Aerosol Jet-Printed High-Aspect Ratio Micro-Needle Electrode Arrays Applied for Human Cerebral Organoids and 3D Neurospheroid Networks.

The human brain is a complex and poorly accessible organ. Thus, new tools are required for studying the neural function in a controllable environment that preserves multicellular interaction and neuronal wiring. In particular, high-throughput methods that alleviate the need for animal experiments are essential for future studies. Recent developments of induced pluripotent stem cell technologies have enabled in vitro modeling of the human brain by creating three-dimensional brain tissue mimic structures. To leverage these new technologies, a systematic and versatile approach for evaluating neuronal activity at larger tissue depths within the regime of tens to hundreds of micrometers is required. Here, we present an aerosol-jet- and inkjet-printing-based method to fabricate microelectrode arrays, equipped with high-aspect ratio µ-needle electrodes that penetrate 3D neural network assemblies. The arrays have been successfully applied for electrophysiological recordings on interconnected neurospheroids formed on an engineered substrate and on cerebral organoids, both derived from human induced pluripotent stem cells.

On-chip real-time impedance monitoring of hiPSC-derived and artificial basement membrane-supported endothelium.

Recent advances have shown the high sensibility of electrochemical impedance spectroscopy in real-time monitoring of cell barriers on a chip. Here, we applied this method to the investigation of human induced pluripotent stem cell (hiPSC) derived and artificial basement membrane (ABM) supported endothelial barrier. The ABM was obtained by self-assembling type IV collagen and laminin with a monolayer of crosslinked gelatin nanofibers. The hiPSCs were differentiated into brain microvascular endothelial cells (BMECs) and then plated on the ABM. After incubation for two days, the ABM-BMEC assembly was placed as a tissue insert into a microfluidic device for culture and real-time impedance monitoring over days. We found a significantly enhanced stability of the BMEC barrier in a serum-free and bromodeoxyuridine (BrdU) containing culture medium compared to the conventional culture due to the restricted cell proliferation. We also found that the BMEC barrier was sensitive to stimuli such as thrombin and that the change of the barrier impedance was mainly due to the change of the cell layer resistance. We can thus advocate this method to investigate the integrity of the cell barrier and the barrier-based assays.

S100A8-mediated metabolic adaptation controls HIV-1 persistence in macrophages in vivo.

HIV-1 eradication is hindered by viral persistence in cell reservoirs, established not only in circulatory CD4+T-cells but also in tissue-resident macrophages. The nature of macrophage reservoirs and mechanisms of persistence despite combined anti-retroviral therapy (cART) remain unclear. Using genital mucosa from cART-suppressed HIV-1-infected individuals, we evaluated the implication of macrophage immunometabolic pathways in HIV-1 persistence. We demonstrate that ex vivo, macrophage tissue reservoirs contain transcriptionally active HIV-1 and viral particles accumulated in virus-containing compartments, and harbor an inflammatory IL-1R+S100A8+MMP7+M4-phenotype prone to glycolysis. Reactivation of infectious virus production and release from these reservoirs in vitro are induced by the alarmin S100A8, an endogenous factor produced by M4-macrophages and implicated in "sterile" inflammation. This process metabolically depends on glycolysis. Altogether, inflammatory M4-macrophages form a major tissue reservoir of replication-competent HIV-1, which reactivate viral production upon autocrine/paracrine S100A8-mediated glycolytic stimulation. This HIV-1 persistence pathway needs to be targeted in future HIV eradication strategies.

Automatic differentiation of human induced pluripotent stem cells toward synchronous neural networks on an arrayed monolayer of nanofiber membrane.

Automatic differentiation of human-induced pluripotent stem cells (hiPSCs) facilitates the generation of cortical neural networks and studies of brain functions. Here, we present a method of directed differentiation of hiPSCs with a substrate made of a honeycomb microframe and a monolayer of crosslinked gelatin nanofibers in the form of an array of nanofiber membranes. Neural precursor cells (NPCs) were firstly derived from hiPSCs and then placed on the nanofiber membranes for automatically controlled neural differentiation over a long period. Due to the strong modulation of the substrate stiffness and permeability, most cells were found in the center area of the honeycomb compartments, giving rise to regular and inter-connected cortical neural clusters. More importantly, the neural activities of the clusters were synchronized proving the reliability of the method. Our results showed that the self-organization, as well as the neural activities of differentiating neural cells, were more efficient in the nanofiber membrane compared to the types of the substrate such as glass and nanofiber-covered glass. In addition to the inherent advantages such as manpower saving and fewer risks of contamination and human error, automatic differentiation avoided undesired shaking which might have critical effects on the formation of synchronous neural clusters. STATEMENT OF SIGNIFICANCE: Synchronization of cortical neural activities is essential for information processing and human cognition. By automated differentiation of human induced pluripotent stem cells on arrayed monolayer of nanofiber membrane, synchronous neural clusters could be formed. Such an approach would allow creating a variety of neural networks with regular and interconnected clusters for systematic studies of human cortical functions.

Sars-Cov-2 Spike Protein-Induced Damage of hiPSC-Derived Cardiomyocytes.

