Human in vitro models of brain neurophysiology are needed to investigate molecular and cellular mechanisms associated with neurological disorders and neurotoxicity. We have developed a reproducible iPSC-derived human 3D brain microphysiological system (BMPS), comprised of differentiated mature neurons and glial cells (astrocytes and oligodendrocytes) that reproduce neuronal-glial interactions and connectivity. BMPS mature over eight weeks and show the critical elements of neuronal function: synaptogenesis and neuron-to-neuron (e.g., spontaneous electric field potentials) and neuronal-glial interactions (e.g., myelination), which mimic the microenvironment of the central nervous system, rarely seen in vitro before. The BMPS shows 40% overall myelination after 8 weeks of differentiation. Myelin was observed by immunohistochemistry and confirmed by confocal microscopy 3D reconstruction and electron microscopy. These findings are of particular relevance since myelin is crucial for proper neuronal function and development. The ability to assess oligodendroglial function and mechanisms associated with myelination in this BMPS model provide an excellent tool for future studies of neurological disorders such as multiple sclerosis and other demyelinating diseases. The BMPS provides a suitable and reliable model to investigate neuron-neuroglia function as well as pathogenic mechanisms in neurotoxicology.

Migration of neural progenitors in the complex tissue environment of the central nervous system is not well understood. Progress in this area has the potential to drive breakthroughs in neuroregenerative therapies, brain cancer treatments, and neurodevelopmental studies. To a large extent, advances have been limited due to a lack of controlled environments recapitulating characteristics of the central nervous system milieu. Reductionist cell culture models are frequently too simplistic, and physiologically more relevant approaches such as ex vivo brain slices or in situ experiments provide little control and make information extraction difficult. Here, we present a brain-on-chip model that bridges the gap between cell culture and ex vivo/in vivo conditions through recapitulation of self-organized neural differentiation. We use a new multi-layer silicone elastomer device, over the course of four weeks to differentiate pluripotent human (NTERA2) cells into neuronal clusters interconnected with thick axonal bundles and interspersed with astrocytes, resembling the brain parenchyma. Neurons within the device express the neurofilament heavy (NF200) mature axonal marker and the microtubule-associated protein (MAP2ab) mature dendritic marker, demonstrating that the devices are sufficiently biocompatible to allow neuronal maturation. This neuronal-glial environment is interfaced with a layer of human brain microvascular endothelial cells showing characteristics of the blood–brain barrier including the expression of zonula occludens (ZO1) tight junctions and increased trans-endothelial electrical resistance. We used this device to model migration of human neural progenitors in response to chemotactic cues within a brain-tissue setting. We show that in the presence of an environment mimicking brain conditions, neural progenitor cells show a significantly enhanced chemotactic response towards shallow gradients of CXCL12, a key chemokine expressed during embryonic brain development and in pathological tissue regions of the central nervous system. Our brain-on-chip model thus provides a convenient and scalable model of neural differentiation and maturation extensible to analysis of complex cell and tissue behaviors.

Human induced pluripotent stem cells (iPSCs), together with 21st century cell culture methods, have the potential to better model human physiology with applications in toxicology, disease modeling, and the study of host-pathogen interactions. Several models of the human brain have been developed recently, demonstrating cell-cell interactions of multiple cell types with physiologically relevant 3D structures. Most current models, however, lack the ability to represent the inflammatory response in the brain because they do not include a microglial cell population. Microglia, the resident immunocompetent phagocytes in the central nervous system (CNS), are not only important in the inflammatory response and pathogenesis; they also function in normal brain development, strengthen neuronal connections through synaptic pruning, and are involved in oligodendrocyte and neuronal survival. Here, we have successfully introduced a population of human microglia into 3D human iPSC-derived brain spheres (BrainSpheres, BS) through co-culturing cells of the Immortalized Human Microglia – SV40 cell line with the BS model (μBS). We detected an inflammatory response to lipopolysaccharides (LPS) and flavivirus infection, which was only elicited in the model when microglial cells were present. A concentration of 20 ng/mL of LPS increased gene expression of the inflammatory cytokines interleukin-6 (IL-6), IL-10, and IL-1β, with maximum expression at 6–12 h post-exposure. Increased expression of the IL-6, IL-1β, tumor necrosis factor alpha (TNF-α), and chemokine (C-C motif) ligand 2 (CCL2) genes was observed in μBS following infection with Zika and Dengue Virus, suggesting a stronger inflammatory response in the model when microglia were present than when only astrocyte, oligodendrocyte, and neuronal populations were represented. Microglia innately develop within cerebral organoids (Nature communications)¹, our findings suggest that the μBS model is more physiologically relevant and has potential applications in infectious disease and host-pathogen interactions research.

A major reason for the current reproducibility crisis in the life sciences is the poor implementation of quality control measures and reporting standards. Improvement is needed, especially regarding increasingly complex in vitro methods. Good Cell Culture Practice (GCCP) was an effort from 1996 to 2005 to develop such minimum quality standards also applicable in academia. This paper summarizes recent key developments in in vitro cell culture and addresses the issues resulting for GCCP, e.g. the development of induced pluripotent stem cells (iPSCs) and gene-edited cells. It further deals with human stem-cell-derived models and bioengineering of organo-typic cell cultures, including organoids, organ-on-chip and human-on-chip approaches. Commercial vendors and cell banks have made human primary cells more widely available over the last decade, increasing their use, but also requiring specific guidance as to GCCP. The characterization of cell culture systems including high-content imaging and high-throughput measurement technologies increasingly combined with more complex cell and tissue cultures represent a further challenge for GCCP. The increasing use of gene editing techniques to generate and modify in vitro culture models also requires discussion of its impact on GCCP. International (often varying) legislations and market forces originating from the commercialization of cell and tissue products and technologies are further impacting on the need for the use of GCCP. This report summarizes the recommendations of the second of two workshops, held in Germany in December 2015, aiming map the challenge and organize the process or developing a revised GCCP 2.0.

The desire to develop and evaluate drugs as potential countermeasures for biological and chemical threats requires test systems that can also substitute for the clinical trials normally crucial for drug development. Current animal models have limited predictivity for drug efficacy in humans as the large majority of drugs fails in clinical trials. We have limited understanding of the function of the central nervous system and the complexity of the brain, especially during development and neuronal plasticity. Simple in vitro systems do not represent physiology and function of the brain. Moreover, the difficulty of studying interactions between human genetics and environmental factors leads to lack of knowledge about the events that induce neurological diseases. Microphysiological systems (MPS) promise to generate more complex in vitro human models that better simulate the organ’s biology and function. MPS combine different cell types in a specific three-dimensional (3D) configuration to simulate organs with a concrete function. The final aim of these MPS is to combine different “organoids” to generate a human-on-a-chip, an approach that would allow studies of complex physiological organ interactions. The recent discovery of induced pluripotent stem cells (iPSCs) gives a range of possibilities allowing cellular studies of individuals with different genetic backgrounds (e.g., human disease models). Application of iPSCs from different donors in MPS gives the opportunity to better understand mechanisms of the disease and can be a novel tool in drug development, toxicology, and medicine. In order to generate a brain-on-a-chip, we have established a 3D model from human iPSCs based on our experience with a 3D rat primary aggregating brain model. After four weeks of differentiation, human 3D aggregates stain positive for different neuronal markers and show higher gene expression of various neuronal differentiation markers compared to 2D cultures. Here we present the applications and challenges of this emerging technology.