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Enthesitis, the inflammation of the enthesis, which is the point of attachment of tendons and ligaments to bones, is a common musculoskeletal disease. The inflammation often originates from the fibrocartilage region of the enthesis as a consequence of mechanical overuse or -load and consequently tissue damage. During enthesitis, waves of inflammatory cytokines propagate in(to) the fibrocartilage, resulting in detrimental, heterotopic bone formation. Understanding of human enthesitis and its treatment options is limited, also because of lacking in vitro model systems that can closely mimic the pathophysiology of the enthesis and can be used to develop therapies. In this study, an enthes(it)is-on-chip model is developed. On opposite sides of a porous culture membrane separating the chip's two microfluidic compartments, human mesenchymal stromal cells are selectively differentiated into tenocytes and fibrochondrocytes. By introducing an inflammatory cytokine cocktail into the fibrochondrocyte compartment, key aspects of acute and chronic enthesitis, measured as increased expression of inflammatory markers, can be recapitulated. Upon inducing chronic inflammatory conditions, hydroxyapatite deposition, enhanced osteogenic marker expression and reduced secretion of tissue-related extracellular matrix components are observed. Adding the anti-inflammatory drug celecoxib to the fibrochondrocyte compartment mitigates the inflammatory state, demonstrating the potential of the enthesitis-on-chip model for drug testing., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Tendon is a highly organized tissue that transmits forces between muscle and bone. The architecture of the extracellular matrix of tendon, predominantly from collagen type I, is important for maintaining tenocyte phenotype and function. Therefore, in repair and regeneration of damaged and diseased tendon tissue, it is crucial to restore the aligned arrangement of the collagen type I fibers of the original matrix. To this end, a novel, user-friendly microfluidic piggyback platform is developed allowing the controlled patterned formation and alignment of collagen fibers simply on the bottom of culture dishes. Rat tenocytes cultured on the micropatterns of aligned fibrous collagen exhibit a more elongated morphology. The cells also show an increased expression of tenogenic markers at the gene and protein level compared to tenocytes cultured on tissue culture plastic or non-fibrillar collagen coatings. Moreover, using imprinted polystyrene replicas of aligned collagen fibers, this work shows that the fibrillar structure of collagen per se affects the tenocyte morphology, whereas the biochemical nature of collagen plays a prominent role in the expression of tenogenic markers. Beyond the controlled provision of aligned collagen, the microfluidic platform can aid in developing more physiologically relevant in vitro models of tendon and its regeneration., (© 2023 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

A challenge in regenerative medicine is creating the three-dimensional organic and inorganic in vitro microenvironment of bone, which would allow the study of musculoskeletal disorders and the generation of building blocks for bone regeneration. This study presents a microwell-based platform for creating spheroids of human mesenchymal stromal cells, which are then mineralized using ionic calcium and phosphate supplementation. The resulting mineralized spheroids promote an osteogenic gene expression profile through the influence of the spheroids' biophysical environment and inorganic signaling and require less calcium or phosphate to achieve mineralization compared to a monolayer culture. We found that mineralized spheroids represent an in vitro model for studying small molecule perturbations and extracellular mediated calcification. Furthermore, we demonstrate that understanding pathway signaling elicited by the spheroid environment allows mimicking these pathways in traditional monolayer culture, enabling similar rapid mineralization events. In sum, this study demonstrates the rapid generation and employment of a mineralized cell model system for regenerative medicine applications., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Stefan Giselbrecht reports a relationship with 300MICRONS GmbH that includes: board membership. Roman Truckenmuller reports a relationship with 300MICRONS GmbH that includes: board membership., (© 2023 The Authors.)

