Corrigendum: Spatiotemporal analysis of 3D human iPSC-derived neural networks using a 3D multi-electrode array.

[This corrects the article DOI: 10.3389/fncel.2023.1287089.].

Spatiotemporal analysis of 3D human iPSC-derived neural networks using a 3D multi-electrode array.

While there is a growing appreciation of three-dimensional (3D) neural tissues (i.e., hydrogel-based, organoids, and spheroids), shown to improve cellular health and network activity to mirror brain-like activity in vivo, functional assessment using current electrophysiology techniques (e.g., planar multi-electrode arrays or patch clamp) has been technically challenging and limited to surface measurements at the bottom or top of the 3D tissue. As next-generation MEAs, specifically 3D MEAs, are being developed to increase the spatial precision across all three dimensions (X, Y, Z), development of improved computational analytical tools to discern region-specific changes within the Z dimension of the 3D tissue is needed. In the present study, we introduce a novel computational analytical pipeline to analyze 3D neural network activity recorded from a "bottom-up" 3D MEA integrated with a 3D hydrogel-based tissue containing human iPSC-derived neurons and primary astrocytes. Over a period of ~6.5 weeks, we describe the development and maturation of 3D neural activity (i.e., features of spiking and bursting activity) within cross sections of the 3D tissue, based on the vertical position of the electrode on the 3D MEA probe, in addition to network activity (identified using synchrony analysis) within and between cross sections. Then, using the sequential addition of postsynaptic receptor antagonists, bicuculline (BIC), 2-amino-5-phosphonovaleric acid (AP-5), and 6-cyano-5-nitroquinoxaline-2,3-dione (CNQX), we demonstrate that networks within and between cross sections of the 3D hydrogel-based tissue show a preference for GABA and/or glutamate synaptic transmission, suggesting differences in the network composition throughout the neural tissue. The ability to monitor the functional dynamics of the entire 3D reconstructed neural tissue is a critical bottleneck; here we demonstrate a computational pipeline that can be implemented in studies to better interpret network activity within an engineered 3D neural tissue and have a better understanding of the modeled organ tissue.

Cell-free Scaled Production and Adjuvant Addition to a Recombinant Major Outer Membrane Protein from Chlamydia muridarum for Vaccine Development.

Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard manufacturing. To achieve an effective protein-based subunit vaccine, the protein antigen often needs to adopt a native-like conformation. This is particularly important for pathogen-surface antigens that are membrane-bound proteins. Cell-free methods have been successfully used to produce correctly folded functional membrane protein through the co-translation of nanolipoprotein particles (NLPs), commonly known as nanodiscs. This strategy can be used to produce subunit vaccines consisting of membrane proteins in a lipid-bound environment. However, cell-free protein production is often limited to small scale (<1 mL). The amount of protein produced in small-scale production runs is usually sufficient for biochemical and biophysical studies. However, the cell-free process needs to be scaled up, optimized, and carefully tested to obtain enough protein for vaccine studies in animal models. Other processes involved in vaccine production, such as purification, adjuvant addition, and lyophilization, need to be optimized in parallel. This paper reports the development of a scaled-up protocol to express, purify, and formulate a membrane-bound protein subunit vaccine. Scaled-up cell-free reactions require optimization of plasmid concentrations and ratios when using multiple plasmid expression vectors, lipid selection, and adjuvant addition for high-level production of formulated nanolipoprotein particles. The method is demonstrated here with the expression of a chlamydial major outer membrane protein (MOMP) but may be widely applied to other membrane protein antigens. Antigen effectiveness can be evaluated in vivo through immunization studies to measure antibody production, as demonstrated here.

Characterization of Bacillus anthracis Spore Proteins Using a Nanoscaffold Vaccine Platform.

