Neuronal circuits on a chip for biological network monitoring

Cultured neuronal networks (CNNs) are a robust model to closely investigate neuronal circuits’ formation and monitor their structural properties evolution. Typically, neurons are cultured in plastic plates or, more recently, in microfluidic platforms with potentially a wide variety of neuroscience applications. As a biological protocol, cell culture integration with a microfluidic system provides benefits such as accurate control of cell seeding area, culture medium renewal, or lower exposure to contamination. The objective of this report is to present a novel neuronal network on a chip device, including a chamber, fabricated from PDMS, vinyl and glass connected to a microfluidic platform to perfuse the continuous flow of culture medium. Network growth is compared in chips and traditional Petri dishes to validate the microfluidic chip performance. The network assessment is performed by computing relevant topological measures like the number of connected neurons, the clustering coefficient, and the shortest path between any pair of neurons throughout the culture's life. The results demonstrate that neuronal circuits on a chip have a more stable network structure and lifespan than developing in conventional settings, and therefore this setup is an advantageous alternative to current culture methods. This technology could lead to challenging applications such as batch drug testing of in vitro cell culture models. From the engineering perspective, a device's advantage is the chance to develop custom designs more efficiently than other microfluidic systems.     

Validation of long-term primary neuronal cultures and network activity through the integration of reversibly bonded microbioreactors and MEA substrates

In vitro recording of neuronal electrical activity is a widely used technique to understand brain functions and to study the effect of drugs on the central nervous system. The integration of microfluidic devices with microelectrode arrays (MEAs) enables the recording of networks activity in a controlled microenvironment. In this work, an integrated microfluidic system for neuronal cultures was developed, reversibly coupling a PDMS microfluidic device with a commercial flat MEA through magnetic forces. Neurons from mouse embryos were cultured in a 100 μm channel and their activity was followed up to 18 days in vitro. The maturation of the networks and their morphological and functional characteristics were comparable with those of networks cultured in macro-environments and described in literature. In this work, we successfully demonstrated the ability of long-term culturing of primary neuronal cells in a reversible bonded microfluidic device (based on magnetism) that will be fundamental for neuropharmacological studies.     

Microfluidic device‐assisted etching of p‐HEMA for cell or protein patterning

The construction of biomaterials with which to limit the growth of cells or to limit the adsorption of proteins is essential for understanding biological phenomena. Here, we describe a novel method to simply and easily create thin layers of poly (2‐hydroxyethyl methacrylate) (p‐HEMA) for protein and cellular patterning via etching with ethanol and microfluidic devices. First, a cell culture surface or glass coverslip is coated with p‐HEMA. Next, a polydimethylsiloxane (PDMS) microfluidic is placed onto the p‐HEMA surface, and ethanol is aspirated through the device. The PDMS device is removed, and the p‐HEMA surface is ready for protein adsorption or cell plating. This method allows for the fabrication of 0.3 µm thin layers of p‐HEMA, which can be etched to 10 µm wide channels. Furthermore, it creates regions of differential protein adhesion, as shown by Coomassie staining and fluorescent labeling, and cell adhesion, as demonstrated by C2C12 myoblast growth. This method is simple, versatile, and allows biologists and bioengineers to manipulate regions for cell culture adhesion and growth. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:243–248, 2018     

Probing morphological,genetic and metabolomic changes of in vitro embryo development in a microfluidic device

Assisted reproduction technologies for clinical and research purposes rely on a brief in vitro embryo culture which, despite decades of progress, remain suboptimal in comparison to the physiological environment. One promising tool to improve this technique is the development of bespoke microfluidic chambers. Here we present and validate a new microfluidic device in polydimethylsiloxane (PDMS) for the culture of early mouse embryos. Device material and design resulted embryo compatible and elicit minimal stress. Blastocyst formation, hatching, attachment and outgrowth formation on fibronectin-coated devices were similar to traditional microdrop methods. Total blastocyst cell number and allocation to the trophectoderm and inner cell mass lineages were unaffected. The devices were designed for culture of 10–12 embryos. Development rates, mitochondrial polarization and metabolic turnover of key energy substrates glucose, pyruvate and lactate were consistent with groups of 10 embryos in microdrop controls. Increasing group size to 40 embryos per device was associated with increased variation in development rates and altered metabolism. Device culture did not perturb blastocyst gene expression but did elicit changes in embryo metabolome, which can be ascribed to substrate leaching from PDMS and warrant further investigation.     

PDMS device for patterned application of microfluids to neuronal cells arranged by microcontact printing.

