Neural Cell Cultures Research Articles

Film interface for drug testing for delivery to cells in culture and in the brain

Brain access remains a major challenge in drug testing. The nearly ‘impermeable’ blood-brain-barrier (BBB) prevents most drugs from gaining access to brain cells via systematic intravenous (IV) injection. In this study, silk fibroin films were used as drug carrier as well as cell culture substrate to simulate the in vivo interface between drug reservoir and brain cells for testing drug delivery in the brain. In in vitro studies, film-released arabinofuranosyl cytidine (AraC), a mitotic inhibitor, selectively killed glial cells in film-supported mixed neural cell cultures; with widened dosage windows for drug efficacy and tolerance compared to drugs in solution. In the brain, the presence of silk films was well tolerated with no signs of acute neuroinflammation, cell death, or altered brain function. Topical application of silk films on the cortical surface delivered Evans blue, a BBB-impenetrable fluorescent marker, through the intact dura matter into the parenchyma of the ipsilateral hemisphere as deep as the hippocampal region, but not the contralateral hemisphere. In a mouse traumatic brain injury (TBI) model, necrosis markers by film delivery accessed more cells in the lesion core than by con-current IV delivery; whereas the total coverage including the peri-lesional area appeared to be comparable between the two routes. The complementary distribution patterns of co-delivered markers provided direct evidence of the partial confinement of either route’s access to brain cells by a restrictive zone near the lesion border. Finally, film-delivered necrostatin-1 reduced overall cell necrosis by approximately 40% in the TBI model. These findings from representative small molecules of delivery route-dependent drug access are broadly applicable for evaluating drug actions both in vitro and in vivo. Combined with its demonstrated role of supporting neuron-electrode interfaces, the film system can be further developed for testing a range of neuromodulation approaches (i.e., drug delivery, electrical stimulation, cell graft) in the brain. Statement of SignificanceThis study demonstrated that silk fibroin films can be used to evaluate drug actions both in vitro and in vivo, partially overcoming the significant delivery barriers of the brain. This system can be adapted for efficient drug access to specific brain regions and/or cell types. The film system can be further developed for testing a range of interventions with drugs, electrical signals or cell graft for analysis of treatment outcomes including cell responses and brain function.

CNS-derived extracellular vesicles from superoxide dismutase 1 (SOD1)G93A ALS mice originate from astrocytes and neurons and carry misfolded SOD1

Extracellular vesicles (EVs) are secreted by myriad cells in culture and also by unicellular organisms, and their identification in mammalian fluids suggests that EV release also occurs at the organism level. However, although it is clearly important to better understand EVs' roles in organismal biology, EVs in solid tissues have received little attention. Here, we modified a protocol for EV isolation from primary neural cell culture to collect EVs from frozen whole murine and human neural tissues by serial centrifugation and purification on a sucrose gradient. Quantitative proteomics comparing brain-derived EVs from nontransgenic (NTg) and a transgenic amyotrophic lateral sclerosis (ALS) mouse model, superoxide dismutase 1 (SOD1)G93A, revealed that these EVs contain canonical exosomal markers and are enriched in synaptic and RNA-binding proteins. The compiled brain EV proteome contained numerous proteins implicated in ALS, and EVs from SOD1G93A mice were significantly depleted in myelin-oligodendrocyte glycoprotein compared with those from NTg animals. We observed that brain- and spinal cord-derived EVs, from NTg and SOD1G93A mice, are positive for the astrocyte marker GLAST and the synaptic marker SNAP25, whereas CD11b, a microglial marker, was largely absent. EVs from brains and spinal cords of the SOD1G93A ALS mouse model, as well as from human SOD1 familial ALS patient spinal cord, contained abundant misfolded and nonnative disulfide-cross-linked aggregated SOD1. Our results indicate that CNS-derived EVs from an ALS animal model contain pathogenic disease-causing proteins and suggest that brain astrocytes and neurons, but not microglia, are the main EV source.

