NeuroCult™ SM1 Neuronal Supplement

Serum-free supplement for the culture and differentiation of neural cells

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NeuroCult™ SM1 Neuronal Supplement

Serum-free supplement for the culture and differentiation of neural cells

10 mL
Catalog #05711
102 CAD

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Overview

NeuroCult™ SM1 Neuronal Supplement is a standardized serum-free supplement for the culture of mouse and rat primary neurons and the differentiation and maturation of human ES/iPS cell-derived neurons. NeuroCult™ SM1 can also be used as a versatile serum-replacement supplement for various applications. The formulation of NeuroCult™ SM1 Neuronal Supplement is based on the published B27 formulation (Brewer et al.), but optimized to more reproducibly support the survival of mature neurons in long-term culture. This supplement may be used in combination with BrainPhys™ Neuronal Medium or NeuroCult™ Neuronal Basal Medium.

Also available: NeuroCult™ SM1 Without Vitamin A.
Advantages:
• Formulated to support improved cell survival in long-term primary neuronal culture
• Cultures feature increased neurite outgrowth and branching in short- and long-term cultures
• Product undergoes rigorous performance testing
Contains:
• Antioxidants
• Vitamin A
• Insulin
• Other ingredients
Subtype:
Supplements
Cell Type:
Neural Cells, PSC-Derived; Neurons; Pluripotent Stem Cells
Species:
Human; Mouse; Rat
Application:
Cell Culture; Differentiation; Maintenance
Brand:
NeuroCult
Area of Interest:
Neuroscience; Stem Cell Biology
Formulation:
Defined; Serum-Free

Technical Resources

Educational Materials

(13)
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Data and Publications

Data

Morphology of Neurons in Representative NeuroCult™ SM1 Cultures at 7 and 21 Days in Vitro

Figure 1. Morphology of Neurons in Representative NeuroCult™ SM1 Cultures at 7 and 21 Days in Vitro

Primary rat E18 cortical neurons were cultured for 7 (A) and 21 (B) DIV in NeuroCult™ SM1-supplemented NeuroCult™ Neuronal Basal Medium. (A) Phase contrast imaging at 7 DIV shows large numbers of viable neurons, with minimal cell clumping and extensive neurite outgrowth and branching. (B) After 21 DIV, large numbers of viable neurons with developed dendritic arbors remain in culture. Magnification 20x.

Number of Neurons in NeuroCult™ SM1 and TSFM Cultures After 7 and 21 Days in Vitro

Figure 2. Number of Neurons in NeuroCult™ SM1 and TSFM Cultures After 7 and 21 Days in Vitro

(A) Comparable numbers of neurons are obtained when cells are cultured for 7 days in NeuroCult™ SM1-supplemented NeuroCult™ Neuronal Basal Medium compared to a traditional serum-free medium (TSFM; n = 25; mean ± 95% CI; p > 0.05). (B) Significantly higher numbers of neurons are obtained when cells are cultured for 21 days in a NeuroCult™ SM1-supplemented medium compared to TSFM (n = 25; mean ± 95% CI; ***p < 0.001).

Neurite Outgrowth of Primary Neurons Cultured in NeuroCult™ SM1 and TSFM for 7 and 21 Days

Figure 3. Neurite Outgrowth of Primary Neurons Cultured in NeuroCult™ SM1 and TSFM for 7 and 21 Days

Significantly longer neurite outgrowth was observed for cells cultured for 7 (A) and 21 (B) days in NeuroCult™ SM1-supplemented NeuroCult™ Neuronal Basal Medium compared to a traditional serum-free medium (TSFM; n = 240 independent measures, mean ± 95% CI, ***p < 0.001).

Neurite Branching of Primary Neurons Cultured in NeuroCult™ SM1 and TSFM for 7 and 21 Days

Figure 4. Neurite Branching of Primary Neurons Cultured in NeuroCult™ SM1 and TSFM for 7 and 21 Days

Significantly more neurite branch points were observed for cells cultured for 7 (A) and 21 (B) days in NeuroCult™ SM1-supplemented NeuroCult™ Neuronal Basal Medium compared to a traditional serum-free medium (TSFM; n = 240 independent measures, mean ± 95% CI, ***p < 0.001).

Expression of Pre- and Post-Synaptic Markers in Neurons Cultured for 21 Days in NeuroCult™ SM1

Figure 5. Expression of Pre- and Post-Synaptic Markers in Neurons Cultured for 21 Days in NeuroCult™ SM1

Neurons cultured in NeuroCult™ SM1-supplemented NeuroCult™ Neuronal Basal Medium for 21 days are phenotypically mature as indicated by the presence of an extensive dendritic arbor and appropriate expression and localization of pre- (Synapsin) and post-synaptic (PSD-95) markers. (A-C) Synapsin (green) staining is concentrated in discrete puncta distributed along the somata and dendritic processes, as defined by MAP2 (red) staining. (D-F) Dendritic staining observed for MAP2 and punctate staining for the postsynaptic marker PSD-95. Nuclei were counter-stained with DAPI. Scale bar 10 µm.

