Publications – Connect

2023

Microenvironments Matter: Advances in Brain-on-Chip

Summary
To highlight the particular needs with respect to modeling the unique and complex organization of the human brain structure, we reviewed the state-of-the-art in devising brain models with engineered instructive microenvironments. To acquire a better perspective on the brain’s working mechanisms, we first summarize the importance of regional stiffness gradients in brain tissue, varying per layer and the cellular diversities of the layers. Through this, one can acquire an understanding of the essential parameters in emulating the brain in vitro. In addition to the brain’s organizational architecture, we addressed also how the mechanical properties have an impact on neuronal cell responses. In this respect, advanced in vitro platforms emerged and profoundly changed the methods of brain modeling efforts from the past, mainly focusing on animal or cell line research. The main challenges in imitating features of the brain in a dish are with regard to composition and functionality. In neurobiological research, there are now methods that aim to cope with such challenges by the self-assembly of human-derived pluripotent stem cells (hPSCs), i.e., brainoids. Alternatively, these brainoids can be used stand-alone or in conjunction with Brain-on-Chip (BoC) platform technology, 3D-printed gels, and[…]

2022

Ascorbic acid does not necessarily interfere with the electrochemical detection of dopamine

Summary

It is widely stated that ascorbic acid (AA) interferes with the electrochemical detection of neurotransmitters, especially dopamine, because of their overlapping oxidation potentials on typical electrode materials. As the concentration of AA is several orders of magnitude higher than the concentration of neurotransmitters, detection of neurotransmitters is difficult in the presence of AA and requires either highly stable AA concentration or highly selective neurotransmitter sensors. In contrast to the common opinion, we show that AA does not always interfere electrochemical detection of neurotransmitters. The decay of AA is rapid in cell culture medium, having a half-time of 2.1 hours, according to which the concentration decreases by 93% in 8 hours and by 99.75% in 18 hours. Thus, AA is eventually no longer detected by electrodes and the concentration of neurotransmitters can be effectively monitored. To validate this claim, we used unmodified single-wall carbon nanotube electrode to measure dopamine at physiologically relevant concentration range (25–1000 nM) from human midbrain organoid medium with highly linear response. Finally, AA is known to affect dopamine oxidation current through regeneration of dopamine, which complicates precise detection of small amounts of dopamine. By designing experiments as described here, this complication can be completely eliminated.

Read more: https://www.nature.com/articles/s41598-022-24580-0

2022

Early anteroposterior regionalisation of human neural crest is shaped by a pro-mesodermal factor

Summary

The neural crest (NC) is an important multipotent embryonic cell population and its impaired specification leads to various developmental defects, often in an anteroposterior (A-P) axial level-specific manner. The mechanisms underlying the correct A-P regionalisation of human NC cells remain elusive. Recent studies have indicated that trunk NC cells, the presumed precursors of childhood tumour neuroblastoma, are derived from neuromesodermal-potent progenitors of the postcranial body. Here we employ human embryonic stem cell differentiation to define how neuromesodermal progenitor (NMP)-derived NC cells acquire a posterior axial identity. We show that TBXT, a pro-mesodermal transcription factor, mediates early posterior NC/spinal cord regionalisation together with WNT signalling effectors. This occurs by TBXT-driven chromatin remodelling via its binding in key enhancers within HOX gene clusters and other posterior regulator-associated loci. This initial posteriorisation event is succeeded by a second phase of trunk HOX gene control that marks the differentiation of NMPs toward their TBXT-negative NC/spinal cord derivatives and relies predominantly on FGF signalling. Our work reveals a previously unknown role of TBXT in influencing posterior NC fate and points to the existence of temporally discrete, cell type-dependent modes of posterior axial identity control.