Sars-Cov-2 may trigger molecular and functional alterations of cardiomyocytes (CMs) of the heart due to the presence of receptor angiotensin-converting enzyme 2 (ACE2) of the host cells. While the endocytic itinerary of the virus via cleavage of the spike protein of Sars-Cov-2 is well understood, the role of the remaining part of the spike protein subunit and ACE2 complex is still elusive. Herein, the possible effects of this complex are investigated by using synthetic spike proteins of Sars-Cov-2, human-induced pluripotent stem cells (hiPSC), and a culture device made of an arrayed monolayer of cross-linked nanofibers. hiPSCs are first differentiated into CMs that form cardiac tissue-like constructs with regular beating and expression of both ACE2 and gap junction protein Connexin 43. When incubated with the spike proteins, the hiPSC-CMs undergo a rhythmic fluctuation with overstretched sarcomere structures and dispersed gap junction proteins. When incubated with the spike proteins and supplementary angiotensin II, the damage of the spike protein on hiPSC-CMs is enhanced due to downregulated ACE2, chromatin margination, altered Connexin 43 expression, sarcomere disruption, and beating break. This discovery may imply latent effects of the spike proteins on the heart.

Reconstituted basement membrane enables airway epithelium modeling and nanoparticle toxicity testing.

Basement membrane (BM) acts as a sheet-like extracellular matrix to support and promote the formation of epithelial and endothelial cell layers. The in vitro reconstruction of the BM is however not easy due to its ultrathin membrane features. This difficulty is overcome by self-assembling type IV collagen and laminin in the porous areas of a monolayer of crosslinked gelatin nanofibers deposited on a honeycomb microframe. Herein, a method is presented to generate airway epithelium by using such an artificial basement membrane (ABM) and human-induced pluripotent stem cells (hiPSCs). Bipolar primordial lung progenitors are firstly induced from hiPSCs and then replated on the ABM for differentiation toward matured airway epithelium under submerged and air-liquid interface culture conditions. As a result, a pseudostratified airway epithelium consisting of several cell types is achieved, showing remarkable apical secretion of MUC5AC proteins and clear advantages over other types of substrates. As a proof of concept, the derived epithelium is used for toxicity test of cadmium telluride (CdTe) nanoparticles (NPs), demonstrating the applicability of ABM-based assays involving hiPSC-derived epithelial cells-based assays.

Generation of Alveolar Epithelium Using Reconstituted Basement Membrane and hiPSC-Derived Organoids.

In vitro modeling of alveolar epithelium needs to recapitulate features of both cellular and noncellular components of the lung tissues. Herein, a method is presented to generate alveolar epithelium by using human induced pluripotent stem cells (hiPSCs) and reconstituted or artificial basement membrane (ABM). The ABM is obtained by self-assembling type IV collagen and laminin with a monolayer of crosslinked gelatin nanofibers as backbone and a patterned honeycomb microframe for handling. Alveolar organoids are obtained from hiPSCs and then dissociated into single cells. After replating the alveolar cells on the ABM and a short-period incubation under submerged and air-liquid interface culture conditions, an alveolar epithelium is achieved, showing high-level expressions of both alveolar cell-specific proteins and characteristic tight junctions. Besides, endothelial cells derived from the same hiPSCs are cocultured on the backside of the epithelium, forming an air-blood barrier. The method is generic and can potentially be applied to other types of artificial epithelium and endothelium.

Generation of Interconnected Neural Clusters in Multiscale Scaffolds from Human-Induced Pluripotent Stem Cells.

The development of in vitro neural networks depends to a large extent on the scaffold properties, including the scaffold stiffness, porosity, and dimensionality. Herein, we developed a method to generate interconnected neural clusters in a multiscale scaffold consisting of a honeycomb microframe covered on both sides with a monolayer of cross-linked gelatin nanofibers. Cortical neural precursor cells were first produced from human-induced pluripotent stem cells and then loaded into the scaffold for a long period of differentiation toward cortical neural cells. As a result, neurons and astrocytes self-organized in the scaffold to form clusters in each of the honeycomb compartments with remarkable inter-cluster connections. These cells highly expressed neuron- and astrocyte-specific proteins, including NF200, tau, synapsin I, and glial fibrillary acidic protein, and showed spatially correlated neural activities. Two types of neural clusters, that is, spheroid-like and hourglass-like clusters, were found, indicating the complexity of neural-scaffold interaction and the variability of three-dimensional neural organization. Furthermore, we incorporated a reconstituted basement membrane into the scaffold and performed co-culture of the neural network with brain microvascular endothelial cells. As a proof of concept, an improved neurovascular unit model was tested, showing large astrocytic end-feet on the back side of the endothelium.

Three-dimensional prostate tumor model based on a hyaluronic acid-alginate hydrogel for evaluation of anti-cancer drug efficacy.

In vitro cell-based assays are widely applied to evaluate anti-cancer drug efficacy. However, the conventional approaches are mostly based on two-dimensional (2D) culture systems, making it difficult to recapitulate the in vivo tumor scenario because of spatial limitations. Here, we develop an in vitro three-dimensional (3D) prostate tumor model based on a hyaluronic acid (HA)-alginate hybrid hydrogel to bridge the gap between in vitro and in vivo anticancer drug evaluations. In situ encapsulation of PCa cells was achieved by mixing HA and alginate aqueous solutions in the presence of cells and then crosslinking with calcium ions. Unlike in 2D culture, cells were found to aggregate into spheroids in a 3D matrix. The expression of epithelial to mesenchyme transition (EMT) biomarkers was found to be largely enhanced, indicating an increased invasion and metastasis potential in the hydrogel matrix. A significant up-regulation of proangiogenic growth factors (IL-8, VEGF) and matrix metalloproteinases (MMPs) was observed in 3D-cultured PCa cells. The results of anti-cancer drug evaluation suggested a higher drug tolerance within the 3D tumor model compared to conventional 2D-cultured cells. Finally, we found that the drug effect within the in vitro 3D cancer model based on HA-alginate matrix exhibited better predictability for in vivo drug efficacy.