Microengineering is increasingly being used for controlling the microenvironment of stem cells. Here, a novel method for fabricating structures with subcellular dimensions in commonly available thermoplastic poly(methyl methacrylate) (PMMA) is shown. Microstructures are produced in PMMA substrates using Deep Ultraviolet lithography, and the effect of different developers is described. Microgrooves fabricated in PMMA are used for the neuronal differentiation of mouse embryonic stem cells (mESCs) directly on the polymer. The fabrication of 3D, curvilinear patterned surfaces is also highlighted. A 3D multilayered microfluidic chip is fabricated using this method, which includes a porous polycarbonate (PC) membrane as cell culture substrate. Besides directly manufacturing PMMA-based microfluidic devices, an application of the novel approach is shown where a reusable PMMA master is created for replicating microstructures with polydimethylsiloxane (PDMS). As an application example, microchannels fabricated in PDMS are used to selectively expose mESCs to soluble factors in a localized manner. The described microfabrication process offers a remarkably simple method to fabricate for example multifunctional topographical or microfluidic culture substrates outside cleanrooms, thereby using inexpensive and widely accessible equipment. The versatility of the underlying process could find various applications also in optical systems and surface modification of biomedical implants., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Stefan Giselbrecht reports a relationship with 300 MICRONS GmbH that includes: board membership and equity or stocks. Roman Truckenmüller and Stefan Giselbrecht are founders and shareholders of 300MICRONS GmbH., (© 2023 The Authors.)

The culture of lung organoids relies on drops of basement membrane matrices. This comes with limitations, for example, concerning the microscopic monitoring and imaging of the organoids in the drops. Also, the culture technique is not easily compatible with micromanipulations of the organoids. In this study, we investigated the feasibility of the culture of human bronchial organoids in defined x-, y- and z-positions in a polymer film-based microwell array platform. The circular microwells have thin round/U-bottoms. For this, single cells are first precultured in drops of basement membrane extract (BME). After they form cell clusters or premature organoids, the preformed structures are then transferred into the microwells in a solution of 50% BME in medium. There, the structures can be cultured toward differentiated and mature organoids for several weeks. The organoids were characterized by bright-field microscopy for size growth and luminal fusion over time, by scanning electron microscopy for overall morphology, by transmission electron microscopy for the existence of microvilli and cilia, by video microscopy for beating cilia and swirling fluid, by live-cell imaging, by fluorescence microscopy for the expression of cell-specific markers and for proliferating and apoptotic cells, and by ATP measurement for extended cell viability. Finally, we demonstrated the eased micromanipulation of the organoids in the microwells by the example of their microinjection., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Roman Truckenmueller reports a relationship with 300MICRONS GmbH that includes: board membership and equity or stocks. Stefan Giselbrecht reports a relationship with 300MICRONS GmbH that includes: board membership and equity or stocks., (© 2023 The Authors.)

Three-dimensional cell culture in engineered hydrogels is increasingly used in tissue engineering and regenerative medicine. The transfer of nutrients, gases, and waste materials through these hydrogels is of utmost importance for cell viability and response, yet the translation of diffusion coefficients into practical guidelines is not well established. Here, we combined mathematical modeling, fluorescent recovery after photobleaching, and hydrogel diffusion experiments on cell culture inserts to provide a multiscale practical approach for diffusion. We observed a dampening effect of the hydrogel that slowed the response to concentration changes and the creation of a diffusion gradient in the hydrogel by media refreshment. Our designed model combined with measurements provides a practical point of reference for diffusion coefficients in real-world culture conditions, enabling more informed choices on hydrogel culture conditions. This model can be improved in the future to simulate more complicated intrinsic hydrogel properties and study the effects of secondary interactions on the diffusion of analytes through the hydrogel.