A microfluidic device in polydimethylsiloxane (PDMS) consisting of an eight lines micro-injection array integrated in a base flow channel has been realized. The device is assembled from multiple PDMS parts, which have been moulded using notably micromachined masters in SU-8 photoresist. In contact with a planar substrate, up to eight independent laminar flow lines with cross-sections of 100 x 200 microm(2) can be generated. Dedicated for the application of pharmaceutical compounds to electrogenic cells in vitro, this device was tested with a neuronal cell line, Mz1-cells. These were cultured on lines of laminin deposited onto polystyrene substrates by microcontact printing. We were able to inject into this culture multiple lines of coloured PBS in parallel to the orientation of cellular growth. No mixing between the individual flow lines did occur.     

Tobacco protoplast culture in a polydimethylsiloxane-based microfluidic channel

Summary. Several advances have been made in the use of microfluidic devices for insect and mammalian cell cultures, but no reports of their use for plant cell cultures have been published. We, therefore, conducted a plant cell culture in a microfluidic device using polydimethylsiloxane. Nicotiana tabacum protoplasts were cultured in a variously shaped polydimethylsiloxane channel containing Nitsch medium supplemented with 0.5 g of NLN-13 vitamin mixture, 2.0 mg of α-naphthaleneacetic acid, and 0.5 mg of 6-benzyladenine per liter and 9% mannitol. Protoplasts in the polydimethylsiloxane channel showed cell division and microcolony formation within 4 weeks. The use of a microfluidic channel is a novel technique in the field of plant cell culture. The results of this study will encourage the utilization of polydimethylsiloxane-based microfluidic devices in plant cell engineering and cell analysis. Correspondence and reprints: Department of Biology, Dankook University, 29 San Anseo-dong, Cheonan 300-714, South Korea.     

Over a Century of Neuron Culture: From the Hanging Drop to Microfluidic Devices

The brain is the most intricate, energetically active, and plastic organ in the body. These features extend to its cellular elements, the neurons and glia. Understanding neurons, or nerve cells, at the cellular and molecular levels is the cornerstone of modern neuroscience. The complexities of neuron structure and function require unusual methods of culture to determine how aberrations in or between cells give rise to brain dysfunction and disease. Here we review the methods that have emerged over the past century for culturing neurons in vitro, from the landmark finding by Harrison (1910) — that neurons can be cultured outside the body — to studies utilizing culture vessels, micro-islands, Campenot and brain slice chambers, and microfluidic technologies. We conclude with future prospects for neuronal culture and considerations for advancement. We anticipate that continued innovation in culture methods will enhance design capabilities for temporal control of media and reagents (chemotemporal control) within sub-cellular environments of three-dimensional fluidic spaces (microfluidic devices) and materials (e.g., hydrogels). They will enable new insights into the complexities of neuronal development and pathology.     

Constraining the connectivity of neuronal networks cultured on microelectrode arrays with microfluidic techniques: a step towards neuron-based functional chips

In vitro culture of small neuronal networks with pre-defined topological features is particularly desirable when the electrical activity of such assemblies can be monitored for long periods of time. Indeed, it is hoped that such networks, with pre-determined connectivity, will provide unique insights into the structure/function relationship of biological neural networks and their properties of self-organization. However, the experimental techniques that have been developed so far for that purpose have either failed to provide very long-term pattern definition and retention, or they have not shown potential for integration into more complex microfluidic devices. To address this problem, three-dimensional microfluidic systems in poly(dimethylsiloxane) (PDMS) were fabricated and used in conjunction with both custom-made and commercially available planar microelectrode arrays (pMEAs). Various types of primary neuronal cell cultures were established inside these systems. Extracellular electrical signals were successfully recorded from all types of cells placed inside the patterns, and this bioelectrical activity was present for several weeks. The advantage of this approach is that it can be further integrated with microfluidic devices and pMEAs to yield, for example, complex neuron-based biosensors or chips for pharmacological screening.     

Cell placement and guidance on substrates for neurochip interfaces

Interface devices such as integrated planar patch‐clamp chips are being developed to study the electrophysiological activity of neuronal networks grown in vitro. The utility of such devices will be dependent upon the ability to align neurons with interface features on the chip by controlling neuronal placement and by guiding cell connectivity. In this paper, we present a strategy to accomplish this goal. Patterned chemical modification of SiN surfaces with poly‐d‐lysine transferred from PDMS stamps was used to promote adhesion and guidance of cryo‐preserved primary rat cortical neurons. We demonstrate that these neurons can be positioned and grown over microhole features which will ultimately serve as patch‐clamp interfaces on the chip. Biotechnol. Bioeng. 2010; 105: 368–373. © 2009 Wiley Periodicals, Inc.     

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.     

