In this study we used differentiated adult human upcyte® cells for the in vitro generation of liver organoids. Upcyte® cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte® process). Proliferating upcyte® cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte® cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel™, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.
Injury to nerve tissue in the peripheral nervous system (PNS) results in long-term impairment of limb function, dysaesthesia and pain, often with associated psychological effects. Whilst minor injuries can be left to regenerate without intervention and short gaps up to 2 cm can be sutured, larger or more severe injuries commonly require autogenous nerve grafts harvested from elsewhere in the body (usually sensory nerves). Functional recovery is often suboptimal and associated with loss of sensation from the tissue innervated by the harvested nerve. The challenges that persist with nerve repair have resulted in development of nerve guides or conduits from non-neural biological tissues and various polymers to improve the prognosis for the repair of damaged nerves in the PNS. This study describes the design and fabrication of a multimodal controlled pore size nerve regeneration conduit using polylactic acid (PLA) and (PLA):poly(lactic-coglycolic) acid (PLGA) fibers within a neurotrophin-enriched alginate hydrogel. The nerve repair conduit design consists of two types of PLGA fibers selected specifically for promotion of axonal outgrowth and Schwann cell growth (75:25 for axons; 85:15 for Schwann cells). These aligned fibers are contained within the lumen of a knitted PLA sheath coated with electrospun PLA nanofibers to control pore size. The PLGA guidance fibers within the nerve repair conduit lumen are supported within an alginate hydrogel impregnated with neurotrophic factors (NT-3 or BDNF with LIF, SMDF and MGF-1) to provide neuroprotection, stimulation of axonal growth and Schwann cell migration. The conduit was used to promote repair of transected sciatic nerve in rats over a period of 4 weeks. Over this period, it was observed that over-grooming and self-mutilation (autotomy) of the limb implanted with the conduit was significantly reduced in rats implanted with the full-configuration conduit compared to rats implanted with conduits containing only an alginate hydrogel. This indicates return of some feeling to the limb via the fully-configured conduit. Immunohistochemical analysis of the implanted conduits removed from the rats after the four-week implantation period confirmed the presence of myelinated axons within the conduit and distal to the site of implantation, further supporting that the conduit promoted nerve repair over this period of time. This study describes the design considerations and fabrication of a novel multicomponent, multimodal bio-engineered synthetic conduit for peripheral nerve repair. AbstractInjury to nerve tissue in the Peripheral Nervous System (PNS) results in long-term impairment of limb function, dysaesthesia and pain, often with associated psychological effects. Whilst minor injuries can be left to regenerate without intervention and short gaps up to 2 cm can be sutured, larger or more severe injuries commonly require autogenous nerve grafts harvested from elsewhere in the body (usually sensory nerves). Functional recovery is often suboptimal and associated with loss ...
Wet–spun composite fibres from graphene and polypyrrole nanoparticles show appropriate mechanical properties, high electrical conductivity and good cytocompatibility.
Tissue engineering scaffolds for nerve regeneration, or artificial nerve conduits, are particularly challenging due to the high level of complexity the structure of the nerve presents. The list of requirements for artificial nerve conduits is long and includes the ability to physically guide nerve growth using physical and chemical cues as well as electrical stimulation. Combining these characteristics into a conduit, while maintaining biocompatibility and biodegradability, has not been satisfactorily achieved by currently employed fabrication techniques. Here we present a method combining pultrusion and wet-spinning techniques facilitating incorporation of pre-formed filaments into ionically crosslinkable hydrogels. This new biofabrication technique allows the incorporation of conducting or drug-laden filaments, controlled guidance channels and living cells into hydrogels, creating new improved conduit designs.
A polyvinyl alcohol (PVA)-stabilized polypyrrole nanodispersion has been optimised for conductivity and processability by decreasing the quantity of PVA before and after synthesis. A reduction of PVA before synthesis leads to the formation of particles with a slight increase in dry particle diameter (51 ± 6 to 63 ± 3 nm), and conversely a reduced hydrodynamic diameter. Conductivity of the dried nanoparticle films was not measureable after a reduction of PVA prior to synthesis. Using filtration of particles after synthesis, PVA content was sufficiently reduced to achieve dried thin film conductivity of 2 S cm -1 , while the electroactivity of the dispersed particles remained unchanged. The as-synthesized and PVA-reduced polypyrrole particles were successfully spun into allnanoparticle fibres using a wet-extrusion approach without the addition of any polymer or gel matrix. Using nanoparticles as a starting material is a novel approach, which allowed the production of macroscale fibres that consisted entirely of polypyrrole nanoparticles. Fibres made from PVA-reduced polypyrrole showed higher electroactivity compared to fibres composed of the dispersion high in PVA. The mechanical properties of the fibres were also improved by reducing the amount of PVA present, resulting in a stronger, more ductile and less brittle fibre, which could find potential application in various fields.
In nerve and muscle regeneration applications, the incorporation of conducting elements into biocompatible materials has gained interest over the last few years, as it has been shown that electrical stimulation of some regenerating cells has a positive effect on their development. A variety of different materials, ranging from graphene to conducting polymers, have been incorporated into hydrogels and increased conductivities have been reported. However, the majority of conductivity measurements are performed in a dry state, even though material blends are designed for applications in a wet state, in vivo environment. The focus of this work is to use polypyrrole nanoparticles to increase the wet-state conductivity of alginate to produce a conducting, easily processable, cell-supporting composite material. Characterization and purification of the conducting polymer nanoparticle dispersions, as well as electrochemical measurements, have been performed to assess conductivity of the nanoparticles and hydrogel composites in the wet state, in order to determine whether filling an ionically conducting hydrogel with electrically conductive nanoparticles will enhance the conductivity. It was determined that the introduction of spherical nanoparticles into alginate gel does not increase, but rather slightly reduces conductivity of the hydrogel in the wet state. ABSTRACTIn nerve and muscle regeneration applications, the incorporation of conducting elements into biocompatible materials has gained interest over the last few years, as it has been shown that electrical stimulation of some regenerating cells has a positive effect on their development. A variety of different materials, ranging from graphene to conducting polymers, have been incorporated into hydrogels and increased conductivities have been reported. However, the majority of conductivity measurements are performed in a dry state, even though material blends are designed for applications in a wet state, in vivo environment. The focus of this work is to use polypyrrole nanoparticles to increase the wet-state conductivity of alginate to produce a conducting, easily processable, cell-supporting composite material. Characterization and purification of the conducting polymer nanoparticle dispersions, as well as electrochemical measurements, have been performed to assess conductivity of the nanoparticles and hydrogel composites in the wet state, in order to determine whether filling an ionically conducting hydrogel with electrically conductive nanoparticles will enhance the conductivity. It was determined that the introduction of spherical nanoparticles into alginate gel does not increase, but rather slightly reduces conductivity of the hydrogel in the wet state.
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