Read more: https://elifesciences.org/articles/74263#content

2022

Nanofabricating neural networks: Strategies, advances, and challenges

Summary

Nanofabrication can help us to emulate natural intelligence. Forward-engineering brain gained enormous momentum but still falls short in human neurodegenerative disease modeling. Here, organ-on-chip (OoC) implementation of tissue culture concepts in microfluidic formats already progressed with the identification of our knowledge gap in toxicology and drug metabolism studies. We believe that the self-organization of stem cells and chip technology is a key to advance such complex in vitro tissue models, including models of the human nervous system as envisaged in this review. However, current cultured networks of neurons show limited resemblance with the biological functions in the real nervous system or brain tissues. To take full advantage of scaling in the engineering domain of electron-, ion-, and photon beam technology and nanofabrication methods, more research is needed to meet the requirements of this specific field of chip technology applications. So far, surface topographies, microfluidics, and sensor and actuator integration concepts have all contributed to the patterning and control of neural network formation processes in vitro. However, when probing the state of the art for this type of miniaturized three-dimensional tissue models in PubMed, it was realized that there is very little systematic cross-disciplinary research with biomaterials originally formed for tissue engineering purposes translated to on-chip solutions for in vitro modeling. Therefore, this review contributes to the formulation of a sound design concept based on the understanding of the existing knowledge and the technical challenges toward finding better treatments and potential cures for devastating neurodegenerative diseases, like Parkinson’s disease. Subsequently, an integration strategy based on a modular approach is proposed for nervous system-on-chip (NoC) models that can yield efficient and informative optical and electronic NoC readouts in validating and optimizing these conceptual choices in the innovative process of a fast growing and exciting new OoC industry.

Read more: https://doi.org/10.1116/6.0001649

2022

Summary

The production of the tissues that make up the mammalian embryonic trunk takes place in a head-tail direction, via the differentiation of posteriorly-located axial progenitor populations. These include bipotent neuromesodermal progenitors (NMPs), which generate both spinal cord neurectoderm and presomitic mesoderm, the precursor of the musculoskeleton. Over the past few years, a number of studies have described the derivation of NMP-like cells from mouse and human pluripotent stem cells (PSCs). In turn, these have greatly facilitated the establishment of PSC differentiation protocols aiming to give rise efficiently to posterior mesodermal and neural cell types, which have been particularly challenging to produce using previous approaches. Moreover, the advent of 3-dimensional-based culture systems incorporating distinct axial progenitor-derived cell lineages has opened new avenues toward the functional dissection of early patterning events and cell vs non-cell autonomous effects. Here, we provide a brief overview of the applications of these cell types in disease modelling and cell therapy and speculate on their potential uses in the future.

2022

Nanoscale geometry determines mechanical biocompatibility of vertically aligned nanofibers

Summary

Vertically aligned carbon nanofibers (VACNFs) are promising material candidates for neural biosensors due to their ability to detect neurotransmitters in physiological concentrations. However, the expected high rigidity of CNFs could induce mechanical mismatch with the brain tissue, eliciting formation of a glial scar around the electrode and thus loss of functionality. We have evaluated mechanical biocom- patibility of VACNFs by growing nickel-catalyzed carbon nanofibers of different lengths and inter-fiber distances. Long nanofibers with large inter-fiber distance prevented maturation of focal adhesions, thus constraining cells from obtaining a highly spread morphology that is observed when astrocytes are be- ing contacted with stiff materials commonly used in neural implants. A silicon nanopillar array with 500 nm inter-pillar distance was used to reveal that this inhibition of focal adhesion maturation occurs due to the surface nanoscale geometry, more precisely the inter-fiber distance. Live cell atomic force mi- croscopy was used to confirm astrocytes being significantly softer on the long Ni-CNFs compared to other surfaces, including a soft gelatin hydrogel. We also observed hippocampal neurons to mature and form synaptic contacts when being cultured on both long and short carbon nanofibers, without having to use any adhesive proteins or a glial monoculture, indicating high cytocompatibility of the material also with neuronal population. In contrast, neurons cultured on a planar tetrahedral amorphous carbon sample showed immature neurites and indications of early-stage apoptosis. Our results demonstrate that me- chanical biocompatibility of biomaterials is greatly affected by their nanoscale surface geometry, which provides means for controlling how the materials and their mechanical properties are perceived by the cells.

2021

Shaping axial identity during human pluripotent stem cell differentiation to neural crest cells

Summary

The neural crest (NC) is a multipotent cell population which can give rise to a vast array of derivatives including neurons and glia of the peripheral nervous system, cartilage, cardiac smooth muscle, melanocytes and sympathoadrenal cells. An attractive strategy to model human NC development and associated birth defects as well as produce clinically relevant cell populations for regenerative medicine applications involves the in vitro generation of NC from human pluripotent stem cells (hPSCs). However, in vivo, the potential of NC cells to generate distinct cell types is determined by their position along the anteroposterior (A–P) axis and, therefore the axial identity of hPSC-derived NC cells is an important aspect to consider. Recent advances in understanding the developmental origins of NC and the signalling pathways involved in its specification have aided the in vitro generation of human NC cells which are representative of various A–P positions. Here, we explore recent advances in methodologies of in vitro NC specification and axis patterning using hPSCs.

