Challenges and Future Perspectives in Modeling Neurodegenerative Diseases Using Organ-on-a-Chip Technology.

Neurodegenerative diseases (NDDs) affect more than 50 million people worldwide, posing a significant global health challenge as well as a high socioeconomic burden. With aging constituting one of the main risk factors for some NDDs such as Alzheimer's disease (AD) and Parkinson's disease (PD), this societal toll is expected to rise considering the predicted increase in the aging population as well as the limited progress in the development of effective therapeutics. To address the high failure rates in clinical trials, legislative changes permitting the use of alternatives to traditional pre-clinical in vivo models are implemented. In this regard, microphysiological systems (MPS) such as organ-on-a-chip (OoC) platforms constitute a promising tool, due to their ability to mimic complex and human-specific tissue niches in vitro. This review summarizes the current progress in modeling NDDs using OoC technology and discusses five critical aspects still insufficiently addressed in OoC models to date. Taking these aspects into consideration in the future MPS will advance the modeling of NDDs in vitro and increase their translational value in the clinical setting.

N2FXm, a method for joint nuclear and cytoplasmic volume measurements, unravels the osmo-mechanical regulation of nuclear volume in mammalian cells.

In eukaryotes, cytoplasmic and nuclear volumes are tightly regulated to ensure proper cell homeostasis. However, current methods to measure cytoplasmic and nuclear volumes, including confocal 3D reconstruction, have limitations, such as relying on two-dimensional projections or poor vertical resolution. Here, to overcome these limitations, we describe a method, N2FXm, to jointly measure cytoplasmic and nuclear volumes in single cultured adhering human cells, in real time, and across cell cycles. We find that this method accurately provides joint size over dynamic measurements and at different time resolutions. Moreover, by combining several experimental perturbations and analyzing a mathematical model including osmotic effects and tension, we show that N2FXm can give relevant insights on how mechanical forces exerted by the cytoskeleton on the nuclear envelope can affect the growth of nucleus volume by biasing nuclear import. Our method, by allowing for accurate joint nuclear and cytoplasmic volume dynamic measurements at different time resolutions, highlights the non-constancy of the nucleus/cytoplasm ratio along the cell cycle.

Accelerating the in vitro emulation of Alzheimer's disease-associated phenotypes using a novel 3D blood-brain barrier neurosphere co-culture model.

High failure rates in clinical trials for neurodegenerative disorders such as Alzheimer's disease have been linked to an insufficient predictive validity of current animal-based disease models. This has created an increasing demand for alternative, human-based models capable of emulating key pathological phenotypes in vitro. Here, a three-dimensional Alzheimer's disease model was developed using a compartmentalized microfluidic device that combines a self-assembled microvascular network of the human blood-brain barrier with neurospheres derived from Alzheimer's disease-specific neural progenitor cells. To shorten microfluidic co-culture times, neurospheres were pre-differentiated for 21 days to express Alzheimer's disease-specific pathological phenotypes prior to the introduction into the microfluidic device. In agreement with post-mortem studies and Alzheimer's disease in vivo models, after 7 days of co-culture with pre-differentiated Alzheimer's disease-specific neurospheres, the three-dimensional blood-brain barrier network exhibited significant changes in barrier permeability and morphology. Furthermore, vascular networks in co-culture with Alzheimer's disease-specific microtissues displayed localized ß-amyloid deposition. Thus, by interconnecting a microvascular network of the blood-brain barrier with pre-differentiated neurospheres the presented model holds immense potential for replicating key neurovascular phenotypes of neurodegenerative disorders in vitro.

Accelerated epithelial layer healing induced by tactile anisotropy in surface topography.

Mammalian cells respond to tactile cues from topographic elements presented by the substrate. Among these, anisotropic features distributed in an ordered manner give directionality. In the extracellular matrix, this ordering is embedded in a noisy environment altering the contact guidance effect. To date, it is unclear how cells respond to topographical signals in a noisy environment. Here, using rationally designed substrates, we report morphotaxis, a guidance mechanism enabling fibroblasts and epithelial cells to move along gradients of topographic order distortion. Isolated cells and cell ensembles perform morphotaxis in response to gradients of different strength and directionality, with mature epithelia integrating variations of topographic order over hundreds of micrometers. The level of topographic order controls cell cycle progression, locally delaying or promoting cell proliferation. In mature epithelia, the combination of morphotaxis and noise-dependent distributed proliferation provides a strategy to enhance wound healing as confirmed by a mathematical model capturing key elements of the process.

A novel bistable device to study mechanosensitive cell responses to instantaneous stretch.