Collagen Based Multicomponent Interpenetrating Networks as Promising Scaffolds for 3D Culture of Human Neural Stem Cells, Human Astrocytes, and Human Microglia.

This work describes for the first time the fabrication and characterization of multicomponent interpenetrating networks composed of collagenI, hyaluronic acid, and poly(ethylene glycol) diacrylate for the 3D culture of human neural stem cells, astrocytes, and microglia. The chemical composition of the scaffolds can be modulated while maintaining values of complex moduli within the range of the mechanical performance of brain tissue (∼6.9 kPa) and having cell viability exceeding 84%. The developed scaffolds are a promising new family of biomaterials that can potentially serve as 3Din vitro models for studying the physiology and physiopathology of the central nervous system.

Label-free classification of neurons and glia in neural stem cell cultures using a hyperspectral imaging microscopy combined with machine learning

Due to a growing demand for a viable label-free observation method in the biomedical field, many techniques, such as quantitative phase imaging and Raman spectroscopy, have been studied, and a complementary approach, hyperspectral imaging, has also been introduced. We developed a high-speed hyperspectral imaging microscopy imaging method with commercially available apparatus, employing a liquid crystal tunable bandpass filter combined with a pixel-wise machine learning classification. Next, we evaluated the feasibility of the application of this method for stem cell research utilizing neural stem cells. Employing this microscopy method, with a 562 × 562 μm2 field of view, 2048 × 2048 pixel resolution images containing 63 wavelength pixel-wise spectra could be obtained in 30 seconds. The neural stem cells were differentiated into neurons and astroglia (glia), and a four-class cell classification evaluation (including neuronal cell body, glial cell body, process and extracellular region) was conducted under co-cultured conditions. As a result, an average of 88% of the objects of interest were correctly classified, with an average precision of 94%, and more than 99% of the extracellular pixels were correctly segregated. These results indicated that the proposed hyperspectral imaging microscopy is feasible as a label-free observation method for stem cell research.

Blockade of microglial adenosine A2A receptor suppresses elevated pressure-induced inflammation, oxidative stress, and cell death in retinal cells.

Glaucoma is a retinal degenerative disease characterized by the loss of retinal ganglion cells and damage of the optic nerve. Recently, we demonstrated that antagonists of adenosine A2A receptor (A2AR) control retinal inflammation and afford protection to rat retinal cells in glaucoma models. However, the precise contribution of microglia to retinal injury was not addressed, as well as the effect of A2AR blockade directly in microglia. Here we show that blocking microglial A2AR prevents microglial cell response to elevated pressure and it is sufficient to protect retinal cells from elevated pressure‐induced death. The A2AR antagonist SCH 58261 or the knockdown of A2AR expression with siRNA in microglial cells prevented the increase in microglia response to elevated hydrostatic pressure. Furthermore, in retinal neural cell cultures, the A2AR antagonist decreased microglia proliferation, as well as the expression and release of pro‐inflammatory mediators. Microglia ablation prevented neural cell death triggered by elevated pressure. The A2AR blockade recapitulated the effects of microglia depletion, suggesting that blocking A2AR in microglia is able to control neurodegeneration in glaucoma‐like conditions. Importantly, in human organotypic retinal cultures, A2AR blockade prevented the increase in reactive oxygen species and the morphological alterations in microglia triggered by elevated pressure. These findings place microglia as the main contributors for retinal cell death during elevated pressure and identify microglial A2AR as a therapeutic target to control retinal neuroinflammation and prevent neural apoptosis elicited by elevated pressure.