2021

Investigating the pattern transfer fidelity of Norland Optical Adhesive 81 for nanogrooves by microtransfer molding.

Summary

We demonstrated the microtransfer molding of Norland Optical Adhesive 81 (NOA81) thin films. NOA81 nanogrooves and flat thin films were transferred from a flexible polydimethylsiloxane (PDMS) working mold. In the case of nanogrooves, the mold’s feature area of 15 × 15 mm² contains a variety of pattern dimensions in a set of smaller nanogroove fields of a few mm² each. We demonstrated that at least six microtransfers can be performed from the same PDMS working mold. Within the restriction of our atomic force microscopy measurement technique, nanogroove height varies with 82 ± 11 nm depending on the pattern dimensions of the measured fields. Respective micrographs of two of these fields, i.e., one field designated with narrower grooves (D1000L780, case 1) and the other designated with wider grooves (D1000L230, case 2) but with the same periodicity values, demonstrate faithful transfer of the patterns. The designated pattern dimensions refer to the periodicity (D) and the ridge width (L) in the original design process of the master mold (dimensional units are nm). In addition, neither NOA81 itself (flat films) nor NOA81 nanogroove thin films with a thickness of 1.6 μm deteriorate the imaging quality in optical cell microscopy.

Read more:

https://doi.org/10.1116/6.0001333

In Vitro Generation of Posterior Motor Neurons from Human Pluripotent Stem Cells

The ability to generate spinal cord motor neurons from human pluripotent stem cells (hPSCs) is of great use for modelling motor neuron–based diseases and cell-replacement therapies. A key step in the design of hPSC differentiation strategies aiming to produce motor neurons involves induction of the appropriate anteroposterior (A-P) axial identity, an important factor influencing motor neuron subtype specification, functionality, and disease vulnerability. Most current protocols for induction of motor neurons from hPSCs produce predominantly cells of a mixed hindbrain/cervical axial identity marked by expression of Hox paralogous group (PG) members 1-5, but are inefficient in generating high numbers of more posterior thoracic/lumbosacral Hox PG(8-13)⁺ spinal cord motor neurons. Here, we describe a protocol for efficient generation of thoracic spinal cord cells and motor neurons from hPSCs. This step-wise protocol relies on the initial generation of a neuromesodermal-potent axial progenitor population, which is differentiated first to produce posterior ventral spinal cord progenitors and subsequently to produce posterior motor neurons exhibiting a predominantly thoracic axial identity. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.

Generating Enteric Nervous System Progenitors from Human Pluripotent Stem Cells

The intrinsic innervation of the gastrointestinal (GI) tract is comprised of enteric neurons and glia, which are buried within the wall of the bowel and organized into two concentric plexuses that run along the length of the gut forming the enteric nervous system (ENS). The ENS regulates vital GI functions including gut motility, blood flow, fluid secretion, and absorption and thus maintains gut homeostasis. During vertebrate development it originates predominantly from the vagal neural crest (NC), a multipotent cell population that emerges from the caudal hindbrain region, migrates to and within the gut to ultimately generate neurons and glia in response to gut-derived signals. Loss of GI innervation due to congenital or acquired defects in ENS development causes enteric neuropathies which lack curative treatment. Human pluripotent stem cells (hPSCs) offer a promising in vitro source of enteric neurons for modeling human ENS development and pathology and potential use in cell therapy applications. Here we describe in detail a differentiation strategy for the derivation of enteric neural progenitors and neurons from hPSCs through a vagal NC intermediate. Using a combination of instructive signals and retinoic acid in a dose/time dependent manner, vagal NC cells commit into the ENS lineage and develop into enteric neurons and glia upon culture in neurotrophic media. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.

