 1 &#176;C) and relative humidity (50%) under a regular light&#8211;dark schedule (light-on 7 AM&#8211;7 PM) in the animal facility. Ensure that food and water are freely available.</li>
	<li>Anesthetize a pregnant female rat after 18 days of development (E18) using 3% isoflurane. Then, sacrifice the animal by cervical dislocation.</li>
	<li>Remove hippocampi from each rat embryo and place them into ice cold Hank&#8217;s balanced salt solution without Ca2+ and Mg2+. At day 18 of development hippocampus and cortex tissues are very soft and do not need to be cut into small pieces. Further details can be found18,19.</li>
	<li>Dissociate the tissue in 0.125% of Trypsin/Hank&#8217;s solution containing 0.05% of DNAse for 18-20 min at 37 &#176;C.</li>
	<li>Remove the supernatant solution with a Pasteur pipette and stop the enzymatic digestion by adding medium with 10% fetal bovine serum (FBS) for 5 min.</li>
	<li>Remove the medium with FBS and wash once with the medium with its supplement, 1% L-glutamine, gentamicin 10 &#181;g/ml. Remove again the medium with a Pasteur pipette and refill it again with a small amount (500 &#181;l) of growth medium with its supplement, 1% L-glutamine, gentamicin 10 &#181;g/ml.</li>
	<li>Dissociate the tissue pellet mechanically with a narrow Pasteur pipette until a milky suspension of cells is apparent. It is not necessary to centrifuge the cell suspension.</li>
	<li>Dilute the small volume of cell suspension with the growth medium to obtain a final volume of 2.0 ml. Count the obtained cellular concentration with a hemocytometer chamber. Dilute this concentration at 1:5 in order to obtain the desired cell concentration of 600-700 cells/&#181;l.</li>
</ol>
3. Cell Plating
<ol>
	<li>Plate cells at a density of about 2,000 cells/mm2 onto the active area of the MEA, defined by the PDMS constraint to create a 2D neuronal network. 
	NOTE: Each hippocampus contains about 5 x 105 cells, (1 x 106 for a single embryo). Dissect 6 embryos to get a total amount of 6 x 10 6 cells in 2 ml, and an estimated concentration of 3,000 cells/&#181;l. Dilute this concentration at 1:5 in order to obtain the desired cell concentration and plate about 600-700 cells/&#181;l. If the MEA area delimited by the PDMS constraint is about 7.065 mm2, and the total number of plated cells 600 cells/&#181;l x 24 &#181;l = 14,400 cells, the final density of 2,038 cells/mm2.</li>
	<li>Place the MEA devices into the incubator with humidified CO2 atmosphere (5%) at 37 &#176;C.</li>
	<li>Distribute 160 &#181;l of the suspension with a cell concentration of 600-700 cells/&#181;l (about 100,000 cells) onto the surface of the microbeads monolayer positioned inside the multiwell plates to complete the preliminary step for the construction of three-dimensional culture. Put the multiwell plates in the incubator with humidified CO2 atmosphere (5%) at 37 &#176;C.</li>
</ol>
4. 3D Neuronal Network Construction
<ol>
	<li>6-8 hr&#160;after the plating, transfer the suspension (microbeads with neurons) from the multiwell plates very carefully inside the area delimited by the PDMS constraint by using a pipette set for a volume of about 30-40 &#181;l. After each transfer, wait for about half a minute, to allow the microbeads to self-assemble in a hexagonal compact structure.</li>
	<li>Once all the layers are deposited and spontaneously assembled, refill with a large drop of about 300 &#181;l of medium the top of the area delimited by the PDMS constraint.</li>
	<li>Put the 3D structure coupled to the MEA in incubator (T = 37.0 oC, CO2 = 5%) for 48 hr&#160;before adding a final volume (about 1 ml) of growth medium culture with its supplement. 
	NOTE: Consider the total number of beads on the multiwell plates with membrane insert (30,000) and the number of beads on a single layer onto the MEA device (6,000). The resulting 3D structure is composed of 5 layers of microbeads and cells. Taking into account that the 3D neuronal network is not geometrically perfect, the resulting 3D structure could be composed of 5-8 layers.</li>
	<li>The day after plating, carefully add the final volume of the medium (about 900 &#181;l) inside the MEA ring. Maintain the 3D cultures in a humidified CO2 atmosphere (5%) at 37 &#176;C for 4-5 weeks. Replace half of the medium once a week.</li>
</ol>
5. Confocal Microscopy Acquisition
<ol>
	<li>Fix the biological samples to the stage of the microscope in a holder. Set the laser (Argon, 496-555 nm), the scan speed (400 Hz) at which to acquire the image, the gain and offset (-0.3%) of PMT for each image to capture the correct bandwidth emission filter and in order to avoid saturation and to increase the signal to noise ratio. The Results Section reports the values used to acquire the images of Figure 4.</li>
	<li>For the acquisition of the z-stack sequence, first select, the value of z position of the top and the bottom layer of the sample, and then select the step size to make the acquisition.</li>
</ol>

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The authors declare that they have no competing financial interests.