Intestinal organoids recapitulate many features of the in vivo gastrointestinal tract and have revolutionized in vitro studies of intestinal function and disease. However, the restricted accessibility of the apical surface of the organoids facing the central lumen (apical-in) limits studies related to nutrient uptake and drug absorption and metabolism. Here, we demonstrate that pluripotent stem cell (PSC)-derived intestinal organoids with reversed epithelial polarity (apical-out) can successfully recapitulate tissue-specific functions. In particular, these apical-out organoids show strong epithelial barrier formation with all the major junctional complexes, nutrient transport and active lipid metabolism. Furthermore, the organoids express drug-metabolizing enzymes and relevant apical and basolateral transporters. The scalable and robust generation of functional, apical-out intestinal organoids lays the foundation for a completely new range of organoid-based high-throughput/high-content in vitro applications in the fields of nutrition, metabolism and drug discovery., Competing Interests: RT and SG are founders and shareholders of 300MICRONS GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kakni, López-Iglesias, Truckenmüller, Habibović and Giselbrecht.)

Microbiome is an integral part of the gut and is essential for its proper function. Imbalances of the microbiota can be devastating and have been linked with several gastrointestinal conditions. Current gastrointestinal models do not fully reflect the in vivo situation. Thus, it is important to establish more advanced in vitro models to study host-microbiome/pathogen interactions. Here, we developed for the first time an apical-out human small intestinal organoid model in hypoxia, where the apical surface is directly accessible and exposed to a hypoxic environment. These organoids mimic the intestinal cell composition, structure and functions and provide easy access to the apical surface. Co-cultures with the anaerobic strains Lactobacillus casei and Bifidobacterium longum showed successful colonization and probiotic benefits on the organoids. These novel hypoxia-tolerant apical-out small intestinal organoids will pave the way for unraveling unknown mechanisms related to host-microbiome interactions and serve as a tool to develop microbiome-related probiotics and therapeutics., Competing Interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: R.T and S.G are founders and shareholders of 300MICRONS GmbH., (© The Author(s) 2023.)

The mammalian intestinal epithelium contains more immune cells than any other tissue, and this is largely because of its constant exposure to pathogens. Macrophages are crucial for maintaining intestinal homeostasis, but they also play a central role in chronic pathologies of the digestive system. We developed a versatile microwell-based intestinal organoid-macrophage co-culture system that enables us to recapitulate features of intestinal inflammation. This microwell-based platform facilitates the controlled positioning of cells in different configurations, continuous in situ monitoring of cell interactions, and high-throughput downstream applications. Using this novel system, we compared the inflammatory response when intestinal organoids were co-cultured with macrophages versus when intestinal organoids were treated with the pro-inflammatory cytokine TNF-α. Furthermore, we demonstrated that the tissue-specific response differs according to the physical distance between the organoids and the macrophages and that the intestinal organoids show an immunomodulatory competence. Our novel microwell-based intestinal organoid model incorporating acellular and cellular components of the immune system can pave the way to unravel unknown mechanisms related to intestinal homeostasis and disorders.

For over a decade, organoids mimicking the development, physiology, and disease of the digestive system have been a topic of broad interest and intense study. Establishing organoid models that recapitulate all distinct regions of the gastrointestinal tract (GIT) has proven challenging since each tissue surrogate requires tailor-made modifications of the original protocol to generate intestinal organoids. In this review, we discuss the challenges and current advances of the GIT organoid models. Moreover, we envision the next-generation GIT organoids as integrated organoid models, able to recapitulate structural and functional characteristics of multiple regions of the digestive tube in a single in vitro model. We discuss these new trends and provide an outlook for the future of GIT in vitro models., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Tissue-culture-ware polystyrene is the gold standard for in vitro cell culture. While microengineering techniques can create advanced cell microenvironments in polystyrene, they require specialized equipment and reagents, which hinder their accessibility for most biological researchers. An economical and easily accessible method is developed and validated for fabricating microstructures directly in polystyrene with sizes approaching subcellular dimensions while requiring minimal processing time. The process involves deep ultraviolet irradiation through a shadow mask or ink pattern using inexpensive, handheld devices followed by selective chemical development with common reagents to generate micropatterns with depths/heights between 5 and 10 µm, which can be used to guide cell behavior. The remarkable straightforwardness of the process enables this class of microengineering techniques to be broadly accessible to diverse research communities., (© 2022 The Authors. Advanced Materials published by Wiley-VCH GmbH.)