Publications

(5)
ASSAY and Drug Development Technologies 2015 September

Phenotypic assays to identify agents that induce reactive gliosis: a counter-screen to prioritize compounds for preclinical animal studies.

Beckerman SR et al.

Abstract

Astrocyte phenotypes change in a process called reactive gliosis after traumatic central nervous system (CNS) injury. Astrogliosis is characterized by expansion of the glial fibrillary acidic protein (GFAP) cytoskeleton, adoption of stellate morphologies, and differential expression of some extracellular matrix molecules. The astrocytic response immediately after injury is beneficial, but in the chronic injury phase, reactive astrocytes produce inhibitory factors (i.e., chondroitin sulfate proteoglycans [CSPGs]) that limit the regrowth of injured axons. There are no drugs that promote axon regeneration or functional recovery after CNS trauma in humans. To develop novel therapeutics for the injured CNS, we screened various libraries in a phenotypic assay to identify compounds that promote neurite outgrowth. However, the effects these compounds have on astrocytes are unknown. Specifically, we were interested in whether compounds could alter astrocytes in a manner that mimics the glial reaction to injury. To test this hypothesis, we developed cell-based phenotypic bioassays to measure changes in (1) GFAP morphology/localization and (2) CSPG expression/immunoreactivity from primary astrocyte cultures. These assays were optimized for six-point dose-response experiments in 96-well plates. The GFAP morphology assay is suitable for counter-screening with a Z-factor of 0.44±0.03 (mean±standard error of the mean; N=3 biological replicates). The CSPG assay is reproducible and informative, but does not satisfy common metrics for a "screenable" assay. As proof of principle, we tested a small set of hit compounds from our neurite outgrowth bioassay and identified one that can enhance axon growth without exacerbating the deleterious characteristics of reactive gliosis.
PloS one 2012 January

The ADNP derived peptide, NAP modulates the tubulin pool: implication for neurotrophic and neuroprotective activities.

Oz S et al.

Abstract

Microtubules (MTs), key cytoskeletal elements in living cells, are critical for axonal transport, synaptic transmission, and maintenance of neuronal morphology. NAP (NAPVSIPQ) is a neuroprotective peptide derived from the essential activity-dependent neuroprotective protein (ADNP). In Alzheimer's disease models, NAP protects against tauopathy and cognitive decline. Here, we show that NAP treatment significantly affected the alpha tubulin tyrosination cycle in the neuronal differentiation model, rat pheochromocytoma (PC12) and in rat cortical astrocytes. The effect on tubulin tyrosination/detyrosination was coupled to increased MT network area (measured in PC12 cells), which is directly related to neurite outgrowth. Tubulin beta3, a marker for neurite outgrowth/neuronal differentiation significantly increased after NAP treatment. In rat cortical neurons, NAP doubled the area of dynamic MT invasion (Tyr-tubulin) into the neuronal growth cone periphery. NAP was previously shown to protect against zinc-induced MT/neurite destruction and neuronal death, here, in PC12 cells, NAP treatment reversed zinc-decreased tau-tubulin-MT interaction and protected against death. NAP effects on the MT pool, coupled with increased tau engagement on compromised MTs imply an important role in neuronal plasticity, protecting against free tau accumulation leading to tauopathy. With tauopathy representing a major pathological hallmark in Alzheimer's disease and related disorders, the current findings provide a mechanistic basis for further development. NAP (davunetide) is in phase 2/3 clinical trial in progressive supranuclear palsy, a disease presenting MT deficiency and tau pathology.
The Journal of neuroscience : the official journal of the Society for Neuroscience 2011 July

Inhibitory synapse dynamics: coordinated presynaptic and postsynaptic mobility and the major contribution of recycled vesicles to new synapse formation.

Dobie F et al.

