extracellular electrophysiology in modular neuronal cell culture

Toepassingen van 3D-printen voor neuronale celkweekapparaten op microschaal

Investigating neuronal activity in behaving organisms presents several challenges. For instance, physical access to the brain tissue is limited by the need to maintain its integrity, so the brain&#39;s superficial regions are more easily considered. Isolating specific targets within the intact tissue is often a daunting task and sometimes impossible. Although dissociated homogeneous neuronal cultures offer convenient access to the molecular, biochemical, and biophysical properties of individual (sub)cellular components of a neural circuit, a realistic connectivity and anatomical organization of the intact brain is lost. These fundamental constraints inspired research efforts to achieve a middle ground, where in vivo complexity is avoided while the structure can be constructed in vitro, which is on demand1,2,3,4,5,6,7,8,9. In particular, modular neuronal cultures have been a subject of extensive research over the past decades, aiming to tackle key questions of brain physiology as described below.
Organization:&#160;In vivo studies show that the brain is structured anatomically in layers with precise cell types and arrays of projections. Functional assays revealed the organization of the neuronal networks in node assemblies and modules, with precise connectivity schemes10,11. The role of connectivity and microcircuit motifs cannot, however, be studied adequately in vivo due to the sheer number of synapses that are involved, as well as the interwoven effects of development and activity-dependent plasticity.
Signal transfer: In in vivo or random in vitro cultures, it is challenging to assess signal transfer. Examining the axonal conduction and action potential along its length requires guiding neurite outgrowth by surface functionalization or chemical patterning, providing a high signal-to-noise ratio in extracellular readouts of electrical activity12.
Translational relevance: Deciphering the exclusive role of pre- versus post-synaptic elements across pathological conditions requires having access to these elements individually. Modular cultures with constrained connectivity, effectively segregating the pre-and post-synaptic elements, are indispensable tools to this end13.
Several methods exist to obtain some form of structure in neuronal culture. They can be broadly categorized as chemical and physical surface manipulation9. The former methods14,15 rely on the propensity of neuronal cells to attach to certain (bio)chemical compounds. This requires depositing adhesive or attractive molecules on a surface with micro-scale precision and following a detailed pattern. While this allows a partial coverage of the surface of the cells, following the desired pattern, chemical methods are inherently limited and have a relatively low success rate in neurite growth guidance16. Full control over axon directionality requires establishing a spatial gradient of ad-hoc chemicals to shape axonal guidance17. The latter methods involve physical surface manipulation and are more commonly used to structure the neuronal networks in vitro. Neuronal cells are physically constrained at desired locations by geometrical confinements, such as microscopic chambers, walls, channels, etc., shaping a biocompatible polymer such as polydimethylsiloxane (PDMS)3,5,6,7,18,19,20 cured and solidified into a microfluidic device. The de facto method for PDMS microfluidic fabrication is soft photolithography21, where a two-dimensional mask is patterned at a micro-scale and employed to selectively etch a silicon-based material upon UV exposure. In a nutshell, a UV-curable resin (i.e., photoresist) is coated onto a silicon wafer through spin-coating, reaching a specific height determined by its viscosity and spinning speed. Then, the patterned mask is positioned over the photoresist and exposed to UV light. Transparent areas within the mask, corresponding to regions of interest, will let UV light induce localized crosslinking of the photoresist molecules. The areas of unexposed photoresist are washed away using a solvent, resulting in the formation of a master mold. This is repeatedly used to bake an elastomer of the choice (i.e., PDMS), which is then engraved with the desired geometries in as many replicas as desired. Such a manufacturing method is the most common method to fabricate microfluidic devices22. Perhaps the main limitations of soft photolithography are the prerequisite of notable capital investment and the unfamiliarity of biological labs with the required techniques and expertise. The preparation of the mask and the steps of soft photolithography required to design complex multiple-height high aspect ratio geometries are non-trivial23 and often require outsourcing. Even though alternative and low-budget methods have been proposed, they do not always satisfy the high precision requirements of biological prototyping24.
