Enhanced neurite outgrowth by human neurons grown on solid three-dimensional scaffolds (original) (raw)
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Growth of human stem cell-derived neurons on solid three-dimensional polymers
Journal of Biochemical and Biophysical Methods, 2005
Understanding neural differentiation and the development of complex neurite networks in threedimensional matrices is critical for neural tissue engineering in vitro. In this study we describe for the first time the growth of human stem cell-derived neurons on solid polystyrene matrices coated with bioactive molecules. Highly porous foams were prepared from poly(styrene/divinylbenzene) using a high internal phase emulsion (HIPE) as a template to create the porous structure. The resulting polyHIPE matrices were readily coated with aqueous-based solutions including poly-d-lysine and laminin. Human neurons adhered well to poly-d-lysine coated surfaces and extended neural processes, however, neurite outgrowth was particularly enhanced when polymers also received a coating of laminin. These data clearly demonstrate the potential use of solid polystyrene scaffolds to create three-dimensional environments for cell growth and differentiation. We propose that these robust and stable matrices can be conveniently and routinely used in the tissue culture laboratory to study the behaviour of cells grown in three-dimensions. D
Scaffolds for 3D in vitro culture of neural lineage cells
Acta biomaterialia, 2017
Understanding how neurodegenerative disorders develop is not only a key challenge for researchers but also for the wider society, given the rapidly aging populations in developed countries. Advances in this field require new tools with which to recreate neural tissue in vitro and produce realistic disease models. This in turn requires robust and reliable systems for performing 3D in vitro culture of neural lineage cells. This review provides a state of the art update on three-dimensional culture systems for in vitro development of neural tissue, employing a wide range of scaffold types including hydrogels, solid porous polymers, fibrous materials and decellularised tissues as well as microfluidic devices and lab-on-a-chip systems. To provide some context with in vivo development of the central nervous system (CNS), we also provide a brief overview of the neural stem cell niche, neural development and neural differentiation in vitro. We conclude with a discussion of future directions...
Three-dimensional growth matrix for human embryonic stem cell-derived neuronal cells
Journal of Tissue Engineering and Regenerative Medicine, 2014
The future of tissue engineering applications for neuronal cells will require a supportive 3D matrix. This particular matrix should be soft, elastic and supportive for cell growth. In this study, we characterized the suitability of a 3D synthetic hydrogel matrix, PuraMatrix ™ , as a growth platform for human embryonic stem cell (hESC)-derived neural cells. The viability of the cells grown on top of, inside and under the hydrogel was monitored. The maturation and electrical activity of the neuronal networks inside the hydrogel were further characterized. We showed that cells stayed viable on the top of the PuraMatrix ™ surface and growth of the neural cells and neural processes was good. Further, hESC-derived neurons, astrocytes and oligodendrocytes all grew, matured and migrated when cultured inside the hydrogel. Importantly, neuronal cells were able to form electrically active connections that were verified using microelectrode array. Thus, PuraMatrix is a good supportive growth matrix for human neural cells and may serve as a matrix for neuronal scaffolds in neural tissue engineering.
The International Journal of Artificial Organs, 2011
In this study, a highly porous poly (D, L-lactic acid) (PDLLA) scaffold was designed and fabricated using dioxane and thermal-induced phase separation (TIPS) methods (liquid-liquid and solid-liquid). Additionally, we characterized the ability of mouse embryonic stem cells (ESCs) to differentiate into neural cells in PDLLA scaffold with uniform porosity, interconnectivity, and high porosity, and then compared them with cells seeded under conventional two-dimensional (2D) culture conditions. Histochemistry staining showed the migration of differentiated cells through the scaffold. Immunofluorescence analysis of the differentiated cells by counting positive cells revealed that the PDLLA scaffold resulted in a significantly greater number of neural markers, microtubule associated protein-2, β-tubulin III, neurofilament protein, and glial fibrillary acidic protein (the astrocyte marker) when compared to those in 2D culture condition. Moreover, the expression of Nestin, Mash1, Pax6, and H...