In this work, a novel experimental in vitro platform made up of 3D engineered neuronal cultures coupled to MEAs for network electrophysiology has been presented. The use of microbeads as scaffold to allow the neuritic outgrowth along the z-axis has been tailored to be integrated with the planar MEA. In this way, the obtained micro-system results in a valid and reliable in vitro 3D model to study the emergent electrophysiological dynamics16.
MEA recording s...

In vitro two-dimensional (2D) neural networks coupled to Micro-Electrode Arrays (MEAs)arethe gold-standard experimental model adopted to study the interplay between neuronal dynamics and the underlying connectivity. During the development, neurons recreate complex networks which display well definedspatio-temporalpatternsof activity1,2 (i.e., bursts, network bursts, random spiking activity). MEAs record the electrophysiological activity from many sites (from tens to thousands of microelectrodes), allowing a detailed investigation of the expressed dynamics at the network level. In addition, the use of dissociated cultures makes possibile to design engineered-networks. It is easier to understand in this way the functional relationships between the recorded electrophysiological activity and the parameters of the network organization like cell density3, degree of modularity4,5, presence of heterogeneous neuronal populations6, etc. However, all in vitro studies on dissociated cultured cells are based on 2D neuronal networks. This approach leads to oversimplifications with respect to the in vivo (intrinsically 3-dimensional, 3D) system: (i) in a 2D model, somata and growth cones are flattened and the axons-dendrites outgrowth cannot spread in all directions7. (ii) 2D in vitro networks exhibit stereotyped electrophysiological dynamics dominated by bursting activity involving most of the neurons of the network8.
Recently, different solutions have been developed to allow the construction of in vitro 3D dissociated neuronal networks. The common idea consists in creating a scaffold where neurons can grow in a 3D fashion. Such a scaffold can be realized with polymer gels and solid porous matrices9-13. By exploiting the mechanical properties of the polymers, it is possible to embed cells inside these structures by defining a uniform block of 3D cultures of neurospheres11. The main feature of this approach is the rigid mechanical property of the neurospheres9,12. However, these materials have limited porosity, and they do not guarantee cell migration inside the matrix. To overcome this drawback, a possible solution consists in slicing the matrix into &#8216;unit&#8217; modules. Unfortunately, the size and shape diversity of the particles could hamper the packing into regular layered structures. In7, Cullen and coworkers designed a 3D neuronal construct made up of neurons and/or astrocytes within a bioactive extracellular matrix-based scaffold. Such an engineered neural tissue allowed in vitro investigations to study and manipulate neurobiological responses within 3D micro-environments. This model consisted of neurons and glia distributed throughout the extracellular matrix (ECM) and/or hydrogel scaffolds (500-600 &#181;m thick). In this condition, an optimum cell viability (greater than 90%) was found by plating cells at a final density of about 3,750 - 5,000 cells/mm3. It must be noted that such a density value is far lower than the one in the in vivo condition, where the cell density of the mouse brain cortex is about 90,000 cells/mm314. To overcome this limitation Pautot and coworkers15 realized a 3D in vitro system where cell density and network connectivity are controlled to resemble in vivo conditions while enabling real-time imaging of the network. Practically, this method is based on the concept that dissociated cultured neurons are able to grow on silica microbeads. These beads provide a growth surface large enough for neuronal cell bodies to adhere and for their arborizations to grow, mature, extend, and define synaptic contacts to other neurons. This method exploits the spontaneous assembly properties of mono-dispersed beads to form 3D layered hexagonal arrays containing distinct subsets of neurons on different layers with constrained connectivity among neurons on different beads. The achieved cell density with this method was about 75,000 cells/mm3.
Recently, we have adapted Pautot&#8217;s method to MEAs16: the obtained results show that the 3D electrophysiological activity presents a wider repertoire of activities than the one expressed by 2D networks.&#160;3D mature cultures exhibit an enhanced dynamic in which both network burst and random spike activity coexist. Similarly, Tang-Schomer and coworkers17 realized a silk protein-based porous scaffold which maintains a primary cortical culture in vitro for some months, and recorded the electrophysiological activity by means of a tungsten electrode.
In this work, the experimental procedures to build 3D neuronal networks coupled to MEAs will be described.