The behavior of cells and tissues in vivo is determined by the integration of multiple biochemical and mechanical signals. Of the mechanical signals, stretch has been studied for decades and shown to contribute to pathophysiological processes. Several different stretch devices have been developed for in vitro investigations of cell stretch. In this work, we describe a new 3D-printed uniaxial stretching device for studying cell response to rapid deformation. The device is a bistable compliant mechanism holding two equilibrium states-an unstretched and stretched configuration-without the need of an external actuator. Furthermore, it allows multiple simultaneous measurements of different levels of stretch on a single substrate and is compatible with standard immunofluorescence imaging of fixed cells as well as live-cell imaging. To demonstrate the effectiveness of the device to stretch cells, a test case using aligned myotubes is presented. Leveraging material area changes associated with deformation of the substrate, changes in nuclei density provided evidence of affine deformation between cells and substrate. Furthermore, intranuclear deformations were also assessed and shown to deform non-affinely. As a proof-of-principle of the use of the device for mechanobiological studies, we uniaxially stretched aligned healthy and dystrophic myotubes that displayed different passive mechanical responses, consistent with previous literature in the field. We also identified a new feature in the mechanoresponse of dystrophic myotubes, which is of potential interest for identifying the diseased cells based on a quick mechanical readout. While some applications of the device for elucidating passive mechanical responses are demonstrated, the simplicity of the device allows it to be potentially used for other modes of deformation with little modifications.

Optimized Topological and Topographical Expansion of Epithelia.

Autologous epidermis grafts generated in vitro represent a promising option for the treatment of burn wounds. The procedure relies on of a sufficient number of cells harvested from healthy tissue, which are then sparsely seeded on a target surface. The time required to reconstitute a fully confluent and mature monolayer and the limited availability of cell seeds hinder the broad clinical application of this procedure. Here, a novel engineering approach to enhance the in vitro expansion of epithelial tissues is designed and experimentally validated. The method combines three independent elements supporting fast epithelialization. First, the tactical seeding of epithelial cells at high density in confined channels generated by means of magnetic silicon stencils. Second, the implementation of a curved interface along the channels, increasing the edge interface length. Third, a rationally developed and oriented anisotropic topography, in the form of gratings, aligned perpendicularly to the channels. Upon removal of the stencil, unconfined cell monolayers are free to expand and invade the open space, in a process of epithelialization that fully exploits the directional migration of epithelial collectives. As compared to sparse seeding, this approach attains an almost three times faster full epithelialization of a target surface with the same number of cells. Molecular signals triggered by cell-cell and cell-substrate contacts supported this enhanced response. In summary, we introduce a facile and scalable approach yielding fast in vitro epithelial tissue expansion with optimized yield.

Cell cycle-dependent force transmission in cancer cells.

The generation of traction forces and their transmission to the extracellular environment supports the disseminative migration of cells from a primary tumor. In cancer cells, the periodic variation of nuclear stiffness during the cell cycle provides a functional link between efficient translocation and proliferation. However, the mechanical framework completing this picture remains unexplored. Here, the Fucci2 reporter was expressed in various human epithelial cancer cells to resolve their cell cycle phase transition. The corresponding tractions were captured by a recently developed reference-free confocal traction-force microscopy platform. The combined approach was conducive to the analysis of phase-dependent force variation at the level of individual integrin contacts. Detected forces were invariably higher in the G1 and early S phases than in the ensuing late S/G2, and locally colocalized with high levels of paxillin phosphorylation. Perturbation of paxillin phosphorylation at focal adhesions, obtained through the biochemical inhibition of focal adhesion kinase (FAK) or the transfection of nonphosphorylatable or phosphomimetic paxillin mutants, significantly diminished the force transmitted to the substrate. These data demonstrate a reproducible modulation of force transmission during the cell cycle progression of cancer cells, instrumental to their invasion of dense environments. In addition, they delineate a model in which paxillin phosphorylation supports the mechanical maturation of adhesions relaying forces to the substrate.

Pore Shape Defines Paths of Metastatic Cell Migration.

Invasion of dense tissues by cancer cells involves the interplay between the penetration resistance offered by interstitial pores and the deformability of cells. Metastatic cancer cells find optimal paths of minimal resistance through an adaptive path-finding process, which leads to successful dissemination. The physical limit of nuclear deformation is related to the minimal cross section of pores that can be successfully penetrated. However, this single biophysical parameter does not fully describe the architectural complexity of tissues featuring pores of variable area and shape. Here, employing laser nanolithography, we fabricate pore microenvironment models with well-controlled pore shapes, through which human breast cells (MCF10A) and their metastatic offspring (MCF10CA1a.cl1) could pervade. In these experimental settings, we demonstrate that the actual pore shape, and not only the cross section, is a major and independent determinant of cancer penetration efficiency. In complex architectures containing pores demanding large deformations from invading cells, tall and narrow rectangular openings facilitate cancer migration. In addition, we highlight the characteristic traits of the explorative behavior enabling metastatic cells to identify and select such pore shapes in a complex multishape pore environment, pinpointing paths of least resistance to invasion.