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In vitro fabrication of vascularized three-dimensional (3D) human cardiomyocyte (CM) tissues derived from human induced pluripotent stem cells (iPSCs) has attracted much attention owing to their requirement of much amount of nutrition and oxygen, but not yet published. In this manuscript, we report construction of vascularized 3D-iPSC-CM tissues by a newly-discovered filtration-Layer-by-Layer (LbL) technique. The filtration-LbL fabricates nanometer-sized fibronectin and gelatin (FN-G) films onto iPSC-CM surfaces. The FN-G nanofilms induce cell-cell interactions via integrin molecules on cell surfaces, leading to construction of 3D-tissues. The constructed vascularized 3D-iPSC-CM tissues would be a promising tool for tissue regeneration and drug development. We believe that this manuscript has a strong impact and offers important suggestions to researchers concerned with biomaterials and tissue engineering.

There is a growing interest in the concept of four-dimensional (4D) printing that combines a three-dimensional (3D) manufacturing process with dynamic modulation for bioinspired soft materials exhibiting more complex functionality. However, conventional approaches have drawbacks of low resolution, control of internal micro/nanostructure, and creation of fast, complex actuation due to a lack of high-resolution fabrication technology and suitable photoresist for soft materials. Here, we report an approach of 4D printing that develops a bioinspired soft actuator with a defined 3D geometry and programmed printing density. Multiphoton lithography (MPL) allows for controlling printing density in gels at pixel-by-pixel with a resolution of a few hundreds of nanometers, which tune swelling behaviors of gels in response to external stimuli. We printed a 3D soft actuator composed of thermoresponsive poly(N-isopropylacrylamide) (PNIPAm) and gold nanorods (AuNRs). To improve the resolution of printing, we synthesized a functional, thermoresponsive macrocrosslinker. Through plasmonic heating by AuNRs, nanocomposite-based soft actuators undergo nonequilibrium, programmed, and fast actuation. Light-mediated manufacture and manipulation (MPL and photothermal effect) offer the feasibility of 4D printing toward adaptive bioinspired soft materials.

The in vitro fabrication of vascularized tissue is a key challenge in tissue engineering, but little is known about the mechanisms of blood-capillary formation. Here we investigated the mechanisms of in vitro vascularization using precisely-controlled 3D-microenvironments constructed by a sandwich culture using the cell-accumulation technique. 3D-microenvironments controlled at the single layer level showed that sandwich culture between more than 3 fibroblast-layers induced tubule formation. Moreover, the secretion of angiogenic factors increased upon increasing the number of sandwiching layers, which induced highly dense tubular networks. We found that not only angiogenic factors, but also the 3D-microenvironments of the endothelial cells, especially apical side, played crucial roles in tubule formation in vitro. Based on this knowledge, the introduction of blood and lymph capillaries into mesenchymal stem cell (MSC) tissues was accomplished. These findings would be useful for the in vitro vascularization of various types of engineered organs and studies on angiogenesis.

In vitro reconstruction of an alveolar barrier for modeling normal lung functions and pathological events serve as reproducible, high-throughput pharmaceutical platforms for drug discovery, diagnosis, and regenerative medicine. Despite much effort, the reconstruction of organ-level alveolar barrier functions has failed due to the lack of structural similarity to the natural basement membrane, functionalization with specific ligands for alveolar cell function, the use of primary cells and biodegradability. Here we report a bipolar cultured alveolar-capillary barrier model of human primary cells supported by a basement membrane mimics of fully synthetic bifunctional nanofibers. One-step electrospinning process using a bioresorbable polyester and multifunctional star-shaped polyethylene glycols (sPEG) enables the fabrication of an ultrathin nanofiber mesh with interconnected pores. The nanofiber mesh possessed mechanical stability against cyclic expansion as seen in the lung in vivo. The sPEGs as an additive provide biofunctionality to fibers through the conjugation of peptide to the nanofibers and hydrophilization to prevent unspecific protein adsorption. Biofunctionalized nanofiber meshes facilitated bipolar cultivation of endothelial and epithelial cells with fundamental alveolar functionality and showed higher permeability for molecules compared to microporous films. This nanofiber mesh for a bipolar cultured barrier have the potential to promote growth of an organ-level barrier model for modeling pathological conditions and evaluating drug efficacy, environmental pollutants, and nanotoxicology.