Stem Cells, 2009
Researches on neural differentiation using embryonic stem cells (ESC) require analysis of neurogenesis in conditions mimicking physiological cellular interactions as closely as possible. In this study, we report an air-liquid interfacebased culture of human ESC. This culture system allows three-dimensional cell expansion and neural differentiation in the absence of added growth factors. Over a 3-month period, a macroscopically visible, compact tissue developed. Histological coloration revealed a dense neural-like neural tissue including immature tubular structures. Electron microscopy, immunochemistry, and electrophysiological recordings demonstrated a dense network of neurons, astrocytes, and oligodendrocytes able to propagate signals. Within this tissue, tubular structures were niches of cells resembling germinal layers of human fetal brain. Indeed, the tissue contained abundant proliferating cells expressing markers of neural progenitors. Finally, the capacity to generate neural tissues on air-liquid interface differed for different ESC lines, confirming variations of their neurogenic potential. In conclusion, this study demonstrates in vitro engineering of a human neural-like tissue with an organization that bears resemblance to early developing brain. As opposed to previously described methods, this differentiation (a) allows three-dimensional organization, (b) yields dense interconnected neural tissue with structurally and functionally distinct areas, and (c) is spontaneously guided by endogenous developmental cues. STEM
Biomaterials, 2010
In the present in vitro study, the axon growth promoting effects of human neural progenitor-derived astrocytes (hNP-AC) were investigated in simple 2D-as well as in more complex 3D-culture systems. The interactions of the hNP-AC with migrating Schwann cells and fibroblasts were also studied. hNP-AC were found to promote extensive dorsal root ganglion axon regeneration in 2D cultures, being even greater than that observed on the positive control, laminin-coated substrate. Contact-mediated mechanisms and the release of substances into the medium both played a role in supporting axon regeneration. Following seeding onto 3D collagen scaffolds, hNP-AC also promoted significantly greater axon regeneration from DRG explants than was seen on non-seeded scaffolds. The highly orientated, porous microstructure of the scaffold also supported substantial intermixing of hNP-AC and migrating Schwann cells/fibroblasts from the DRG explant, cell populations that are normally mutually repulsive. This suggests that the topography of 3D scaffolds may not only influence cellesubstrate interactions but also cellecell interactions within the scaffold. This opens the possibility that the design of future scaffolds could be optimised to enhance cell integration as well as modulating complex cellecell interactions.
Development and Characterization of scaffold based three-dimensional neuronal cultures
2017
Culture of primary neurons, and especially hippocampal neurons, is important for understanding cellular mechanisms in neurobiology. Actually, this is achieved by using culture dish or glass slide with surface coated proteins. Here, we proposed a patch method for culture of primary neurons on a monolayer of gelatin nanofibers electrospun and crosslinked on a honeycomb microframe of poly (ethylene glycol) diacrylate (PEGDA). This method allows us to minimize exogenous material contact of cells and largely increase the exposure area of cells to the culture medium. We found that neurons, and especially astrocytes, have a more in vivo like morphology comparing to that on culture dish or on glass slide. We also found that neurons were preferentially located in the suspended areas of the monolayer nanofibers. Finally, calcium imaging revealed that primary neurons have a higher degree of neural activity on the patch than on glass. These results suggest that crosslinked and monolayer gelatin nanofibers closely mimic the extracellular matrix structure and allow more effective culture of primary neurons than conventional methods, thus facilitating advanced studies of neural functions as well as cell-based assays.
Neural differentiation of pluripotent cells in 3D alginate-based cultures
2014
Biomaterial-supported culture methods, allowing for directed three-dimensional differentiation of stem cells are an alternative to canonical two-dimensional cell cultures. In this paper, we evaluate the suitability of alginate for three-dimensional cultures to enhance differentiation of mouse embryonic stem cells (mESCs) towards neural lineages. We tested whether encapsulation of mESCs within alginate beads could support and/or enhance neural differentiation with respect to two-dimensional cultures. We encapsulated cells in beads of alginate with or without modification by fibronectin (Fn) or hyaluronic acid (HA). Gene expression analysis showed that cells grown in alginate and alginate-HA present increased differentiation toward neural lineages with respect to the two-dimensional control and to Fn group. Immunocytochemistry analyses confirmed these results, further showing terminal differentiation of neurons as seen by the expression of synaptic markers and markers of different neuronal subtypes. Our data show that alginate, alone or modified, is a suitable biomaterial to promote in vitro differentiation of pluripotent cells toward neural fates.