3-dimensional (3D) cell cultures better mimic natural environment of cells than 2-dimensional (2D) cell cultures to obtain in vivo like inter and intracellular responses. However, third dimension brings complexity to cell culture. Therefore, high-resolution/high-content screening in 3D is one of the most important challenges with this type of cell cultures. Although optical monitoring techniques, well-established in 2D area, are enhanced to monitor 3D cell cultures, they are generally endpoint, static, time inefficient, and labor intensive. Alternatively, electrical sensing can become a solution to achieve dynamic, real-time, and label-free monitoring of cells in both 2D and 3D cell cultures. Developments in electrical monitoring of cell culture have led to novel approaches, proposed by adapting fundamentals of 2D electrical techniques to 3D to obtain high spatiotemporal systems. In this review, we classified these approaches into five main groups: (i) 3D impedance measurement approach (ii) electrical impedance tomography, (iii) 3D microelectrode array approach, (iv) 3D nanoelectronics scaffold approach, and (v) microphysiometry. We also defined the challenges in the adaptation of electrical monitoring techniques to 3D cultures and explained possible solutions in terms of specific applications and technical point of views, including methods particular to our group. In conclusion, 3D electrical monitoring in cell cultures is considerably challenging but highly accelerated recently by significant advances of microfabrication technology, bioengineering, and material science. Novel approaches reviewed here have a lot of potential and offer opportunities for further developments to find solutions, fit to serve the (bio)medical needs.

Analyzing Developing Brain-On-Chip Cultures with the CALIMA Calcium Imaging Tool

Brain-on-chip (BoC) models are tools for reproducing the native microenvironment of neurons, in order to study the (patho)physiology and drug-response of the brain. Recent developmentsin BoC techniques focus on steering neurons in their activity via microfabrication and via computer-steered feedback mechanisms. These cultures are often studied through calcium imaging (CI), a method for visualizing the cellular activity through infusing cells with a fluorescent dye. CAlciumImagingAnalyser 2.0 (CALIMA 2.0) is an updated version of a software tool that detects and analyzes fluorescent signals and correlates cellular activity to identify possible network formation in BoC cultures. Using three previous published data sets, it was demonstrated that CALIMA 2.0 can analyze large data sets of CI-data and interpret cell activity to help study the activity and maturity of BoC cultures. Last, an analysis of the processing speed shows that CALIMA 2.0 is sufficiently fast to process data sets with an acquisition rate up to 5 Hz in real-time on a medium-performance computer.

Defining the signalling determinants of a posterior ventral spinal
cord identity in human neuromesodermal progenitor derivatives

The anteroposterior axial identity of motor neurons (MNs) determines their functionality and vulnerability to neurodegeneration. Thus, it is a crucial parameter in the design of strategies aiming to produce MNs from human pluripotent stem cells (hPSCs) for regenerative medicine/disease modelling applications. However, the in vitro generation of posterior MNs corresponding to the thoracic/lumbosacral spinal cord has been challenging. Although the induction of cells resembling neuromesodermal progenitors (NMPs), the bona fide precursors of the spinal cord, offers a promising solution, the progressive specification of posterior MNs from these cells is not well defined. Here, we determine the signals guiding the transition of human NMP-like cells toward thoracic ventral spinal cord neurectoderm. We show that combined WNT-FGF activities drive a posterior dorsal pre-/early neural state, whereas suppression of TGFβ-BMP signalling pathways promotes a ventral identity and neural commitment. Based on these results, we define an optimised protocol for the generation of thoracic MNs that can efficiently integrate within the neural tube of chick embryos. We expect that our findings will facilitate the comparison of hPSC-derived spinal cord cells of distinct axial identities.

Stiff-to-Soft Transition from Glass to 3D Hydrogel Substrates in Neuronal Cell Culture

Summary

Over the past decade, hydrogels have shown great potential for mimicking three- dimensional (3D) brain architectures in vitro due to their biocompatibility, biodegradability, and wide range of tunable mechanical properties. To better comprehend in vitro human brain models and the mechanotransduction processes, we generated a 3D hydrogel model by casting photo-polymerized gelatin methacryloyl (GelMA) in comparison to poly (ethylene glycol) diacrylate (PEGDA) atop of SH-SY5Y neuroblastoma cells seeded with 150,000 cells/cm² according to our previous experience in a microliter-sized polydimethylsiloxane (PDMS) ring serving for confinement. 3D SH-SY5Y neuroblastoma cells in GelMA demonstrated an elongated, branched, and spreading morphology resembling neurons, while the cell survival in cast PEGDA was not supported. Confocal z-stack microscopy confirmed our hypothesis that stiff-to-soft material transitions promoted neuronal migration into the third dimension. Unfortunately, large cell aggregates were also observed. A subsequent cell seeding density study revealed a seeding cell density above 10,000 cells/cm² started the formation of cell aggregates, and below 1500 cells/cm² cells still appeared as single cells on day 6. These results allowed us to conclude that the optimum cell seeding density might be between 1500 and 5000 cells/cm². This type of hydrogel construct is suitable to design a more advanced layered mechanotransduction model toward 3D microfluidic brain-on-a-chip applications.