Abstract

Dynamics of GABAergic synaptic components have been studied previously over milliseconds to minutes, revealing mobility of postsynaptic scaffolds and receptors. Here we image inhibitory synapses containing fluorescently tagged postsynaptic scaffold Gephyrin, together with presynaptic vesicular GABA transporter (VGAT) or postsynaptic GABA(A) receptor γ2 subunit (GABA(A)Rγ2), over seconds to days in cultured rat hippocampal neurons, revealing modes of inhibitory synapse formation and remodeling. Entire synapses were mobile, translocating rapidly within a confined region and exhibiting greater nonstochastic motion over multihour periods. Presynaptic and postsynaptic components moved in unison, maintaining close apposition while translocating distances of several micrometers. An observed flux in the density of synaptic puncta partially resulted from the apparent merging and splitting of preexisting clusters. De novo formation of inhibitory synapses was observed, marked by the appearance of stably apposed Gephyrin and VGAT clusters at sites previously lacking either component. Coclustering of GABA(A)Rγ2 supports the identification of such new clusters as synapses. Nascent synapse formation occurred by gradual accumulation of components over several hours, with VGAT clustering preceding that of Gephyrin and GABA(A)Rγ2. Comparing VGAT labeling by active uptake of a luminal domain antibody with post hoc immunocytochemistry indicated that recycling vesicles from preexisting boutons significantly contribute to vesicle pools at the majority of new inhibitory synapses. Although new synapses formed primarily on dendrite shafts, some also formed on dendritic protrusions, without apparent interconversion. Altogether, the long-term imaging of GABAergic presynaptic and postsynaptic components reveals complex dynamics and perpetual remodeling with implications for mechanisms of assembly and synaptic integration.
PloS one 2011 January

NMDA receptors mediate synaptic competition in culture.

She K et al.

Abstract

BACKGROUND: Activity through NMDA type glutamate receptors sculpts connectivity in the developing nervous system. This topic is typically studied in the visual system in vivo, where activity of inputs can be differentially regulated, but in which individual synapses are difficult to visualize and mechanisms governing synaptic competition can be difficult to ascertain. Here, we develop a model of NMDA-receptor dependent synaptic competition in dissociated cultured hippocampal neurons. METHODOLOGY/PRINCIPAL FINDINGS: GluN1 -/- (KO) mouse hippocampal neurons lacking the essential NMDA receptor subunit were cultured alone or cultured in defined ratios with wild type (WT) neurons. The absence of functional NMDA receptors did not alter neuron survival. Synapse development was assessed by immunofluorescence for postsynaptic PSD-95 family scaffold and apposed presynaptic vesicular glutamate transporter VGlut1. Synapse density was specifically enhanced onto minority wild type neurons co-cultured with a majority of GluN1 -/- neighbour neurons, both relative to the GluN1 -/- neighbours and relative to sister pure wild type cultures. This form of synaptic competition was dependent on NMDA receptor activity and not conferred by the mere physical presence of GluN1. In contrast to these results in 10% WT and 90% KO co-cultures, synapse density did not differ by genotype in 50% WT and 50% KO co-cultures or in 90% WT and 10% KO co-cultures. CONCLUSIONS/SIGNIFICANCE: The enhanced synaptic density onto NMDA receptor-competent neurons in minority coculture with GluN1 -/- neurons represents a cell culture paradigm for studying synaptic competition. Mechanisms involved may include a retrograde 'reward' signal generated by WT neurons, although in this paradigm there was no 'punishment' signal against GluN1 -/- neurons. Cell culture assays involving such defined circuits may help uncover the rules and mechanisms of activity-dependent synaptic competition in the developing nervous system.
Journal of neuroscience research 1993 August

Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination.

Brewer G et al.

Abstract

We have systematically optimized the concentrations of 20 components of a previously published serum-free medium (Brewer and Cotman, Brain Res 494: 65-74, 1989) for survival of rat embryonic hippocampal neurons after 4 days in culture. This serum-free medium supplement, B27, produced neuron survival above 60%, independent of plating density above 160 plated cells/mm2. For isolated cells (< 100 cells/mm2), survival at 4 days was still above 45%, but could be rescued to the 60% level at 40 cells/mm2 by simply applying a coverslip on top of the cells. This suggests a need for additional trophic factors. High survival was achieved with osmolarity lower than found in Dulbecco's Modified Eagle's Medium (DMEM), and by reducing cysteine and glutamine concentrations and by the elimination of toxic ferrous sulphate found in DME/F12. Neurobasal is a new medium that incorporates these modifications to DMEM. In B27/Neurobasal, glial growth is reduced to less than 0.5% of the nearly pure neuronal population, as judged by immunocytochemistry for glial fibrillary acidic protein and neuron-specific enolase. Excellent long-term viability is achieved after 4 weeks in culture with greater than 90% viability for cells plated at 640/mm2 and greater than 50% viability for cells plated at 160/mm2. Since the medium also supports the growth of neurons from embryonic rat striatum, substantia nigra, septum, and cortex, and neonatal dentate gyrus and cerebellum (Brewer, in preparation), support for other neuron types is likely. B27/Neurobasal should be useful for in vitro studies of neuronal toxicology, pharmacology, electrophysiology, gene expression, development, and effects of growth factors and hormones.
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