Zika virus induces abnormal cranial osteogenesis by negatively affecting cranial neural crest development

Zika virus (ZIKV) infection during gestation is deemed to be coupled to birth defects through direct impairment of the nervous system during neurogenesis. However, in this study, our data showed that ZIKV infection dramatically suppressed cranial osteogenesis, shown by Safranin O/Fast Green and alizarin red staining, in chick embryos, which provides another possibility that craniofacial bone malformation caused by ZIKV may be a major cause of ZIKV-mediated birth defects. By immunofluorescent staining and electron microcopy, we confirmed ZIKV infection in chick embryo neural tubes and sites of neural crest. Next, in vivo (chick embryos) and in vitro [primary culture of neural crest cells (NCC)] ZIKV and HNK-1 double immunofluorescent staining demonstrated that ZIKV infection inhibited the production of migratory NCC. The reduction of both AP-2α- and Pax7-positive NCC in HH10 chick embryos infected by ZIKV confirmed that abnormal development of cranial NCC also occurred in the migratory process. Whole mount in situ hybridization demonstrated that cadherin 6B expression was elevated and Slug, FoxD3, and BMP4/Msx1 expressions decreased in ZIKV-infected HH10 chick embryos, implying that epithelial–mesenchymal transition (EMT) of neural crest production was blocked by ZIKV infection. Moreover, in vivo and in vitro pHIS3 and Pax7 double immunofluorescent staining showed that NCC proliferation was repressed by ZIKV infection. C-caspase-3 and AP-2α double immunofluorescent staining in HH10 chick embryos and western blotting showed that NCC apoptosis increased following ZIKV infection. Finally, electron microscopy showed multiple autophagosomes in ZIKV-infected embryos, and western blot and LC3B immunofluorescent staining demonstrated that autophagy-related genes were activated by ZIKV infection. Taken together, our data first showed that ZIKV infection during embryogenesis could interfere with cranial neural crest development, which in turn causes aberrant cranial osteogenesis. Our results provided new insights into brain malformations induced by ZIKV infection.

Ex Vivo Culture and Manipulation of Mouse Neural Crest Cells from Primary Embryonic Tissue Explants.

In vitro culture of neural crest cells allows for the manipulation and study of neural crest cell function in a cell-autonomous manner. While several stable neural crest cell lines exist, the transformed nature of these cells may not closely reflect the in vivo properties of neural crest cells, hence making molecular and functional analyses using these cell lines difficult to interpret. Here we describe a robust method to culture primary mouse neural crest cells ex vivo for several days to weeks in culture. We further describe a method for siRNA knockdown in these cells to study gene function. This culture method can also be adapted for other molecular analyses, including addition of small-molecule inhibitors and/or growth factors to the culture media, as well as culturing neural crest cells from knockout or genetically modified mice.

Engineering hydrogels with affinity-bound laminin as 3D neural stem cell culture systems.

Laminin incorporation into biological or synthetic hydrogels has been explored to recapitulate the dynamic nature and biological complexity of neural stem cell (NSC) niches. However, the strategies currently explored for laminin immobilization within three-dimensional (3D) matrices do not address a critical aspect influencing cell-matrix interactions, which is the control over laminin conformation and orientation upon immobilization. This is a key feature for the preservation of the protein bioactivity. In this work, we explored an affinity-based approach to mediate the site-selective immobilization of laminin into a degradable synthetic hydrogel. Specifically, a four-arm maleimide terminated poly(ethylene glycol) (PEG-4MAL) macromer was functionalized with a mono-PEGylated recombinant human N-terminal agrin (NtA) domain, to promote high affinity binding of laminin. Different NtA concentrations (10, 50 and 100 μM) were used to investigate the impact of NtA density on laminin incorporation, hydrogel biophysical properties, and biological outcome. Laminin was efficiently incorporated for all the conditions tested (laminin incorporation >95%), and the developed hydrogels revealed mechanical properties (average storage modulus (G') ranging from 187 to 256 Pa) within the values preferred for NSC proliferation and neurite branching and extension. Affinity-bound laminin PEG-4MAL hydrogels better preserve laminin bioactivity, compared to unmodified hydrogels and hydrogels containing physically entrapped laminin, this effect being dependent on NtA concentration. This was evidenced by the 10 μM NtA-functionalized PEG-4MAL gels incorporating laminin that support enhanced human NSC proliferation and neurite extension, compared to the latter. Overall, this work highlights the potential of the proposed engineered matrices to be used as defined 3D platforms for the establishment of artificial NSC niches and as extracellular matrix-mimetic microenvironments to support human NSC transplantation.