<table><tbody><tr><td>Laminin</td><td>Sigma-Aldrich</td><td>L2020</td><td>0.05 &amp;micro;g/ml</td></tr><tr><td>Poly-D-lysine</td><td>Sigma-Aldrich</td><td>P6407</td><td>0.05 &amp;micro;g/ml</td></tr><tr><td>Trypsin</td><td>Gibco</td><td>25050-014</td><td>0.125% diluted 1:2 in HBSS w\o CA&lt;sup&gt;++&lt;/sup&gt;, MG&lt;sup&gt;++&lt;/sup&gt;</td></tr><tr><td>DNAase</td><td>Sigma-Aldrich</td><td>D5025</td><td>0.05% diluted&amp;nbsp; in Hanks solution</td></tr><tr><td>Neurobasal</td><td>Gibco Invitrogen</td><td>21103049</td><td>culture medium</td></tr><tr><td>B27</td><td>Gibco Invitrogen</td><td>17504044</td><td>2% medium supplent</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Sigma-Aldrich</td><td>F-2442</td><td>10%</td></tr><tr><td>Glutamax-I&amp;nbsp;</td><td>Gibco</td><td>35050038</td><td>0.5 mM</td></tr><tr><td>gentamicin</td><td>Sigma-Aldrich</td><td>G.1272</td><td>5 mg/L</td></tr><tr><td>Poly-Dimethyl-Siloxane (PDMS)</td><td>Corning Sigma</td><td>481939</td><td>curing agent and the polymer</td></tr><tr><td>Micro-Electrode Arrays</td><td>Multi Channel Systems (MCS)</td><td>60MEA200/30-Ti-pr</td><td>MEA with: Electrode grid 8x8; Electrode spacing and diameter 200 and 30 &amp;micro;m, respectively; plastic ring without thread</td></tr><tr><td>Microbead</td><td>Distrilab-Duke Scientific</td><td>9040</td><td>&amp;nbsp;1 gr Glass Part. Size Stds 40 &amp;micro;m</td></tr><tr><td>Transwell</td><td>Costar Sigma</td><td>CLS 3413</td><td>multiwell plates with membrane insert (6.5 mm diameter porous 0.4 &amp;micro;m)</td></tr><tr><td>HBSS w\o Ca&lt;sup&gt;++&lt;/sup&gt; ,Mg&lt;sup&gt;++&lt;/sup&gt;&amp;nbsp;</td><td>Gibco Invitrogen</td><td>14175-052</td><td></td></tr><tr><td>Hanks Buffer Solution&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>H8264</td><td></td></tr><tr><td>Teflon</td><td>Sigma</td><td>430935-5G</td><td>polytetrafluoroethylene</td></tr><tr><td>Rat</td><td>Sprague Dawley</td><td>Wistar Rat</td><td></td></tr><tr><td>Confocal Microscopy Upright</td><td>Leica</td><td>TCS SP5 AOBS</td><td></td></tr><tr><td>20.0x0.50 WATER objective</td><td>Leica</td><td>Leica HCX APO L U-V-I&amp;nbsp;</td><td></td></tr><tr><td>40.0x0.80 WATER objective</td><td>Leica</td><td>Leica HCX APO L U-V-I&amp;nbsp;</td><td></td></tr><tr><td>25.0x0.95 WATER objective</td><td>Leica</td><td>HCX IRAPO L</td><td></td></tr></tbody></table>

interfacing 3d engineered neuronal cultures to micro-electrode arrays: an innovative in vitro experimental model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 &#181;m in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.

In this experimental procedure, a relevant role is played by the microbeads that define the mechanical scaffold for the growth of the 3D neuronal network. Figures 1A and B display the arrangement of the microbeads (nominal diameter of 40&#177;2 &#181;m; certified mean diameter of 42.3&#177; 1.1 &#181;m) in the plane. The peculiarity of such structures is that microbeads spontaneously self-assemble defining a compact hexagonal geometry (Figure 1B and C). The so generated scaffold guarante...

Watch this Scientific Journal Video about Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model at JoVE.com

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

interfacing-3d-engineered-neuronal-cultures-to-micro-electrode-arrays

Research

JoVE Journal

Neuroscience

10.0K Views.  University of Genova. In this work, a novel experimental model in which 3D neuronal cultures are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are built by seeding neurons in a scaffold made up of glass microbeads on which neurons grow and form interconnected 3D structures.