Here, an alternative manufacturing method is presented, relying on two-photon polymerization (2PP) and additive manufacturing. It is straightforward and does not require per se advanced microfabrication and microphotolitography expertise. The research field of 2PP micromanufacture emerged in the late 90&#39;s25, and since then, it has witnessed exponential growth26. More about the fundamental principles of this technique can be found elsewhere26. Briefly, by focusing the excitation light impulse in three-dimensional space, 2PP leverages the nonlinear dependence of multiphoton absorption on intensity. This grants the capability of confined absorption, ensuring precise and selective excitation within very localized regions. In essence, a negative-tone photoresist, a material with decreased solubility upon light exposure, is subjected to a focused beam of femtosecond laser pulses at a low duty-cycle27. This allows for impulses with high intensities at low average powers, enabling polymerization without harming the material. The interaction of photo-induced radical monomers gives rise to radical oligomers, initiating polymerization that extends throughout the photoresist up to a distinct volume, i.e., voxel, whose size depends on the intensity and duration of the laser pulses28.
In this work, two components are presented: A) the design and rapid fabrication of a 3D-printed mold, reusable many times to produce disposable polymeric neuronal cell culture devices (Figure 1), and B) their mechanical coupling onto the surface of planar neuronal cell culture substrates, or even of substrate-integrated microelectrode arrays capable of multisite recordings of bioelectric signals.
Computer Assisted Design of a 3D mechanical model is very briefly described here and accompanied by the steps leading to a 3D-printed mold and fabricating PDMS devices is also detailed.
A variety of computer-aided design software applications can be used to generate the starting 3D object model and produce a STL file to control the 2PP printing process. Within the Table of Materials, the first and the last applications listed are free-of-charge or provided with a free license. Constructing a 3D model always requires creating a 2D sketch, which is then extruded in subsequent modelling steps. To demonstrate this concept, a generic 3D CAD software design process is highlighted in protocol section, leading to a structure made of overlapping cubes. For more comprehensive information, a number of online tutorials and free training resources are available, as indicated in the Table of Materials.
The resulting STL file is then translated into a series of commands to be executed by the 3D-printer (i.e., slicing procedure). For the specific 2PP 3D-printer in use, the software DeScribe is used to import the STL file and convert it into the proprietary General Writing Language (GWL) format. The success of 2PP printing process hinges on various parameters, notably, laser power and its scan speed, stitching, and hatching-slicing distances. The choice of these parameters, along with the selection of objective and photoresist, depends on the design&#39;s smallest features, as well as the intended application. Thus, parameter optimization becomes essential to meet the requirements of different design scenarios and use cases. For this work, the recommended recipe IP-S 25x ITO Shell (3D MF) has been considered as a configuration for the printing parameters. Ultimately, a mechanically stable printed part is printed with the necessary resolution while minimizing its 3D-printing time.
The mold design and related STL file, demonstrated in this work, comprises a square frame to segregate the space of a cell culture in two compartments: an outer area (i.e., referred to as Source subsequently) and an inner area (i.e., referred to as Target subsequently). These two compartments are connected through sets of microchannels, each characterized by sharp-angle borders, designed to specifically hinder the growth of neurites from the Target to the Source, but not vice versa, and as such promote a directional synaptic connectivity between neurons growing on the two areas.
Earlier studies employed different geometries of microchannels to encourage the directional growth of neurites. Examples include triangular shapes18, channel barbed structures19, and tapering channels20. Here, a design featuring sharp angle barriers across the microchannel&#39;s borders is employed, characterized also by asymmetric entrances. These microchannels serve to establish continuity between an enclosed interior, the Target compartment, and the external area, the Source compartment. The funnel shape of the initial part of the microchannels, from the Source side, is designed to promote the formation of axonal bundles and their growth along the shortest, i.e., straight line, path connecting the Source to the Target. The triangular space realized by facing sharp angles have larger volume at the Target side to aim at effectively delaying neurites&#39; pathfinding while favoring the rapid shooting of bundles originating from the Source and occupation of the available space. The choice of 540 &#181;m for the length of the microchannels effectively filters out the generally shorter dendritic outgrowth39. In addition, their 5 &#181;m height prevents cell somata from penetrating through the microchannels. Overall, this configuration proved to promote unidirectional connectivity between the outer, Source, and inner, Target modules, and it is presented here as a proof-of-principle among the many alternative choices.
While the PDMS devices, fabricated by the 2PP mold, can be attached to the surface of common cell culture substrates, such as glass coverslips or Petri dishes, in this work commercially available substrate-integrated microelectrode arrays were used. No effort has been made to optimize the 3D design to the microelectrode array layout, and the mechanical coupling was performed under stereomicroscopy guidance aiming only at positioning the device across the array, leaving some microelectrode uncovered in both sides, the Source and the Target. This enables the preliminary assessment of the functional consequences of the constrained connectivity in neuronal cell cultures.