Three-Dimensional Differentiation of Human Pluripotent Stem Cell-Derived Neural Precursor Cells Using Tailored Porous Polymer Scaffolds

This study investigates the utility of a novel poly(ethylene glycol) diacrylate-crosslinked porous polymeric tissue engineering scaffold, with mechanical properties specifically optimised to be comparable to that of mammalian brain tissue for 3D human neural cell culture. Results obtained here demonstrate the attachment, proliferation and terminal differentiation of both human induced pluripotent stem cell- and embryonic stem cell-derived neural precursor cells (hPSC-NPCs) throughout the interconnected porous network within laminin-coated scaffolds. Phenotypic data and functional analyses are presented demonstrating that this material supports terminal in vitro neural differentiation of hPSC-NPCs to a mixed population of viable neuronal and glial cells for periods of up to 49 days. This is evidenced by the upregulation of TUBB3, MAP2, SYP and GFAP gene expression, as well as the presence of the proteins βIII-TUBULIN, NEUN, MAP2 and GFAP. Functional maturity of neural cells following 49 days 3D differentiation culture was tested via measurement of intracellular calcium. These analyses revealed spontaneously active, synchronous and rhythmic calcium flux, as well as response to the neurotransmitter glutamate. This novel construct has potential application as an improved in vitro human neurogenesis model with utility in platform drug discovery programs.

Peptide Nanosponges Designed for the Delivery of Perillyl Alcohol to Glioma Cells.

Peptide nanosponges of low polydispersity are spontaneously formed from trigonal supramolecular building blocks in aqueous buffers, which feature cationic and/or anionic oligopeptides (n = 5-20) and a hydrophobic unit. In contrast to classical liposomes/vesicles, nanosponges feature interwoven hydrophilic and hydrophobic nanodomains and are readily taken up by mammalian cells. Perillyl alcohol is known to be a simple, but effective small molecule drug against glioma multiforme. However, its efficacy is limited by a poor bioavailability. In order to make perillyl alcohol bioavailable, two nanosponges consisting of 10 aspartates, to which perillyl alcohol is attached by means of an ester bond, and 20 lysines or arginines (type (D-POH)10K20 and (D-POH)10R20) were synthesized, purified, and characterized by dynamic light scattering (DLS) and atomic force microscopy (AFM). These nanosponges were then tested in cell cultures of murine glioma cells (GL26) and murine neural progenitor cells (NPC) because the latter was previously utilized in cell-based cancer therapy. The two nanosponges exhibited significantly different biophysical properties (size distribution and ζ potentials). Consequently, different efficacies in killing GL26 and NPC were observed in serum-containing culture media. The results from these experiments confirmed that the type (D-POH)10K20 nanosponge is a promising candidate for the (cell-mediated) cytotherapy of glioblastoma.

Comet assay in neural cells as a tool to monitor DNA damage induced by chemical or physical factors relevant to environmental and occupational exposure

Biomonitoring of the effects of environmental and occupational exposure relevant chemical or physical factors on central nervous system is difficult due to the problems with sampling of biological material. Thus, surrogate systems allowing for the estimation of effect intensity are necessary to evaluate a potential risk of exposure. Cancerous neural cells in culture seem to be a reliable trustworthy alternative to ex vivo primary cells culture, where brain tissue is hardly available. In this review we summarized attempts to test genotoxicity of environmentally related xenobiotics or physical factors. Different neural cells of human and non-human origin are described in respect to their use in genotoxicity testing using the comet assay. Surprisingly, despite the large number of commercially available neural cells of different type and origin, only twelve were used for genotoxicity testing by the comet assay. We also recapitulate the environmentally relevant chemical and physical factors tested on neural cell lines in vitro by the comet assay. The most prevalent were fire retardants, plant protection agents, nanoparticles and magnetic field.

Isolation and Culture of Embryonic Mouse Neural Stem Cells.