Video: Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

Developmental Biology

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays (MEAs)

For the last century, many neuroscientists around the world have dedicated their lives to understanding how neuronal networks work and why they stop working in various diseases. Studies have included neuropathological observation, fluorescent microscopy with genetic labeling, and intracellular recording in both dissociated neurons and slice preparations. This protocol discusses another technology, which involves growing dissociated neuronal cultures on micro-electrode arrays (also called multi-electrode arrays, MEAs).
There are multiple advantages to using this system over other technologies. Dissociated neuronal cultures on MEAs provide a simplified model in which network activity can be manipulated with electrical stimulation sequences through the array's multiple electrodes. Because the network is small, the impact of stimulation is limited to observable areas, which is not the case in intact preparations. The cells grow in a monolayer making changes in morphology easy to monitor with various imaging techniques. Finally, cultures on MEAs can survive for over a year in vitro which removes any clear time limitations inherent with other culturing techniques.1
Our lab and others around the globe are utilizing this technology to ask important questions about neuronal networks. The purpose of this protocol is to provide the necessary information for setting up, caring for, recording from and electrically stimulating cultures on MEAs. In vitro networks provide a means for asking physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction.

How to Culture, Record and Stimulate Neuronal Networks on Micro-electrode Arrays MEAs

This protocol provides the necessary information for setting up, caring for, recording from and electrically stimulating cultures on MEAs. In vitro networks provide a means for asking physiologically relevant questions at the network and cellular levels leading to a better understanding of brain function and dysfunction.

For the last century, many neuroscientists around the world have dedicated their lives to understanding how neuronal networks work and why they stop ...

Biology

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire of dynamical motifs, which underlie most diverse brain functions, can emerge out of a fixed topological and modular organization of brain circuits. Compared to in vivo studies of neuronal circuits which present intrinsic experimental difficulties, in vitro preparations offer a much larger possibility to manipulate and probe the structural, dynamical and chemical properties of experimental neuronal systems. This work describes an in vitro experimental methodology which allows growing of modular networks composed by spatially distinct, functionally interconnected neuronal assemblies. The protocol allows controlling the two-dimensional (2D) architecture of the neuronal network at different levels of topological complexity.
A desired network patterning can be achieved both on regular cover slips and substrate embedded micro electrode arrays. Micromachined structures are embossed on a silicon wafer and used to create biocompatible polymeric stencils, which incorporate the negative features of the desired network architecture. The stencils are placed on the culturing substrates during the surface coating procedure with a molecular layer for promoting cellular adhesion. After removal of the stencils, neurons are plated and they spontaneously redirected to the coated areas. By decreasing the inter-compartment distance, it is possible to obtain either isolated or interconnected neuronal circuits. To promote cell survival, cells are co-cultured with a supporting neuronal network which is located at the periphery of the culture dish. Electrophysiological and optical recordings of the activity of modular networks obtained respectively by using substrate embedded micro electrode arrays and calcium imaging are presented. While each module shows spontaneous global synchronizations, the occurrence of inter-module synchronization is regulated by the density of connection among the circuits.

This manuscript describes a protocol to grow in vitro modular networks consisting of spatially confined, functionally inter-connected neuronal circuits. A polymeric mask is used to pattern a protein layer to promote cellular adhesion over the culturing substrate. Plated neurons grow on coated areas establishing spontaneous connections and exhibiting electrophysiological activity.

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire ...

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. 
We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.

We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device.

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

Bioengineering

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

Multimodal Large-Scale Neural Recordings and Analysis on HD-MEA 

Recording and Analyzing Multimodal Large-Scale Neuronal Ensemble Dynamics on CMOS-Integrated High-Density Microelectrode Array

