Polymer film-based microwell array platform for long-term culture and research of human bronchial organoids.

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.

3D Lung-on-Chip Model Based on Biomimetically Microcurved Culture Membranes.

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.

Development of an In Vitro Airway Epithelial-Endothelial Cell Culture Model on a Flexible Porous Poly(Trimethylene Carbonate) Membrane Based on Calu-3 Airway Epithelial Cells and Lung Microvascular Endothelial Cells.

Due to the continuing high impact of lung diseases on society and the emergence of new respiratory viruses, such as SARS-CoV-2, there is a great need for in vitro lung models that more accurately recapitulate the in vivo situation than current models based on lung epithelial cell cultures on stiff membranes. Therefore, we developed an in vitro airway epithelial-endothelial cell culture model based on Calu-3 human lung epithelial cells and human lung microvascular endothelial cells (LMVECs), cultured on opposite sides of flexible porous poly(trimethylene carbonate) (PTMC) membranes. Calu-3 cells, cultured for two weeks at an air-liquid interface (ALI), showed good expression of the tight junction (TJ) protein Zonula Occludens 1 (ZO-1). LMVECs cultured submerged for three weeks were CD31-positive, but the expression was diffuse and not localized at the cell membrane. Barrier functions of the Calu-3 cell cultures and the co-cultures with LMVECs were good, as determined by electrical resistance measurements and fluorescein isothiocyanate-dextran (FITC-dextran) permeability assays. Importantly, the Calu-3/LMVEC co-cultures showed better cell viability and barrier function than mono-cultures. Moreover, there was no evidence for epithelial- and endothelial-to-mesenchymal transition (EMT and EndoMT, respectively) based on staining for the mesenchymal markers vimentin and α-SMA, respectively. These results indicate the potential of this new airway epithelial-endothelial model for lung research. In addition, since the PTMC membrane is flexible, the model can be expanded by introducing cyclic stretch for enabling mechanical stimulation of the cells. Furthermore, the model can form the basis for biomimetic airway epithelial-endothelial and alveolar-endothelial models with primary lung epithelial cells.

A 3D polydimethylsiloxane microhourglass-shaped channel array made by reflowing photoresist structures for engineering a blood capillary network.

This paper describes an innovative yet straightforward fabrication technique to create three-dimensional microstructures with controllable tapered geometries by combining conventional photolithography and thermal reflow of photoresist. Positive photoresist-based microchannel structures with varying width-to-length ratios were reflowed after their fabrication to generate three-dimensional funnel structures with varying curvatures. A polydimethylsiloxane hourglass-shaped microchannel array was next cast on these photoresist structures, and primary human lung microvascular endothelial cells were cultured in the device to engineer an artificial capillary network. Our work demonstrates that this cost-effective and straightforward fabrication technique has great potential in engineering three-dimensional microstructures for biomedical and biotechnological applications such as blood vessel regeneration strategies, drug screening for vascular diseases, microcolumns for bioseparation, and other fluid dynamic studies at microscale.

Development of Porous and Flexible PTMC Membranes for In Vitro Organ Models Fabricated by Evaporation-Induced Phase Separation.

Polymeric membranes are widely applied in biomedical applications, including in vitro organ models. In such models, they are mostly used as supports on which cells are cultured to create functional tissue units of the desired organ. To this end, the membrane properties, e.g., morphology and porosity, should match the tissue properties. Organ models of dynamic (barrier) tissues, e.g., lung, require flexible, elastic and porous membranes. Thus, membranes based on poly (dimethyl siloxane) (PDMS) are often applied, which are flexible and elastic. However, PDMS has low cell adhesive properties and displays small molecule ad- and absorption. Furthermore, the introduction of porosity in these membranes requires elaborate methods. In this work, we aim to develop porous membranes for organ models based on poly(trimethylene carbonate) (PTMC): a flexible polymer with good cell adhesive properties which has been used for tissue engineering scaffolds, but not in in vitro organ models. For developing these membranes, we applied evaporation-induced phase separation (EIPS), a new method in this field based on solvent evaporation initiating phase separation, followed by membrane photo-crosslinking. We optimised various processing variables for obtaining form-stable PTMC membranes with average pore sizes between 5 to 8 µm and water permeance in the microfiltration range (17,000-41,000 L/m2/h/bar). Importantly, the membranes are flexible and are suitable for implementation in in vitro organ models.

In vitro modelling of alveolar repair at the air-liquid interface using alveolar epithelial cells derived from human induced pluripotent stem cells.

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/ß-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.

From fiber curls to mesh waves: a platform for the fabrication of hierarchically structured nanofibers mimicking natural tissue formation.

Bioinstructive scaffolds for regenerative medicine are characterized by intrinsic properties capable of directing cell response and promoting wound healing. The design of such scaffolds requires the incorporation of well-defined physical properties that mimic the native extracellular matrix (ECM). Here, inspired by epithelial tissue morphogenesis, we present a novel approach to code nanofiber materials with controlled hierarchical wavy structures resembling the configurations of native EMC fibers through using thermally shrinking materials as substrates onto which the fibers are deposited. This approach could serve as a platform for fabricating functional scaffolds mimicking various tissues such as trachea, iris, artery wall and ciliary body. Modeling affirms that the mechanical properties of the fabricated wavy fibers could be regulated through varying their wavy patterns. The nanofibrous scaffolds coded with wavy patterns show an enhanced cellular infiltration. In addition, we further investigated whether the wavy patterns could regulate transforming growth factor-beta (TGF-ß) production, a key signalling pathway involved in connective tissue development. Our results demonstrated that nanofibrous scaffolds coded with wavy patterns could induce TGF-ß expression without the addition of a soluble growth factor. Our new approach could open up new avenues for fabricating bioinstructive scaffolds for regenerative medicine.

Overlooked? Underestimated? Effects of Substrate Curvature on Cell Behavior.

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.

Pre-crosslinked polymeric collagen in 3-D models of mechanically stiff tissues: blended collagen polymer hydrogels for rapid layer fabrication.

Currently one factor hindering the development of collagen hydrogel constructs for tissue engineering is the mismatch between initial cellularity and mechanical strength. The main advantage of collagen hydrogel tissue constructs is their ability to support interstitially seeded cells. However, cells are sensitive to their environment, in particular, substrate stiffness, which cannot easily be replicated within hydrogels without cytotoxic cross-linking treatment. In this study, pre-crosslinked polymeric collagen fibrils are introduced as a starting material, thereby avoiding artificial cross-linking. Shear aggregation of this material in solution results in fibril alignment, but cell addition is only possible when polymeric collagen is blended with its monomeric counterparts to slow the aggregation of collagen fibrils. The hydrogel can then be brought to physiological collagen density by plastic compression. Interstitially seeded fibroblasts were supported for 14days. Although compression of blended gels resulted in some cell death due to increased rate of fluid expulsion, not normally seen in conventional collagen hydrogels, the surviving cell population recovers during subsequent culture. Importantly, the compression process can be controlled and customized to limit cell damage. This is the first report of native polymeric collagen used in a tissue engineering context, for the rapid production of a stiff collagen-cell constructs.