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Graphene-based papers as substrates for cell growth: Characterisation and impact on mammalian cells

Graphene based material (GBM) substrates are promising candidates for tissue engineering and as cell scaffolds owing to their outstanding properties that favour cell adhesion and growth. In this study, we report the fabrication and use of different oxidised graphene-based ‘papers’ as substrates for cell culture, using starting materials of different thickness and lateral dimensions. Graphite oxide (GTO) consisted of thicker sheets than the thinner large graphene oxide (l-GO) and small graphene oxide (s-GO) sheets. The ‘papers’ were prepared by vacuum filtration of the material suspensions and characterised by an array of experimental techniques. The substrates were then evaluated for cellular adhesion and proliferation with two epithelial cell lines that differ in their morphologies, the human lung cell culture (A549) model and the human neural cell culture (SH-SY5Y) model using microscopic analysis. Release of lactate dehydrogenase (LDH) in cell supernatants was measured to assess any cytotoxic responses. Our results revealed that the GO papers had similar physicochemical properties but differing topographies. The papers supported cell growth and no cytotoxicity was observed. These results suggest that substrates based on graphite and graphene oxide papers are suitable biocompatible cellular supports for anchorage-dependent cell growth and can be further explored for tissue engineering, regenerative medicine, substrates for cell growth and bionic applications.

Influence of passage number on the impact of the secretome of adipose tissue stem cells on neural survival, neurodifferentiation and axonal growth

Mesenchymal stem cells (MSCs), and within them adipose tissue derived stem cells (ASCs), have been shown to have therapeutic effects on central nervous system (CNS) cell populations. Such effects have been mostly attributed to soluble factors, as well as vesicles, present in their secretome. Yet, little is known about the impact that MSC passaging might have in the secretion therapeutic profile. Our aim was to show how human ASCs (hASCs) passage number influences the effect of their secretome in neuronal survival, differentiation and axonal growth. For this purpose, post-natal rat hippocampal primary cultures, human neural progenitor cell (hNPCs) cultures and dorsal root ganglia (DRGs) explants were incubated with secretome, collected as conditioned media (CM), obtained from hASCs in P3, P6, P9 and P12. Results showed no differences when comparing percentages of MAP-2 positive cells (a mature neuronal marker) in neuronal cultures or hNPCs, after incubation with hASCs secretome from different passages. The same was observed regarding DRG neurite outgrowth. In order to characterize the secretomes obtained from different passages, a proteomic analysis was performed, revealing that its composition did not vary significantly with passage number P3 to P12. Results allowed us to identify several key proteins, such as pigment epithelium derived factor (PEDF), DJ-1, interleucin-6 (IL-6) and galectin, all of which have already proven to play neuroprotective and neurodifferentiating roles. Proteins that promote neurite outgrowth were also found present, such as semaphorin 7A and glypican-1. We conclude that cellular passaging does not influence significantly hASCs's secretome properties especially their ability to support post-natal neuronal survival, induce neurodifferentiation and promote axonal growth.

Development of a protocol to assess cell internalization and tissue uptake of magnetic nanoparticles by AC Biosusceptometry

Abstract Several applications of nanoparticles rely on the internalization and accumulation of nanocarriers in specific cell compartments and tissues. However, the methods currently employed for characterizing such processes, although well described, are time consuming and do not provide in vivo information, which is a crucial barrier towards translational applications. Here, we hypothesize that the AC Biosusceptometry technique can be employed to assess cell internalization of magnetic nanoparticles, with possible applications in screening assays to track specific biomarkers and cell types. We tested a simpler and easier alternative to study cell internalization and tissue accumulation after perfusion. We utilized citrate coated, manganese ferrite nanoparticles and evaluated the internalization process in mouse macrophages cells (J774.A1) and in an embryonic neural stem cell culture (E14.5) after differentiation in astrocytes and neurons, to assess internalization specificity. Respecting the particles toxicity limits, we tested different concentration of particles, in different incubation times. Sequentially, we imaged the cell cultures to confirm internalization and nanoparticles localization, labeling nucleus and cell body to assure that the particles were inside the cells. Our results showed a linear behavior on internalization for different doses and an optimum incubation time of 2 h.