Three-Dimensional Fine Structure of Nanometer-Scale Nafion Thin
Films

Summary

Nafion is a widely used polymer membrane in various applications ranging from advanced energy solutions to sensing of biomolecules. Despite the intensive research carried out over the years to reveal and understand the fine structure of Nafion, its structural features, especially as nanometer-scale films, are not unambiguously known. In this paper, we use room temperature scanning transmission electron microscopy (STEM) tomography complemented by glancing incidence small-angle X-ray scattering (GISAXS) and TEM at low temperatures to reveal the fine structure of thin (10−100 nm) unannealed Nafion films. The results from the detailed three-dimensional reconstructions obtained show that (i) the phase fractions of the hydrophobic and hydrophilic parts of the polymer are somewhat thickness-dependent, changing from 0.65/0.35 to about 0.7/0.3 when moving from 100 to 10 nm thick films; (ii) the channel diameters show a range of values from 3 to 6 nm in all the films independent of their thickness; (iii) the average distances between the hydrophilic channels inside the film have distributions centered around 12 nm (in 10 nm films), 15 nm (in 30 nm films), and 7 nm (in 100 nm films); (iv) in the thickest films, the hydrophilic channels exhibit higher interconnectivity and some of the channels appear to end within the Nafion film instead of going through the films; and (v) there are some confinement effects caused by the hydrophilic SiO2 surface in the case of 10 and 30 nm thick films shown by the tendency of the hydrophilic channels to move horizontally near the substrate. Furthermore, a stable room temperature STEM tomography imaging method for Nafion films and a sample preparation method that preserves the characteristics of the hydrated morphology of Nafion in the dry state are demonstrated. These results provide a deeper understanding of the fine structure of Nafion thin films and provide a better means to characterize and understand their properties in different applications.

Trends in Carbon, Oxygen, and Nitrogen Core in the X-ray Absorption Spectroscopy of Carbon Nanomaterials: A Guide for the Perplexed

Summary

Successful deployment of carbon nanomaterials in many applications, such as sensing, energy storage, and catalysis, relies on the selection, synthesis, and tailoring of the surface properties. Predictive analysis of the behavior is difficult without detailed knowledge of the differences between various carbon nanomaterials and their surface functionalization, thus leaving the selection process to traditional trial-and-error work. The present characterization fills this knowledge gap for carbon nanomaterial surface properties with respect to chemical states and functionalization. We present an overview of the chemical trends that can be extracted from soft X-ray absorption spectroscopy (XAS) spectra on an extended set of nonideal carbon nanomaterials as a function of sp² bonded carbon and bond ordering. In particular, the surface chemical state, the presence of long-range order in the carbon matrix, and a qualitative estimation of the amount of oxygen and nitrogen and their respective functional group formation on the material surface, together with the detailed material fabrication parameters, are reported. The results expand our understanding of carbon nanomaterial functionalization, which can support material selection in practice, provided that the specifications of the application are known.

Gut innervation and enteric nervous system development: a spatial, temporal and molecular tour de force

Summary

During embryonic development, the gut is innervated by intrinsic (enteric) and extrinsic nerves. Focusing on mammalian ENS development, in this Review we highlight how important the different compartments of this innervation are to assure proper gut function. We specifically address the three-dimensional architecture of the innervation, paying special attention to the differences in development along the longitudinal and circumferential axes of the gut. We review recent information about the formation of both intrinsic innervation, which is fairly well-known, as well as the establishment of the extrinsic innervation, which, despite its importance in gut-brain signaling, has received much less attention. We further discuss how external microbial and nutritional cues or neuroimmune interactions may influence development of gut innervation. Finally, we provide summary tables, describing the location and function of several well-known molecules, along with some newer factors that have more recently been implicated in the development of gut innervation.