Compartmental culture of embryonic stem cell-derived neurons in microfluidic devices for use in axonal biology

Axonal pathology has been clearly implicated in neurodegenerative diseases making the compartmental culture of neurons a useful research tool. Primary neurons have already been cultured in compartmental microfluidic devices but their derivation from an animal is a time-consuming and difficult work and has a limit in their sources. Embryonic stem cell (ESC)-derived neurons (ESC_Ns) overcome this limit, since ESCs can be renewed without limit and can be differentiated into ESC_Ns by robust and reproducible protocols. In this research, ESC_Ns were derived from mouse ESCs in compartmental microfluidic devices, and their axons were isolated from the somal cell bodies. Once embryoid bodies (EBs) were localized in the microfluidic culture chamber, ESC_Ns spread out from the EBs and occupied the cell culture chamber. Their axons traversed the microchannels and finally were isolated from the somata, providing an arrangement comparable to dissociated primary neurons. This ESC_N compartmental microfluidic culture system not only offers a substitute for the primary neuron counterpart system but also makes it possible to make comparisons between the two systems.     

Fabrication of a circular PDMS microchannel for constructing a three-dimensional endothelial cell layer

We describe a simple and efficient fabrication method for generating microfluidic channels with a circular cross-sectional geometry by exploiting the reflow phenomenon of a thick positive photoresist. Initial rectangular shaped positive photoresist micropatterns on a silicon wafer, which were fabricated by a conventional photolithography process, were converted into a half-circular shape by tuning the temperature to around 105 °C. Through optimization of the reflow conditions, we could obtain a perfect circular micropattern of the positive photoresist, and control the diameter in a range from 100 to 400 μm. The resultant convex half-circular photoresist was used as a template for fabricating a concave polydimethylsiloxane (PDMS) through a replica molding process, and a circular PDMS microchannel was produced by bonding two half-circular PDMS layers. A variety of channel dimensions and patterns can be easily prepared, including straight, S-curve, X-, Y-, and T-shapes to mimic an in vivo vascular network. To form an endothelial cell layer, we cultured primary human umbilical vein endothelial cells inside circular PDMS microchannels, and demonstrated successful cell adhesion, proliferation, and alignment along the channel.     

Microfluidic PDMS (polydimethylsiloxane) bioreactor for large-scale culture of hepatocytes

Microfluidics could provide suitable environments for cell culture because of the larger surface-to-volume ratio and fluidic behavior similar to the environments in vivo. Such microfluidic environments are now used to investigate cell-to-cell interactions and behaviors in vitro, emulating situations observed in vivo, for example, microscale blood vessels modeled by microfluidic channels. These emulated situations cannot be realized by conventional technologies. In our previous works, microfluidic channels composed of two PDMS (poly(dimethylsiloxane)) layers were successfully used for Hep G2 cell culture. To achieve physiologically meaningful functions in vitro, a culture with a larger number of cells and higher density must be performed. This will require bioreactors with larger surface areas for cell attachment and sufficient amounts of oxygen and nutrition supply. For those purposes, we fabricated a bioreactor by stacking 10 PDMS layers together, i.e., four cell culture chambers, and a chamber dedicated to the oxygen supply inserted in the middle of the 10-stacked layers. The oxygen supply chamber is separated from the microfluidic channels for the culture medium perfusion by thin 300-microm PDMS walls. The high gas permeability of PDMS allows oxygen supply to the microfluidic channels through the thin walls. On the basis of the measurement of glucose consumption and albumin production, it is shown that cellular activity exhibits a gradual increase and saturation throughout the culture. We clearly observed that in the case of the microfluidic bioreactor for large-scale cultures, the oxygen chamber is indispensable to achieve longer and healthy cultures. In the present bioreactor, the cell density was found to be about 3-4 x 10(7) cells/cm(3), which is in the same order of magnitude as the conventional macroscale bioreactors. Consequently, by stacking single culture chambers and oxygen chambers in between, we could have a scalable method to realize the microfluidic bioreactor for large-scale cultures.     

Determinants of Leukocyte Margination in Rectangular Microchannels

Microfabrication of polydimethylsiloxane (PDMS) devices has provided a new set of tools for studying fluid dynamics of blood at the scale of real microvessels. However, we are only starting to understand the power and limitations of this technology. To determine the applicability of PDMS microchannels for blood flow analysis, we studied white blood cell (WBC) margination in channels of various geometries and blood compositions. We found that WBCs prefer to marginate downstream of sudden expansions, and that red blood cell (RBC) aggregation facilitates the process. In contrast to tubes, WBC margination was restricted to the sidewalls in our low aspect ratio, pseudo-2D rectangular channels and consequently, margination efficiencies of more than 95% were achieved in a variety of channel geometries. In these pseudo-2D channels blood rheology and cell integrity were preserved over a range of flow rates, with the upper range limited by the shear in the vertical direction. We conclude that, with certain limitations, rectangular PDMS microfluidic channels are useful tools for quantitative studies of blood rheology.     