A comparatively straightforward approach to accomplish more physiological realism in organ-on-a-chip (OoC) models is through substrate geometry. There is increasing evidence that the strongly, microscale curved surfaces that epithelial or endothelial cells experience when lining small body lumens, such as the alveoli or blood vessels, impact their behavior. However, the most commonly used cell culture substrates for modeling of these human tissue barriers in OoCs, ion track-etched porous membranes, provide only flat surfaces. Here, we propose a more realistic culture environment for alveolar cells based on biomimetically microcurved track-etched membranes. They recreate the mainly spherical geometry of the cells' native microenvironment. In this feasibility study, the membranes were given the shape of hexagonally arrayed hemispherical microwells by an innovative combination of three-dimensional (3D) microfilm (thermo)forming and ion track technology. Integrated in microfluidic chips, they separated a top from a bottom cell culture chamber. The microcurved membranes were seeded by infusion with primary human alveolar epithelial cells. Despite the pronounced topology, the cells fully lined the alveoli-like microwell structures on the membranes' top side. The confluent curved epithelial cell monolayers could be cultured successfully at the air-liquid interface for 14 days. Similarly, the top and bottom sides of the microcurved membranes were seeded with cells from the Calu-3 lung epithelial cell line and human lung microvascular endothelial cells, respectively. Thereby, the latter lined the interalveolar septum-like interspace between the microwells in a network-type fashion, as in the natural counterpart. The coculture was maintained for 11 days. The presented 3D lung-on-a-chip model might set the stage for other (micro)anatomically inspired membrane-based OoCs in the future.

Cells probe their environment and adapt their shape accordingly via the organization of focal adhesions and the actin cytoskeleton. In an earlier publication, we described the relationship between cell shape and physiology, for example, shape-induced differentiation, metabolism, and proliferation in mesenchymal stem cells and tenocytes. In this study, we investigated how these cells organize their adhesive machinery over time when exposed to microfabricated surfaces of different topographies and adhesive island geometries. We further examined the reciprocal interaction between stress fiber and focal adhesion formation by pharmacological perturbations. Our results confirm the current literature that spatial organization of adhesive sites determines the ability to form focal adhesions and stress fibers. Therefore, cells on roughened surfaces have smaller focal adhesion and fewer stress fibers. Our results further highlight the importance of integrin-mediated adhesion in the adaptive properties of cells and provide clear links to the development of bioactive materials., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Dede Eren, Lucassen, Tuvshindorj, Truckenmüller, Giselbrecht, Eren, Tas, Sudarsanam and de Boer.)

The inner surface of the intestine is a dynamic system, composed of a single layer of polarized epithelial cells. The development of intestinal organoids was a major breakthrough since they robustly recapitulate intestinal architecture, regional specification and cell composition in vitro . However, the cyst-like organization hinders direct access to the apical side of the epithelium, thus limiting their use in functional assays. For the first time, we show an intestinal organoid model from pluripotent stem cells with reversed polarity where the apical side faces the surrounding culture media and the basal side faces the lumen. These inside-out organoids preserve a distinct apico-basolateral orientation for a long period and differentiate into the major intestinal cell types. This novel model lays the foundation for developing new in vitro functional assays particularly targeting the apical surface of the epithelium and thus offers a new research tool to study nutrient/drug uptake, metabolism and host-microbiome/pathogen interactions., Competing Interests: RT and SG are founders and shareholders of 300MICRONS GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kakni, López-Iglesias, Truckenmüller, Habibović and Giselbrecht.)