Gaining Micropattern Fidelity in an NOA81 Microsieve Laser Ablation Process

Summary

We studied the micropattern fidelity of a Norland Optical Adhesive 81 (NOA81) microsieve made by soft-lithography and laser micromachining. Ablation opens replicated cavities, resulting in three-dimensional (3D) micropores. We previously demonstrated that microsieves can capture cells by passive pumping. Flow, capture yield, and cell survival depend on the control of the micropore geometry and must yield high reproducibility within the device and from device to device. We investigated the NOA81 film thickness, the laser pulse repetition rate, the number of pulses, and the beam focusing distance. For NOA81 films spin-coated between 600 and 1200 rpm, the pulse number controls the breaching of films to form the pore’s aperture and dominates the process. Pulse repetition rates between 50 and 200 Hz had no observable influence. We also explored laser focal plane to substrate distance to find the most effective ablation conditions. Scanning electron micrographs (SEM) of focused ion beam (FIB)-cut cross sections of the NOA81 micropores and inverted micropore copies in polydimethylsiloxane (PDMS) show a smooth surface topology with minimal debris. Our studies reveal that the combined process allows for a 3D micropore quality from device to device with a large enough process window for biological studies.

Integrated, automated maintenance, expansion and differentiation of 2D and 3D patient-derived cellular models for high throughput drug screening

Summary

Patient-derived cellular models become an increasingly powerful tool to model human diseases for precision medicine approaches. The identification of robust cellular disease phenotypes in these models paved the way towards high throughput screenings (HTS) including the implementation of laboratory advanced automation. However, maintenance and expansion of cells for HTS remains largely manual work. Here, we describe an integrated, complex automated platform for HTS in a translational research setting also designed for maintenance and expansion of different cell types. The comprehensive design allows automation of all cultivation steps and is flexible for development of methods for variable cell types. We demonstrate protocols for controlled cell seeding, splitting and expansion of human fibroblasts, induced pluripotent stem cells (iPSC), and neural progenitor cells (NPC) that allow for subsequent differentiation into different cell types and image-based multiparametric screening. Furthermore, we provide automated protocols for neuronal differentiation of NPC in 2D culture and 3D midbrain organoids for HTS. The flexibility of this multitask platform makes it an ideal solution for translational research settings involving experiments on different patient-derived cellular models for precision medicine.

2020

Nanogrooves for 2D and 3D Microenvironments of SH-SY5Y Cultures in Brain-on-Chip Technology

Summary

Brain-on-chip (BOC) technology such as nanogrooves and microtunnel structures can advance in vitro neuronal models by providing a platform with better means to maintain, manipulate and analyze neuronal cell cultures. Specifically, nanogrooves have been shown to influence neuronal differentiation, notably the neurite length and neurite direction. Here, we have drawn new results from our experiments using both 2D and 3D neuronal cell culture implementing both flat and nanogrooved substrates. These are used to show a comparison between the number of cells and neurite length as a first indicator for valuable insights into baseline values and expectations that can be generated from these experiments toward design optimization and predictive value of the technology in our BOC toolbox. Also, as a new step toward neuronal cell models with multiple compartmentalized neuronal cell type regions, we fabricated microtunnel devices bonded to both flat and nanogrooved substrates to assess their compatibility with neuronal cell culture. Our results show that with the current experimental protocols using SH-SY5Y cells, we can expect 200 – 400 cells with a total neurite length of approximately 4,000–5,000 μm per 1 mm² within our BOC devices, with a lower total neurite length for 3D neuronal cell cultures on flat substrates only. There is a statistically significant difference in total neurite length between 2D cell culture on nanogrooved substrates versus 3D cell culture on flat substrates. As extension of our current BOC toolbox for which these indicative parameters would be used, the microtunnel devices show that culture of SH-SY5Y was feasible, though a limited number of neurites extended into microtunnels away from the cell bodies, regardless of using nanogrooved or flat substrates. This shows that the novel combination of microtunnel devices with nanogrooves can be implemented toward neuronal cell cultures, with future improvements to be performed to ensure neurites extend beyond the confines of the wells between the microtunnels. Overall, these results will aid toward creating more robust BOC platforms with improved predictive value. In turn, this can be used to better understand the brain and brain diseases.

