Stem cell-based vascularization of microphysiological systems.

Microphysiological systems (MPSs) (i.e., tissue or organ chips) exploit microfluidics and 3D cell culture to mimic tissue and organ-level physiology. The advent of human induced pluripotent stem cell (hiPSC) technology has accelerated the use of MPSs to study human disease in a range of organ systems. However, in the reduction of system complexity, the intricacies of vasculature are an often-overlooked aspect of MPS design. The growing library of pluripotent stem cell-derived endothelial cell and perivascular cell protocols have great potential to improve the physiological relevance of vasculature within MPS, specifically for in vitro disease modeling. Three strategic categories of vascular MPS are outlined: self-assembled, interface focused, and 3D biofabricated. This review discusses key features and development of the native vasculature, linking that to how hiPSC-derived vascular cells have been generated, the state of the art in vascular MPSs, and opportunities arising from interdisciplinary thinking.

Guided assembly of cancer ellipsoid on suspended hydrogel microfibers estimates multi-cellular traction force.

Three-dimensional (3D) multi-cellular aggregates hold important applications in tissue engineering and in vitro biological modeling. Probing the intrinsic forces generated during the aggregation process, could open up new possibilities in advancing the discovery of tissue mechanics-based biomarkers. We use individually suspended, and tethered gelatin hydrogel microfibers to guide multicellular aggregation of brain cancer cells (glioblastoma cell line, U87), forming characteristic cancer 'ellipsoids'. Over a culture period of up to 13 days, U87 aggregates evolve from a flexible cell string with cell coverage following the relaxed and curly fiber contour; to a distinct ellipsoid-on-string morphology, where the fiber segment connecting the ellipsoid poles become taut. Fluorescence imaging revealed the fiber segment embedded within the ellipsoidal aggregate to exhibit a morphological transition analogous to filament buckling under a compressive force. By treating the multicellular aggregate as an effective elastic medium where the microfiber is embedded, we applied a filament post-buckling theory to model the fiber morphology, deducing the apparent elasticity of the cancer ellipsoid medium, as well as the collective traction force inherent in the aggregation process.

Fabrication of Designable and Suspended Microfibers via Low-Voltage 3D Micropatterning.

Building two-dimensional (2D) and three-dimensional (3D) fibrous structures in the micro- and nanoscale will offer exciting prospects for numerous applications spanning from sensors to energy storage and tissue engineering scaffolds. Electrospinning is a well-suited technique for drawing micro- to nanoscale fibers, but current methods of building electrospun fibers in 3D are restrictive in terms of printed height, design of macroscopic fiber networks, and choice of polymer. Here, we combine low-voltage electrospinning and additive manufacturing as a method to pattern layers of suspended mesofibers. Layers of fibers are suspended between 3D-printed supports in situ in multiple fiber layers and designable orientations. We examine the key working parameters to attain a threshold for fiber suspension, use those behavioral observations to establish a "fiber suspension indicator", and demonstrate its utility through design of intricate suspended fiber architectures. Individual fibers produced by this method approach the micrometer/submicrometer scale, while the overall suspended 3D fiber architecture can span over a centimeter in height. We demonstrate an application of suspended fiber architectures in 3D cell culture, utilizing patterned fiber topography to guide the assembly of suspended high-cellular-density structures. The solution-based fiber suspension patterning process we report offers a unique competence in patterning soft polymers, including extracellular matrix-like materials, in a high resolution and aspect ratio. The platform could thus offer new design and manufacturing capabilities of devices and functional products by incorporating functional fibrous elements.

Multi-length scale bioprinting towards simulating microenvironmental cues.