In vivo cells reside in a complex extracellular matrix (ECM) that presents spatially distributed biochemical and -physical cues at the nano- to micrometer scales. Chemical micropatterning is successfully used to generate adhesive islands to control where and how cells attach and restore cues of the ECM in vitro. Although chemical micropatterning has become a powerful tool to study cell-material interactions, only a fraction of the possible micropattern designs was covered so far, leaving many other possible designs still unexplored. Here, a high-throughput screening platform called "Galapagos chip" is developed. It contains a library of 2176 distinct subcellular chemical patterns created using mathematical algorithms and a straightforward UV-induced two-step surface modification. This approach enables the immobilization of ligands in geometrically defined regions onto cell culture substrates. To validate the system, binary RGD/polyethylene glycol patterns are prepared on which human mesenchymal stem cells are cultured, and the authors observe how different patterns affect cell and organelle morphology. As proof of concept, the cells are stained for the mechanosensitive YAP protein, and, using a machine-learning algorithm, it is demonstrated that cell shape and YAP nuclear translocation correlate. It is concluded that the Galapagos chip is a versatile platform to screen geometrical aspects of cell-ECM interaction., (© 2022 The Authors. Small published by Wiley-VCH GmbH.)

There is an increasing need for automated label-free morphometric analysis using brightfield microscopy images of 3D cell culture systems. This requires automated feature detection which can be achieved by improving the image contrast, e.g. by reducing the refractive index mismatch in the light path. Here, a novel microcavity platform fabricated using microthermoforming of thin fluorinated ethylene-propylene (FEP) films which match the refractive index of cell culture medium and provide a homogenous background signal intensity is described. FEP is chemically inert, mechanically stable and has been used as a substrate for light sheet microscopy. The microcavities promote formation of mouse embryonic stem cell (mESC) aggregates, which show axial elongation and germ layer specification similar to embryonic development. A label-free feature extraction pipeline based on a machine-learning plugin for FIJI is used to extract morphometric features from time-lapse imaging in a highly robust and reproducible manner. Lastly, the pipeline is utilized for testing the effect of the drug Latrunculin A on the mESC aggregates, highlighting the platform's potential for high-content screening (HCS) in drug discovery. This new microengineered tool is an important step towards label-free imaging of free-floating stem cell aggregates and paves the way for high-content drug testing and translational studies.

Epithelial membrane transporter kinetics portray an irrefutable role in solute transport in and out of cells. Mechanistic models are used to investigate the transport of solutes at the organ, tissue, cell or membrane scale. Here, we review the recent advancements in using computational models to investigate epithelial transport kinetics on the cell membrane. Various methods have been employed to develop transport phenomena models of solute flux across the epithelial cell membrane. Interestingly, we noted that many models used lumped parameters, such as the Michaelis-Menten kinetics, to simplify the transporter-mediated reaction term. Unfortunately, this assumption neglects transporter numbers or the fact that transport across the membrane may be affected by external cues. In contrast, more recent mechanistic transporter kinetics models account for the transporter number. By creating models closer to reality researchers can investigate the downstream effects of physical or chemical disturbances on the system. Evidently, there is a need to increase the complexity of mechanistic models investigating the solute flux across a membrane to gain more knowledge of transporter-solute interactions by assigning individual parameter values to the transporter kinetics and capturing their dependence on each other. This change results in better pharmacokinetic predictions in larger scale platforms. More reliable and efficient model predictions can be made by creating mechanistic computational models coupled with dedicated in vitro experiments. It is also vital to foster collaborative efforts among transporter kinetics researchers in the modeling, material science and biological fields., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 King, Giselbrecht, Truckenmüller and Carlier.)