Subunit vaccines are theoretically safe and easy to manufacture but require effective adjuvants and delivery systems to yield protective immunity, particularly at critical mucosal sites such as the lung. We investigated nanolipoprotein particles (NLPs) containing the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) as a platform for intranasal vaccination against Bacillus anthracis. Modified lipids enabled attachment of disparate spore and toxin protein antigens. Intranasal vaccination of mice with B. anthracis antigen-MPLA-NLP constructs induced robust IgG and IgA responses in serum and in bronchoalveolar and nasal lavage. Typically, a single dose sufficed to induce sustained antibody titers over time. When multiple immunizations were required for sustained titers, specific antibodies were detected earlier in the boost schedule with MPLA-NLP-mediated delivery than with free MPLA. Administering combinations of constructs induced responses to multiple antigens, indicating potential for a multivalent vaccine preparation. No off-target responses to the NLP scaffold protein were detected. In summary, the NLP platform enhances humoral and mucosal responses to intranasal immunization, indicating promise for NLPs as a flexible, robust vaccine platform against B. anthracis and potentially other inhalational pathogens.

Functional and transcriptional characterization of complex neuronal co-cultures.

Brain-on-a-chip systems are designed to simulate brain activity using traditional in vitro cell culture on an engineered platform. It is a noninvasive tool to screen new drugs, evaluate toxicants, and elucidate disease mechanisms. However, successful recapitulation of brain function on these systems is dependent on the complexity of the cell culture. In this study, we increased cellular complexity of traditional (simple) neuronal cultures by co-culturing with astrocytes and oligodendrocyte precursor cells (complex culture). We evaluated and compared neuronal activity (e.g., network formation and maturation), cellular composition in long-term culture, and the transcriptome of the two cultures. Compared to simple cultures, neurons from complex co-cultures exhibited earlier synapse and network development and maturation, which was supported by localized synaptophysin expression, up-regulation of genes involved in mature neuronal processes, and synchronized neural network activity. Also, mature oligodendrocytes and reactive astrocytes were only detected in complex cultures upon transcriptomic analysis of age-matched cultures. Functionally, the GABA antagonist bicuculline had a greater influence on bursting activity in complex versus simple cultures. Collectively, the cellular complexity of brain-on-a-chip systems intrinsically develops cell type-specific phenotypes relevant to the brain while accelerating the maturation of neuronal networks, important features underdeveloped in traditional cultures.

A flexible 3-dimensional microelectrode array for in vitro brain models.

Three-dimensional (3D) in vitro models have become increasingly popular as systems to study cell-cell and cell-ECM interactions dependent on the spatial, mechanical, and chemical cues within the environment of the tissue, which is limited in traditional two-dimensional (2D) models. Although electrophysiological recordings of neuronal action potentials through 2D microelectrode arrays (MEAs) are a common and trusted method of evaluating neuronal function, network communication, and response to chemicals and biologicals, there are currently limited options for measuring electrophysiological activity from many locations simultaneously throughout a 3D network of neurons in vitro. Here, we have developed a thin-film, 3D flexible microelectrode array (3DMEA) that non-invasively interrogates a 3D culture of neurons and can accommodate 256 channels of recording or stimulation. Importantly, the 3DMEA is straightforward to fabricate and integrates with standard commercially available electrophysiology hardware. Polyimide probe arrays were microfabricated on glass substrates and mechanically actuated to collectively lift the arrays into a vertical position, relying solely on plastic deformation of their base hinge regions to maintain vertical alignment. Human induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes were entrapped in a collagen-based hydrogel and seeded onto the 3DMEA, enabling growth of suspended cells in the matrix and the formation and maturation of a neural network around the 3DMEA probes. The 3DMEA supported the growth of functional neurons in 3D with action potential spike and burst activity recorded over 45 days in vitro. This platform is an important step in facilitating noninvasive electrophysiological characterization of 3D networks of electroactive cells in vitro.

Optimizing cell encapsulation condition in ECM-Collagen I hydrogels to support 3D neuronal cultures.

