Three-dimensional cell cultures are leading the way to the fabrication of tissue-like constructs useful to developmental biology and pharmaceutical screenings. However, their reproducibility and translational potential have been limited by biomaterial and culture media compositions, as well as cellular sources. We developed a construct comprising synthetic multifunctionalized hydrogels, serum-free media, and densely seeded good manufacturing practice protocol-grade human neural stem cells (hNSC). We tracked hNSC proliferation, differentiation, and maturation into GABAergic, glutamatergic, and cholinergic neurons, showing entangled electrically active neural networks. The neuroregenerative potential of the “engineered tissue” was assessed in spinal cord injuries, where hNSC-derived progenitors and predifferentiated hNSC progeny, embedded in multifunctionalized hydrogels, were implanted. All implants decreased astrogliosis and lowered the immune response, but scaffolds with predifferentiated hNSCs showed higher percentages of neuronal markers, better hNSC engraftment, and improved behavioral recovery. Our hNSC-construct enables the formation of 3D functional neuronal networks in vitro, allowing novel strategies for hNSC therapies in vivo.
The development of therapeutic approaches for spinal cord injury (SCI) is still a challenging goal to achieve. The pathophysiological features of chronic SCI are glial scar and cavity formation: an effective therapy will require contribution of different disciplines such as materials science, cell biology, drug delivery and nanotechnology. One of the biggest challenges in SCI regeneration is to create an artificial scaffold that could mimic the extracellular matrix (ECM) and support nervous system regeneration. Electrospun constructs and hydrogels based on self-assembling peptides (SAPs) have been recently preferred. In this work SAPs and polymers were assembled by using a coaxial electrospinning setup. We tested the biocompatibility of two types of coaxially electrospun microchannels: the first one made by a core of poly(ε-caprolactone) and poly(d,l-lactide-co-glycolide) (PCL-PLGA) and a shell of an emulsion of PCL-PLGA and a functionalized self-assembling peptide Ac-FAQ and the second one made by a core of Ac-FAQ and a shell of PCL-PLGA. Moreover, we tested an annealed scaffold by PCL-PLGA microchannel heat-treatment. The properties of coaxial scaffolds were analyzed using scanning electron microscopy (SEM), Fourier transform spectroscopy (FTIR), contact angle measurements and differential scanning calorimetry (DSC). In vitro cytotoxicity was assessed via viability and differentiation assays with neural stem cells (NSCs); whereas in vivo inflammatory response was evaluated following scaffold implantation in rodent spinal cords. Emulsification of the outer shell turned out to be the best choice in terms of cell viability and tissue response: thus suggesting the potential of using functionalized SAPs in coaxial electrospinning for applications in regenerative medicine.
Neural stem cells-based therapies have shown great potential for central nervous system regeneration, with three-dimensional (3D) culture systems representing a key technique for tissue engineering applications, as well as disease modeling and drug screenings. Self-assembling peptides (SAPs), providing biomimetic synthetic microenvironments regulating cellular functionality and tissue repair, constitute a suitable tool for the production of complex tissue-like structures in vitro. However, one of the most important drawbacks in 3D cultures, obtained via animal-derived substrates and serumrich media, is the reproducibility and tunability of a standardized methodology capable to coax neural differentiation of different human cell lines. In this work we cultured four distinct human neural stem cell (hNSC) lines in 3D synthetic multifunctionalized hydrogel (named HYDROSAP) for up to 6 weeks. Three-dimensional cultures of differentiating hNSCs exhibited a progressive differentiation and maturation over time. All hNSCs-derived neurons in 3D culture system exhibited randomly organized entangled networks with increasing expression of GABAergic and glutamatergic phenotypes and presence of cholinergic ones. Oligodendrocytes formed insulating myelin sheaths positive for myelin basic protein (MBP). In summary, results demonstrated a successfully standardized and reproducible 3D cell culture system for hNSC differentiation and maturation in serum-free conditions useful for future therapies.
Human pancreatic islets transplantation is an experimental therapeutic treatment for Type I Diabetes. Limited islets lifespan in culture remains the main drawback, due to the absence of native extracellular matrix as mechanical support after their enzymatic and mechanical isolation procedure. Extending the limited islets lifespan by creating a long-term in vitro culture remains a challenge. In this study, three biomimetic self-assembling peptides were proposed as potential candidates to recreate in vitro a pancreatic extracellular matrix, with the aim to mechanically and biologically support human pancreatic islets, by creating a three-dimensional culture system. The embedded human islets were analyzed for morphology and functionality in long-term cultures (14-and 28-days), by evaluating β-cells content, endocrine component, and extracellular matrix constituents. The three-dimensional support provided by HYDROSAP scaffold, and cultured into MIAMI medium, displayed a preserved islets functionality, a maintained rounded islets morphology and an invariable islets diameter up to 4 weeks, with results analogues to freshly-isolated islets. In vivo efficacy studies of the in vitro 3D cell culture system are ongoing; however, preliminary data suggest that human pancreatic islets pre-cultured for 2 weeks in HYDROSAP hydrogels and transplanted under subrenal capsule may restore normoglycemia in diabetic mice. Therefore, engineered self-assembling peptide scaffolds may provide a useful platform for long-term maintenance and preservation of functional human pancreatic islets in vitro.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.