Engineered extracellular matrices facilitate brain organoids from human pluripotent stem cells.

OBJECTIVE: Brain organoids are miniaturized in vitro brain models generated from pluripotent stem cells, which resemble full-sized brain more closely than conventional two-dimensional cell cultures. Although brain organoids mimic the human brain's cell-to-cell network interactions, they generally fail to faithfully recapitulate cell-to-matrix interactions. Here, an engineered framework, called an engineered extracellular matrix (EECM), was developed to provide support and cell-to-matrix interactions to developing brain organoids. METHODS: We generated brain organoids using EECMs comprised of human fibrillar fibronectin supported by a highly porous polymer scaffold. The resultant brain organoids were characterized by immunofluorescence microscopy, transcriptomics, and proteomics of the cerebrospinal fluid (CSF) compartment. RESULTS: The interstitial matrix-mimicking EECM enhanced neurogenesis, glial maturation, and neuronal diversity from human embryonic stem cells versus conventional protein matrix (Matrigel). Additionally, EECMs supported long-term culture, which promoted large-volume organoids containing over 250 µL of CSF. Proteomics analysis of the CSF found it superseded previous brain organoids in protein diversity, as indicated by 280 proteins spanning 500 gene ontology pathways shared with adult CSF. INTERPRETATION: Engineered EECM matrices represent a major advancement in neural engineering as they have the potential to significantly enhance the structural, cellular, and functional diversity that can be achieved in advanced brain models.

3D jet writing of mechanically actuated tandem scaffolds.

The need for high-precision microprinting processes that are controllable, scalable, and compatible with different materials persists throughout a range of biomedical fields. Electrospinning techniques offer scalability and compatibility with a wide arsenal of polymers, but typically lack precise three-dimensional (3D) control. We found that charge reversal during 3D jet writing can enable the high-throughput production of precisely engineered 3D structures. The trajectory of the jet is governed by a balance of destabilizing charge-charge repulsion and restorative viscoelastic forces. The reversal of the voltage polarity lowers the net surface potential carried by the jet and thus dampens the occurrence of bending instabilities typically observed during conventional electrospinning. In the absence of bending instabilities, precise deposition of polymer fibers becomes attainable. The same principles can be applied to 3D jet writing using an array of needles resulting in complex composite materials that undergo reversible shape transitions due to their unprecedented structural control.

Engineered Fibrillar Fibronectin Networks as Three-Dimensional Tissue Scaffolds.

Extracellular matrix (ECM) proteins, and most prominently, fibronectin (Fn), are routinely used in the form of adsorbed pre-coatings in an attempt to create a cell-supporting environment in both two- and three-dimensional cell culture systems. However, these protein coatings are typically deposited in a form which is structurally and functionally distinct from the ECM-constituting fibrillar protein networks naturally deposited by cells. Here, the cell-free and scalable synthesis of freely suspended and mechanically robust three-dimensional (3D) networks of fibrillar fibronectin (fFn) supported by tessellated polymer scaffolds is reported. Hydrodynamically induced Fn fibrillogenesis at the three-phase contact line between air, an Fn solution, and a tessellated scaffold microstructure yields extended protein networks. Importantly, engineered fFn networks promote cell invasion and proliferation, enable in vitro expansion of primary cancer cells, and induce an epithelial-to-mesenchymal transition in cancer cells. Engineered fFn networks support the formation of multicellular cancer structures cells from plural effusions of cancer patients. With further work, engineered fFn networks can have a transformative impact on fundamental cell studies, precision medicine, pharmaceutical testing, and pre-clinical diagnostics.

3D Jet Writing: Functional Microtissues Based on Tessellated Scaffold Architectures.

The advent of adaptive manufacturing techniques supports the vision of cell-instructive materials that mimic biological tissues. 3D jet writing, a modified electrospinning process reported herein, yields 3D structures with unprecedented precision and resolution offering customizable pore geometries and scalability to over tens of centimeters. These scaffolds support the 3D expansion and differentiation of human mesenchymal stem cells in vitro. Implantation of these constructs leads to the healing of critical bone defects in vivo without exogenous growth factors. When applied as a metastatic target site in mice, circulating cancer cells home in to the osteogenic environment simulated on 3D jet writing scaffolds, despite implantation in an anatomically abnormal site. Through 3D jet writing, the formation of tessellated microtissues is demonstrated, which serve as a versatile 3D cell culture platform in a range of biomedical applications including regenerative medicine, cancer biology, and stem cell biotechnology.

Needleless Electrohydrodynamic Cojetting of Bicompartmental Particles and Fibers from an Extended Fluid Interface.

Electrohydrodynamic cojetting can result in fibers (electrospinning) and particles (electrospraying) with complex, bicompartmental architectures. An important consideration for application of bicompartmental particles and fibers is the limited throughput derived from the use of parallel capillaries, which require laminar flow to form a multifluidic interface. Here, a novel synthesis approach that takes advantage of an extended bicompartmental fluid interface formed at the sharp edge of a 2D plate is reported. Upon application of an electrical potential to the plate, several electrified fluid jets form spontaneously. Depending on the processing conditions, either bicompartmental particles or fibers with well-defined architectures are prepared. Importantly, this needleless process yields production rates that are more than 30 times higher than those of conventional needle-based techniques. Fiber properties, such as morphology or size, are independent of the flow rate, indicating that this process is physically self-regulating by adjusting the number of jets ejecting from the extended fluid interface. The needleless preparation of bicompartmental particles and fibers is an important technological breakthrough that can enable further advances ranging from drug delivery and tissue engineering to industrial applications.

Engineered Human Stem Cell Microenvironments.

Stem cells have the ability to self-renew and differentiate into specialized cell types, and, in the human body, they reside in specialized microenvironments called "stem cell niches." Although several niches have been described and studied in vivo, their functional replication in vitro is still incomplete. The in vitro culture of pluripotent stem cells may represent one of the most advanced examples in the effort to create an artificial or synthetic stem cell niche. A focus has been placed on the development of human stem cell microenvironments due to their significant clinical implications, in addition to the potential differences between animal and human cells. In this concise review we describe the advances in human pluripotent stem cell culture, and explore the idea that the knowledge gained from this model could be replicated to create synthetic niches for other human stem cell populations, which have proven difficult to maintain in vitro.