BACKGROUND: The emergence of three-dimensional (3D) cell culture in neural tissue engineering has significantly elevated the complexity and relevance of in vitro systems. This is due in large part to the incorporation of biomaterials to impart structural dimensionality on the neuronal cultures. However, a comprehensive understanding of how key seeding parameters affect changes in cell distribution and viability remain unreported. NEW METHOD: In this study, we systematically evaluated permutations in seeding conditions (i.e., cell concentration and atmospheric CO2 levels) to understand how these affect key parameters in 3D culture characterization (i.e., cell health and distribution). Primary rat cortical neurons (i.e., 2 × 106, 4 × 106, and 1 × 107 cells/mL) were entrapped in collagen blended with ECM proteins (ECM-Collagen) and exposed to atmospheric CO2 (i.e., 0 vs 5% CO2) during fibrillogenesis. RESULTS: At 14 days in vitro (DIV), cell distribution within the hydrogel was dependent on cell concentration and atmospheric CO2 during fibrillogenesis. A uniform distribution of cells was observed in cultures with 2 × 106 and 4 × 106 cells/mL in the presence of 5% CO2, while a heterogeneous distribution was observed in cultures with 1 × 107 cells/mL or in the absence of CO2. Furthermore, increased cell concentration was proportional to the rise in cell death at 14 DIV, although cells remain viable >30 DIV. COMPARISON WITH EXISTING METHODS: ECM-Collagen gels have been shown to increase cell viability of neurons long-term. CONCLUSION: In using ECM-collagen gels, we highlight the importance of optimizing seeding parameters and thorough 3D culture characterization to understand the neurophysiological responses of these 3D systems.

Tissue-specific extracellular matrix accelerates the formation of neural networks and communities in a neuron-glia co-culture on a multi-electrode array.

The brain's extracellular matrix (ECM) is a macromolecular network composed of glycosaminoglycans, proteoglycans, glycoproteins, and fibrous proteins. In vitro studies often use purified ECM proteins for cell culture coatings, however these may not represent the molecular complexity and heterogeneity of the brain's ECM. To address this, we compared neural network activity (over 30 days in vitro) from primary neurons co-cultured with glia grown on ECM coatings from decellularized brain tissue (bECM) or MaxGel, a non-tissue-specific ECM. Cells were grown on a multi-electrode array (MEA) to enable noninvasive long-term interrogation of neuronal networks. In general, the presence of ECM accelerated the formation of networks without affecting the inherent network properties. However, specific features of network activity were dependent on the type of ECM: bECM enhanced network activity over a greater region of the MEA whereas MaxGel increased network burst rate associated with robust synaptophysin expression. These differences in network activity were not attributable to cellular composition, glial proliferation, or astrocyte phenotypes, which remained constant across experimental conditions. Collectively, the addition of ECM to neuronal cultures represents a reliable method to accelerate the development of mature neuronal networks, providing a means to enhance throughput for routine evaluation of neurotoxins and novel therapeutics.

Lipid composition dictates serum stability of reconstituted high-density lipoproteins: implications for in vivo applications.

Nanolipoprotein particles (NLPs) are reconstituted high-density lipoproteins, consisting of a phospholipid bilayer stabilized by an apolipoprotein scaffold protein. This class of nanoparticle has been a vital tool in the study of membrane proteins, and in recent years has been increasingly used for in vivo applications. Previous work demonstrated that the composition of the lipid bilayer component affects the stability of these particles in serum solutions. In the current study, NLPs assembled with phosphatidylcholine lipids featuring different acyl chain structures were systematically tested to understand the effect that lipid composition has on NLP stability in both neat serum and cell culture media supplemented with 10% serum by volume. The time at which 50% of the particles dissociate, as well as the fraction of the initial population that remains resistant to dissociation, were correlated to key parameters obtained from all-atom simulations of the corresponding lipid bilayers. A significant correlation was observed between the compressibility modulus of the lipid bilayer and particle stability in these complex biological milieu. These results can be used as a reference to tune the stability of these versatile biological nanoparticles for in vitro and in vivo applications.