2019

Nanogroove-enhanced hydrogel scaffolds for 3D neuronal cell culture: an easy access brain-on-chip model

Summary

In order to better understand the brain and brain diseases, in vitro human brain models need to include not only a chemically and physically relevant microenvironment, but also structural network complexity. This complexity reflects the hierarchical architecture in brain tissue. Here, a method has been developed that adds complexity to a 3D cell culture by means of nanogrooved substrates. SH-SY5Y cells were grown on these nanogrooved substrates and covered with Matrigel, a hydrogel. To quantitatively analyze network behavior in 2D neuronal cell cultures, we previously developed an automated image-based screening method. We first investigated if this method was applicable to 3D primary rat brain cortical (CTX) cell cultures. Since the method was successfully applied to these pilot data, a proof of principle in a reductionist human brain cell model was attempted, using the SH-SY5Y cell line. The results showed that these cells also create an aligned network in the 3D microenvironment by maintaining a certain degree of guidance by the nanogrooved topography in the z-direction. These results indicate that nanogrooves enhance the structural complexity of 3D neuronal cell cultures for both CTX and human SH-SY5Y cultures, providing a basis for further development of an easy access brain-on-chip model.

2019

Exploiting nanogroove-induced cell culture anisotropy to advance in vitro brain models

Summary

A new generation of in vitro human brain models is vital to surpass the limitations of current cell culture platforms and animal cell lines in studying brain function and diseases. Brain-on-chip technology can generate well-defined and reproducible platforms to control the cellular microenvironment for in vivo-like, organized brain cell cultures. Previously, the authors investigated differentiation and network organization of the neuroblastoma SH-SY5Y cell line on nanogrooved substrates, showing that nanogroove guidance of neuronal outgrowths is dependent on nanogroove dimensions. Further, increased orientation of neurites was positively correlated to the differentiation of SH-SY5Y cells. However, as mimicking brain structure alone is insufficient, here, the function of the neuronal cell network as dependent on surface topography and material stiffness is investigated. A generalized replication protocol was developed to create similar nanogrooved patterns in cell culture substrates from different materials, specifically polydimethylsiloxane (PDMS) and Ostemer. Experiments using calcium imaging, where calcium fluxes across membranes are visualized as an indication of action potentials in neuronal cells, were performed with differentiated SH-SY5Y cells and human induced pluripotent stem cell-derived neuronal cells (hiPSCNs) on flat versus nanogrooved substrates to study the network function. Calcium live-imaging was performed and results for experiments with SH-SY5Y cells and hiPSCNs showed that nanogrooved PDMS substrates trended toward increased cellular activity and neuronal cell network connectivity. For future investigation of compatible substrate materials in combination with the effect of material stiffness on the cells, nanogrooved Ostemer substrates were demonstrated to faithfully replicate for use in neuronal cell cultures using nanogrooved substrates. First experiments into the neuronal cell function using stem cells described here aid toward elucidating the effect of nanotopographical and mechanical properties and their benefits toward advancing in vitro neuronal cell models both in form and function. Overall, the results indicate, in conjunction with the previous findings on neuronal outgrowth guidance, that anisotropy as introduced by nanogrooved substrates can have a controllable and potentially beneficial influence on neuronal cell cultures.

2019

Retinoic acid accelerates the specification of enteric neural progenitors from in vitro-derived neural crest

Summary

The enteric nervous system (ENS) is derived primarily from the vagal neural crest, a migratory multipotent cell population emerging from the dorsal neural tube between somites 1-7. Defects in the development and function of the ENS give rise to a range of disorders, termed enteric neuropathies and include conditions such as Hirschsprung’s disease. Little is known about the signalling that specifies early ENS progenitors. This has, thus far, limited progress in the generation of enteric neurons from human Pluripotent Stem Cells (hPSCs) that could provide a useful tool for disease modelling and regenerative medicine. We describe the efficient and accelerated generation of ENS progenitors from hPSCs, revealing that retinoic acid is critical for the acquisition of both vagal axial identity and early ENS progenitor specification. These ENS progenitors generate enteric neurons in vitro and following in vivo transplantation, achieving long-term colonisation of the ENS in adult mice. Thus, hPSC-derived ENS progenitors may provide the basis for cell therapy for defects in the ENS.