Multiple effects of the herbicide glufosinate-ammonium and its main metabolite on neural stem cells from the subventricular zone of newborn mice

The globally used herbicide glufosinate-ammonium (GLA) is structurally analogous to the excitatory neurotransmitter glutamate, and is known to interfere with cellular mechanisms involved in the glutamatergic system. In this report, we used an in vitro model of murine primary neural stem cell culture to investigate the neurotoxicity of GLA and its main metabolite, 4-methylphosphinico-2-oxobutanoic acid (PPO). We demonstrated that GLA and PPO disturb ependymal wall integrity in the ventricular-subventricular zone (V-SVZ) and alter the neuro-glial differentiation of neural stem cells. GLA and PPO impaired the formation of cilia, with reduced Celsr2 expression after PPO exposure. GLA promoted the differentiation of neuronal and oligodendroglial cells while PPO increased B1 cell population and impaired neuronal fate of neural stem cells. These results confirm our previous in vivo report that developmental exposure to GLA alters neurogenesis in the SVZ, and neuroblast migration along the rostral migratory stream. They also highlight the importance of investigating the toxicity of pesticide degradation products. Indeed, not only GLA, but also its metabolite PPO disrupts V-SVZ homeostasis and provides a novel cellular mechanism underlying GLA-induced neurodevelopmental toxicity. Furthermore, we were able to demonstrate a neurotoxic activity of a metabolite of GLA different from that of GLA active substance for the very first time.

Culturing and Manipulation of O9-1 Neural Crest Cells

Culturing and Manipulation of O9-1 Neural Crest Cells

Culturing and Manipulation of O9-1 Neural Crest Cells.

Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and organs. The O9-1 cell line is derived from the endogenous mouse embryonic NCCs and maintains its multipotency. However, under specific culture conditions, O9-1 cells can differentiate into different cell types and be utilized in a wide range of research applications. Recently, with the combination of mouse studies and O9-1 cell studies, we have shown that the Hippo signaling pathway effectors Yap and Taz play important roles in neural crest-derived craniofacial development. Although the culturing process for O9-1 cells is more complicated than that used for other cell lines, the O9-1 cell line is a powerful model for investigating NCCs in vitro. Here, we present a protocol for culturing the O9-1 cell line to maintain its stemness, as well as protocols for differentiating O9-1 cells into different cell types, such as smooth muscle cells and osteoblasts. In addition, protocols are described for performing gene loss-of-function studies in O9-1 cells by using CRISPR-Cas9 deletion and small interfering RNA-mediated knockdown.

Around 90° Contact Angle of Dish Surface Is a Key Factor in Achieving Spontaneous Spheroid Formation.

Following the discovery of the primary culture of neural stem cells, the spheroid culture has been recognized as one of the selective culture methods for somatic stem cells. Since then, various methods were reported to generate spheroids, which can enrich the potent stem cell population. However, the fundamental factors affecting spheroid formation remain unclear. In this study, we focused on the surface property of the culture dishes, in particular, hydrophobicity. Primary mouse skin culture cells were prepared with conventional two-dimensional culture, and then, the cells were transferred to culture dishes with varying hydrophobicity, which was confirmed with the water contact angles. Of these, a culture dish possessing an almost 90° water contact angle was the only one that successfully exhibited spheroid formation. The spheroid formation was spontaneous, efficient, and stable. Since this outcome was achieved with a conventional culture medium with serum, but without any additives such as epidermal growth factor, basic fibroblast growth factor, and B27, the spheroid formation from this process was not affected by serum and was also not dependent on additives. The results from immunofluorescence and quantitative real-time polymerase chain reaction testing showed the expression of embryonic stem cell markers such as SSEA-1, SOX2, OCT4, and Nanog, which confirmed that the spheroids with this method are comparable to those from other methods. This outcome was reproducible and could be applied not only to skin-derived cells but also to oral mucosa-derived cells, cortical bone-derived cells, and 3T3 cells, also suggesting the generality and robustness of this phenomenon.