A gradient-generating microfluidic device for cell biology

The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described. A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients. Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles. Here, we developed simple gradient-generating microfluidic devices with three separate inlets. Three microchannels combined into one microchannel to generate concentration gradients. The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF). Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations. The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis. Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.     

Fabrication of a microfluidic device for the compartmentalization of neuron soma and axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80 degrees C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.     

Development of low-cost microfluidic systems for lab-on-a-chip biosensor applications

In this work, we develop low-cost microfluidic systems based on polydimethylsiloxane (PDMS) for lab-on-a-chip applications. PDMS microfluidic structures have been fabricated by micromolding, PDMS casting, and plasma bonding processes. The micromolding technique is used to fabricate PDMS slabs with micro-sized grooves, and the complete microchannel is formed by bonding PDMS slab with glass or PDMS substrate. The molding procedure using SU-8 photoresist patterning on silicon wafer, PDMS microchannel fabrication, and PDMS surface treatment using oxygen plasma and TiO₂ coating, are discussed. The various parameters for oxygen plasma treatment including RF power and treatment time are studied in order to obtain conditions for good bonding with the glass substrate. The best condition for plasma treatment is found to be the low RF power (8 W) with 5 min treatment time. In addition, TiO₂ coating with oxygen plasma treatment has been applied to make PDMS surface more hydrophilic to improve aqueous solution compatilbility. The microfluidic channels for various applications, including sample injection cross channel, micropump channel, T and Y sample mixers, PCR thermocyling chamber and channel, capillary electrophoresis flow channel, and conductimetric systems have been fabricated. Finally, a typical application of the PDMS chip in a flow injection conductimetric system for sodium chloride detection has been demonstrated.     

In vitro peptidergic neurons from the adult locust pars intercerebralis: Morphological and immunocytological studies

Primary cell cultures were prepared from a major neurosecretory center of the adult locust brain, the pars intercerebralis, in order to characterize neurosecretory cells growingin vitro. Individual pars intercerebralis could be removed free of surrounding tissue and dissociated by mechanical treatment. Mature neurosecretory neurons of different sizes regenerate new neurites during the initial three daysin vitro in serum-free medium. They show a tendency to sprout one primary neurite from which fine processes develop. By means of electron microscopy, we observed the integrity of the cellular organelles, indicating that cultured neurons are healthy, and we were able to distinguish three types of neurosecretory neurons on the basis of the ultrastructural aspects of the neurosecretory material. These three types have the same ultrastructural characteristics asin situ neuroparsin, ovary maturing parsin and locust insulin related peptide neurons. Immunogold labelling at the electron microscopic level, using the two available specific antibodies, anti-neuroparsin and anti-ovary maturing parsin, confirms the morphological characterization of neuroparsin and ovary maturing parsin cells. These results show for the first time that cultured locust neurosecretory neurons behave like thosein vivo, in terms of their ultrastructure and immunocytochemistry. Moreover, the presence of recently-formed neurosecretory material both in the Golgi zone of the perikaryon and in the neuronal processes indicates that cultured neurons have functional capacity since they are able to synthesizede novo and to transport the neurosecretory material along the neurite. Thus our well-characterized culture system provides a suitable invitro model to investigate the secretory mechanism of locust neurosecretory neurons.     

Possible roles for cell-to-substratum adhesion in neuronal morphogenesis

The role of cell-to-substratum adhesion in the initiation, elongation, and branching of axons from embryonic sensory neurons was investigated. Cells from sensory ganglia of 4–8-day-old chicken embryos were cultured on several substrata: including collagen; polyornithine-, polylysine-, and polyglutamate-coated surfaces, and tissue culture dishes. The air-blaster method was used to measure growth cone-substratum adhesion.Growth cones adhere much more strongly to polyornithine- or polylysine-coated surfaces and to the upper surfaces of glial cells than to tissue culture plastic. Axons, too, adhere tightly to these substrata, and are crooked, whereas on tissue culture plastic, axons are not adherent and are straight. The fraction of neurons that form axons and the rates of axonal elongation and branching are markedly increased when cells are cultured on polyornithine-coated dishes as compared to tissue culture dishes.This correlation of strong adhesion and enhanced neuronal morphogenesis suggests that adhesive interactions between the growth cone and the microenvironment in an embryo are crucial parts of the initiation and elongation of neuronal processes. Regulation of neuronal morphogenesis may be expressed through the physicochemical properties of the interacting cell surfaces and extracellular environment.