Large-scale neuronal networks and their complex distributed microcircuits are essential to generate perception, cognition, and behavior that emerge from patterns of spatiotemporal neuronal activity. These dynamic patterns emerging from functional groups of interconnected neuronal ensembles facilitate precise computations for processing and coding multiscale neural information, thereby driving higher brain functions. To probe the computational principles of neural dynamics underlying this complexity and investigate the multiscale impact of biological processes in health and disease, large-scale simultaneous recordings have become instrumental. Here, a high-density microelectrode array (HD-MEA) is employed to study two modalities of neural dynamics - hippocampal and olfactory bulb circuits from ex-vivo mouse brain slices and neuronal networks from in-vitro cell cultures of human induced pluripotent stem cells (iPSCs). The HD-MEA platform, with 4096 microelectrodes, enables non-invasive, multi-site, label-free recordings of extracellular firing patterns from thousands of neuronal ensembles simultaneously at high spatiotemporal resolution. This approach allows the characterization of several electrophysiological network-wide features, including single/-multi-unit spiking activity patterns and local field potential oscillations. To scrutinize these multidimensional neural data, we have developed several computational tools incorporating machine learning algorithms, automatic event detection and classification, graph theory, and other advanced analyses. By supplementing these computational pipelines with this platform, we provide a methodology for studying the large, multiscale, and multimodal dynamics from cell assemblies to networks. This can potentially advance our understanding of complex brain functions and cognitive processes in health and disease. Commitment to open science and insights into large-scale computational neural dynamics could enhance brain-inspired modeling, neuromorphic computing, and neural learning algorithms. Furthermore, understanding the underlying mechanisms of impaired large-scale neural computations and their interconnected microcircuit dynamics could lead to the identification of specific biomarkers, paving the way for more accurate diagnostic tools and targeted therapies for neurological disorders.

Author Spotlight: Advancing Large-Scale Neural Dynamics Through HD-MEA Technology

Here, we employ HD-MEA to delve into computational dynamics of large-scale neuronal ensembles, particularly in hippocampal, olfactory bulb circuits, and human neuronal networks. Capturing spatiotemporal activity, combined with computational tools, provides insights into neuronal ensemble complexity. The method enhances understanding of brain functions, potentially identifying biomarkers and treatments for neurological disorders.

Large-scale neuronal networks and their complex distributed microcircuits are essential to generate perception, cognition, and behavior that emerge from ...

Applications of 3D-Printing for Micro-Scale Neuronal Cell Culture Devices

Two-Photon Polymerization 3D-Printing of Micro-scale Neuronal Cell Culture Devices

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, and realistic synaptic connectivity are missing. The latter limits the study of structure and function in the context of compartmentalization and diminishes the significance of cultures in neuroscience. Approximating ex vivo the structured anatomical arrangement of synaptic connectivity is not trivial, despite being key for the emergence of rhythms, synaptic plasticity, and ultimately, brain pathophysiology. Here, two-photon polymerization (2PP) is employed as a 3D printing technique, enabling the rapid fabrication of polymeric cell culture devices using polydimethyl-siloxane (PDMS) at the micrometer scale. Compared to conventional replica molding techniques based on microphotolitography, 2PP micro-scale printing enables rapid and affordable turnaround of prototypes. This protocol illustrates the design and fabrication of PDMS-based microfluidic devices aimed at culturing modular neuronal networks. As a proof-of-principle, a two-chamber device is presented to physically constrain connectivity. Specifically, an asymmetric axonal outgrowth during ex vivo development is favored and allowed to be directed from one chamber to the other. In order to probe the functional consequences of unidirectional synaptic interactions, commercial microelectrode arrays are chosen to monitor the bioelectrical activity of interconnected neuronal modules. Here, methods to 1) fabricate molds with micrometer precision and 2) perform in vitro multisite extracellular recordings in rat cortical neuronal cultures are illustrated. By decreasing costs and future widespread accessibility of 2PP 3D-printing, this method will become more and more relevant across research labs worldwide. Especially in neurotechnology and high-throughput neural data recording, the ease and rapidity of prototyping simplified in vitro models will improve experimental control and theoretical understanding of in vivo large-scale neural systems.

Author Spotlight: Modular Neuronal Networks for Analyzing Brain Functions

3D printing at the micrometer scale enables rapid prototyping of polymeric devices for neuronal cell cultures. As a proof-of-principle, structural connections between neurons were constrained by creating barriers and channels influencing neurite outgrowth, while the functional consequences of such manipulation were observed by extracellular electrophysiology.

Neuronal cultures have been a reference experimental model for several decades. However, 3D cell arrangement, spatial constraints on neurite outgrowth, ...

Building Three-Dimensional Neuronal Networks Coupled to Micro-Electrode Arrays

Source: Tedesco, M., et al., <a href="https://app.jove.com/v/53080">Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model.</a> J. Vis. Exp. (2015)
This video demonstrates the development of a 3D neuronal culture coupled to a planar microelectrode array with hippocampal neurons. The isolated neurons form a 2D network on the microelectrode array&#39;s active area and adhere to glass microbeads in a multi-well plate with a membrane insert. Upon transferring these neuron-coated microbeads to the electrode, they organize into an interconnected 3D neural network.

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

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