It is envisaged that the creation of cellular environments at multiple length scales, that recapitulate in vivo bioactive and structural roles, may hold the key to creating functional, complex tissues in the laboratory. This review considers recent advances in biofabrication and bioprinting techniques across different length scales. Particular focus is placed on 3D printing of hydrogels and fabrication of biomaterial fibres that could extend the feature resolution and material functionality of soft tissue constructs. The outlook from this review discusses how one might create and simulate microenvironmental cues in vitro. A fabrication platform that integrates the competencies of different biofabrication technologies is proposed. Such a multi-process, multiscale fabrication strategy may ultimately translate engineering capability into an accessible life sciences toolkit, fulfilling its potential to deliver in vitro disease models and engineered tissue implants.

Solution fibre spinning technique for the fabrication of tuneable decellularised matrix-laden fibres and fibrous micromembranes.

Recreating tissue-specific microenvironments of the extracellular matrix (ECM) in vitro is of broad interest for the fields of tissue engineering and organ-on-a-chip. Here, we present biofunctional ECM protein fibres and suspended membranes, with tuneable biochemical, mechanical and topographical properties. This soft and entirely biologic membrane scaffold, formed by micro-nano-fibres using low voltage electrospinning, displays three unique characteristics for potential cell culture applications: high-content of key ECM proteins, single-layered mesh membrane, and flexibility for in situ integration into a range of device setups. Extracellular matrix (ECM) powder derived from urinary bladder, was used to fabricate the ECM-laden fibres and membranes. The highest ECM concentration in the dry protein fibre was 50 wt%, with the rest consisting of gelatin. Key ECM proteins, including collagen IV, laminin, and fibronectin, were shown to be preserved post the biofabrication process. The single fibre tensile Young's modulus can be tuned for over two orders of magnitude between ∼600 kPa and 50 MPa depending on the ECM content. Combining the fibre mesh printing with 3D printed or microfabricated structures, culture devices were constructed for endothelial layer formation, and a trans-membrane co-culture formed by glomerular cell types of podocytes and glomerular endothelial cells, demonstrating feasibility of the membrane culture. Our cell culture observation points to the importance of membrane mechanical property and re-modelling ability as a factor for soft membrane-based cell cultures. The ECM-laden fibres and membranes presented here would see potential applications in in vitro assays, and tailoring structure and biological functions of tissue engineering scaffolds. STATEMENT OF SIGNIFICANCE: Recreating tissue-specific microenvironments of the extracellular matrix (ECM) is of broad interest for the fields of tissue engineering and organ-on-a-chip. Both the biochemical and biophysical signatures of the engineered ECM interplay to affect cell response. Currently, there are limited biomaterials processing methods which allow to design ECM membrane properties flexibly and rapidly. Solvents and additives used in many existing processes also induced unwanted ECM protein degradation and toxic residues. This paper presents a solution fibre spinning technique, where careful selection of the solution combination led to well-preserved ECM proteins with tuneable composition. This technique also provides a highly versatile approach to fabricate ECM fibres and membranes, leading to designable fibre Young's modulus for over two orders of magnitude.

Low-Voltage Continuous Electrospinning Patterning.

Electrospinning is a versatile technique for the construction of microfibrous and nanofibrous structures with considerable potential in applications ranging from textile manufacturing to tissue engineering scaffolds. In the simplest form, electrospinning uses a high voltage of tens of thousands volts to draw out ultrafine polymer fibers over a large distance. However, the high voltage limits the flexible combination of material selection, deposition substrate, and control of patterns. Prior studies show that by performing electrospinning with a well-defined "near-field" condition, the operation voltage can be decreased to the kilovolt range, and further enable more precise patterning of fibril structures on a planar surface. In this work, by using solution dependent "initiators", we demonstrate a further lowering of voltage with an ultralow voltage continuous electrospinning patterning (LEP) technique, which reduces the applied voltage threshold to as low as 50 V, simultaneously permitting direct fiber patterning. The versatility of LEP is shown using a wide range of combination of polymer and solvent systems for thermoplastics and biopolymers. Novel functionalities are also incorporated when a low voltage mode is used in place of a high voltage mode, such as direct printing of living bacteria; the construction of suspended single fibers and membrane networks. The LEP technique reported here should open up new avenues in the patterning of bioelements and free-form nano- to microscale fibrous structures.