Toxin removal by the kidney is deficient in a patient suffering from end-stage kidney disease (ESKD), and current dialysis therapies are insufficient in subsidizing this loss. A bioartificial kidney (BAK) aspires to offer ESKD patients a more effective alternative to dialysis. Mathematical models are necessary to support further developments and improve designs for the BAK before clinical trials. The BAK differentiates itself from dialysis by incorporating a living proximal tubule cell monolayer to account for the active transport of protein-bound uremic toxins, namely indoxyl sulfate (IS) in this study. Optimizing such a device is far from trivial due to the non-intuitive spatiotemporal dynamics of the IS removal process. This study used mathematical models to compare two types of active transport kinetics. i.e., two-step binding and lumped parameter. The modeling results indicated that the transporter density is the most influential parameter for the IS clearance. Moreover, a uniform distribution of transporters increases the IS clearance, highlighting the need for a high-quality, functional proximal tubule monolayer in the BAK. In summary, this study contributed to an improved understanding of IS transport in the BAK, which can be used along with laboratory experiments to develop promising renal replacement therapies in the future., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Research has shown that traditional dialysis is an insufficient long-term therapy for patients suffering from end-stage kidney disease due to the high retention of uremic toxins in the blood as a result of the absence of the active transport functionality of the proximal tubule (PT). The PT's function is defined by the epithelial membrane transporters, which have an integral role in toxin clearance. However, the intricate PT transporter-toxin interactions are not fully explored, and it is challenging to decouple their effects in toxin removal in vitro. Computational models are necessary to unravel and quantify the toxin-transporter interactions and develop an alternative therapy to dialysis. This includes the bioartificial kidney, where the hollow dialysis fibers are covered with kidney epithelial cells. In this integrated experimental-computational study, we developed a PT computational model that focuses on indoxyl sulfate (IS) transport by organic anionic transporter 1 (OAT1), capturing the transporter density in detail along the basolateral cell membrane as well as the activity of the transporter and the inward boundary flux. The unknown parameter values of the OAT1 density (1.15×107 transporters µm-2), IS uptake (1.75×10-5 µM-1 s-1), and dissociation (4.18×10-4 s-1) were fitted and validated with experimental LC-MS/MS time-series data of the IS concentration. The computational model was expanded to incorporate albumin conformational changes present in uremic patients. The results suggest that IS removal in the physiological model was influenced mainly by transporter density and IS dissociation rate from OAT1 and not by the initial albumin concentration. While in uremic conditions considering albumin conformational changes, the rate-limiting factors were the transporter density and IS uptake rate, which were followed closely by the albumin-binding rate and IS dissociation rate. In summary, the results of this study provide an exciting avenue to help understand the toxin-transporter complexities in the PT and make better-informed decisions on bioartificial kidney designs and the underlining transporter-related issues in uremic patients.

Cellular and intracellular processes are inherently complex due to the large number of components and interactions, which are often nonlinear and occur at different spatiotemporal scales. Because of this complexity, mathematical modeling is increasingly used to simulate such systems and perform experiments in silico, many orders of magnitude faster than real experiments and often at a higher spatiotemporal resolution. In this article, we will focus on the generic modeling process and illustrate it with an example model of membrane lipid turnover., Competing Interests: The authors have declared that no competing interests exist.