Auteur in de schijnwerpers: modulaire neuronale netwerken voor het analyseren van hersenfuncties

Twee-foton polymerisatie 3D-printen van neuronale celkweekapparaten op microschaal

We are broadly interested in the relationship between structural and functions in the brain, particularly at the level of micro-circuits. We often employ reduced experimental preparations such as dissociated neuron cell culture because they are a precious model to investigate electrical activity, synaptic transmission, and overall, the emergence of collective states. The method we propose is ideal for rapid fabrication of microscale, polymeric devices.This can be immediately used for the experimental study of modular neural networks in a dish, and it do not requires a high level of technical expertise in soft photolithography. This method and our simple proof of concept allow the designing of modular neuronal networks where we can define the structure, and control the flow of biological signals. Drug screening and investigation of pathological conditions immediately benefit from these sophisticated in vitro neuronal networks.

Extracellulaire elektrofysiologie in modulaire neuronale celkweek

Fabriceer om te beginnen een PDMS-apparaat uit een 3D-geprinte mal in gekweekte neuronale cellen in de MEA die op het PDMS is gemonteerd. Monteer de MEA voorzichtig in de hoofdtrap van een meerkanaals elektronische versterker die in een droge 37 graden Celsius is geplaatst en gedurende 10 minuten incubeert. Start de Experimenter-software, stel de bemonsteringsfrequentie in op 25 kilohertz en druk op start data-acquisitie om data-acquisitie te starten.In het gegevensweergavepaneel verschijnen de ruwe sporen van extracellulair geregistreerde activiteit voor elk kanaal. Ga naar het stimulatorpaneel van de software. Kies drie paar naburige micro-elektroden die actief zijn tijdens netwerkbrede bursts en selecteer een bifasische pulsgolfvorm.Stel de piekamplitudes in op 800 millivolt en de pulsduur op 200 microseconden. Om de bipolaire configuratie te bereiken, selecteert en stelt u de waarden van de naburige elektroden in. Stel ten slotte de recorder in op 300 milliseconden voor en 1000 milliseconden na de stimulatie.En druk op start data-acquisitie om de opname te starten. Live beeldvorming onthulde dat fluorescerend gelabelde neurieten uitgroei vertoonden van bron naar doelwit in een microkanaal van het PDMS-apparaat. Elektrofysiologische analyse gaf aan dat de opgewekte respons van de bron en het doelwit in de modulaire neuronale netwerken asymmetrisch is, terwijl de controle zonder PDMS dit patroon niet vertoonde.

Research

JoVE Journal

Neuroscience

Extracellular Electrophysiology in Modular Neuronal Cell Culture

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, ...

Generating CAD Files to Obtain 3D Printed Neuronal Culture Device Using Two-Photon Polymerization

CAD-bestanden genereren om een 3D-geprint neuronaal kweekapparaat te verkrijgen met behulp van polymerisatie van twee fotonen

3D Mold Preparation by Two-Photon Polymerization to Design Neuronal Cell Culture Device

3D-vormvoorbereiding door polymerisatie van twee fotonen om een neuronaal celkweekapparaat te ontwerpen

PDMS Device Fabrication for Culturing Neuronal Cells

Fabricage van PDMS-apparaten voor het kweken van neuronale cellen

Culturing Neuronal Cells in Micro-Scale 3D-Printed PDMS Device for Electrophysiology Studies

Neuronale cellen kweken in 3D-geprint PDMS-apparaat op microschaal voor elektrofysiologische studies