As organoids offer a promising tool to study cell biology and model diseases, organoid technology has rapidly evolved over the last few years. Even though intestinal organoids are one of the most well-established organoid systems, they currently rely on the embedding into an excess amount of poorly defined, tumor-derived extracellular matrix. Here, a novel suspension method is suggested to grow mouse intestinal organoids inside thermoformed microwell arrays. This platform promotes the controlled growth of organoids under matrix-reduced conditions, with Matrigel only used as medium supplement. Hence, this system provides numerous advantages over the previously established methods. Based on the findings, viable and functional mouse intestinal organoids can be preserved for longer periods than in traditional Matrigel domes. Additionally, this microwell-based technique renders a novel organoid culture system in which the heterogeneity of the organoids is significantly reduced. The method paves the way toward more controlled organoid culture systems that can also be beneficial for further downstream applications, such as automated imaging techniques and micromanipulations, which constitute valuable tools for high-throughput applications and translational studies., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Recently developed stem-cell-based in vitro models of morphogenesis can help shed light on the mechanisms involved in embryonic patterning. These models are showcased using traditional cell culture platforms and materials, which allow limited control over the biological system and usually do not support high-content imaging. In contrast, using advanced microengineered tools can help in microscale control, long-term culture, and real-time data acquisition from such biological models and aid in elucidating the underlying mechanisms. Here, a new culturing, manipulation and analysis platform is described to study in vitro morphogenesis using thin polycarbonate film-based microdevices. A pipeline consisting of open-source software to quantify 3D cell movement using 4D image acquisition is developed to analyze cell migration within the multicellular clusters. It is shown that the platform can be used to control and study morphogenesis in non-adherent cultures of the P19C5 mouse stem cell line and mouse embryonic stem cells (mESCs) that show symmetry breaking and axial elongation events similar to early embryonic development. Using the new platform, it is found that localized cell proliferation and coordinated cell migration result in elongation morphogenesis of the P19C5 aggregates. Further, it is found that polarization and elongation of mESC aggregates are dependent on directed cell migration., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Research on acute and chronic lung diseases would greatly benefit from reproducible availability of alveolar epithelial cells (AEC). Primary alveolar epithelial cells can be derived from human lung tissue but the quality of these cells is highly donor dependent. Here, we demonstrated that culture of EpCAM + cells derived from human induced pluripotent stem cells (hiPSC) at the physiological air-liquid interface (ALI) resulted in type 2 AEC-like cells (iAEC2) with alveolar characteristics. iAEC2 cells expressed native AEC2 markers (surfactant proteins and LPCAT-1) and contained lamellar bodies. ALI-iAEC2 were used to study alveolar repair over a period of 2 weeks following mechanical wounding of the cultures and the responses were compared with those obtained using primary AEC2 (pAEC2) isolated from resected lung tissue. Addition of the Wnt/β-catenin activator CHIR99021 reduced wound closure in the iAEC2 cultures but not pAEC2 cultures. This was accompanied by decreased surfactant protein expression and accumulation of podoplanin-positive cells at the wound edge. These results demonstrated the feasibility of studying alveolar repair using hiPSC-AEC2 cultured at the ALI and indicated that this model can be used in the future to study modulation of alveolar repair by (pharmaceutical) compounds.

In bottom-up tissue engineering, small modular units of cells and biomaterials are assembled toward ​larger and more complex ones. In conjunction with a new implementation of this approach, a novel method to fabricate microscale objects from biopolymers by thermal imprinting on water-soluble sacrificial layers is presented. By this means, geometrically well-defined objects could be obtained without involving toxic agents in the form of photoinitiators. The micro-objects were used as cell-adhesive substrates and cell spacers in engineered tissues created by cell-guided assembly of the objects. Such constructs can be applied both for in vitro studies and clinical treatments. Clinically relevantly sized aggregates comprised of cells and micro-objects retained their viability up to 2 weeks of culture. The aggregation behavior of cells and objects showed to depend on the type and number of cells applied. To demonstrate the micro-objects' potential for engineering vascularized tissues, small aggregates of human bone marrow stromal cells (hMSCs) and micro-objects were coated with a layer of human umbilical vein endothelial cells (HUVECs) and fused into larger tissue constructs, resulting in HUVEC-rich regions at the aggregates' interfaces. This three-dimensional network-type spatial cellular organization could foster the establishment of (premature) vascular structures as a vital prerequisite of, for example, bottom-up-engineered bone-like tissue., (© 2019 The Authors.)

In biological systems, form and function are inherently correlated. Despite this strong interdependence, the biological effect of curvature has been largely overlooked or underestimated, and consequently it has rarely been considered in the design of new cell-material interfaces. This review summarizes current understanding of the interplay between the curvature of a cell substrate and the related morphological and functional cellular response. In this context, we also discuss what is currently known about how, in the process of such a response, cells recognize curvature and accordingly reshape their membrane. Beyond this, we highlight state-of-the-art microtechnologies for engineering curved biomaterials at cell-scale, and describe aspects that impair or improve readouts of the pure effect of curvature on cells., (Copyright © 2019 Elsevier Ltd. All rights reserved.)