Neural basal medium

Manufactured by Thermo Fisher Scientific

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Neural basal medium is a cell culture medium specifically formulated to support the growth and maintenance of neural cells. It provides the essential nutrients and growth factors required for the optimal survival and proliferation of neurons, astrocytes, and other neural cell types. The core function of neural basal medium is to create an environment that closely mimics the in vivo conditions of the nervous system, enabling the cultivation of neural cells for various research and experimental applications.

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24 protocols using neural basal medium

Hippocampal Neuron Isolation and Transduction

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The neonatal mice within 24 h were sterilized with 75% medical alcohol, after which the brain of the mice was cut out and the hippocampal tissue was quickly separated, and cut into small pieces. The hippocampal tissue pieces were digested with 0.125% trypsin for 15 min at 37°C, ground and centrifuged. Cells were seeded in 10-mm dishes coated with polylysine (10 mmol/L) at a density of 1×10⁶ cells/mL and maintained in Neural Basal Medium (Gibco, Carlsbad, CA, USA) with 2% B27 (Gibco) and 0.25% Glumax (Gibco). After 3 days, 2.5 μg/mL cytarabine (Sigma-Aldrich) was added to the medium for 24 h to suppress the proliferation of glial cells. Next, 50% of the medium was renewed every three days. Cells were cultured at 37°C with 5% CO₂ for 14 days before experimentations [23 (link)].
Hippocampal neurons were then transduced with lentivirus (Genechem; multiplicity of infection=5) carrying sh-NC, sh-MALAT1-1, sh-MALAT1-2, sh-MALAT1-3, oe-NC, oe-MALAT1, sh-METTL3-1, sh-METTL3-2, sh-METTL3-3, and oe-METTL3. After 48 h, cells were harvested for subsequent experimentations.

Ming Y., Deng Z., Tian X., Jia Y., Ning M, & Cheng S. (2022). m6A Methyltransferase METTL3 Reduces Hippocampal Neuron Apoptosis in a Mouse Model of Autism Through the MALAT1/SFRP2/Wnt/β-catenin Axis. Psychiatry Investigation, 19(10), 771-787.

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Alzheimer's Disease Tau Pathology in Mouse Primary Hippocampal Neurons

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Primary hippocampal neurons were prepared from outbred CD1 E16-E18 mouse embryos. Neurons were digested from tissue with papain (Worthington Biochemical Corporation). The dissociated neurons were resuspended in neural basal medium (Gibco, 21,103) with 2% B27, 1 × Glutamax, and 1 × penicillin/streptomycin (Gibco). Cells were plated with a density 100,000 cells per coverslip in 24-well plates. Coverslips were coated with PDL (poly-D-lysine, 0.1 mg/ml, Sigma-Aldrich) and borate buffer (0.05 M boric acid, pH 8.5). On DIV0, 10% FBS was added to the medium, which was replaced with fresh medium 1 day in vitro (DIV). AD-tau was diluted into PBS and sonicated then added on top of cells. Neurons were treated with 1 µg/well of AD-tau at DIV 7 and 2 µg/well at DIV 14. Cells treated with AD-tau at DIV 7 received CK2 inhibitory post-treatment with the IC₅₀ dose of TBB (0.1 µM) at DIV 14. An equal dose of TBB was added 30 min prior to the addition of AD-tau on DIV 14 as pre-treatment. The IC₅₀ dose of Memantine (0.1 µM) was used as a post-treatment at DIV 14. Immunocytochemistry was conducted at DIV 21 as previously described [30 (link), 87 (link)].

Marshall C.A., McBride J.D., Changolkar L., Riddle D.M., Trojanowski J.Q, & Lee V.M. (2022). Inhibition of CK2 mitigates Alzheimer’s tau pathology by preventing NR2B synaptic mislocalization. Acta Neuropathologica Communications, 10, 30.

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Isolation and Culture of Rat Cortical Neurons

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Primary rat cortical neurons were obtained and cultured as described previously [13 (link)]. Pregnant SD rats (16–18 days) were used to prepare primary-neuron-enriched cultures. In brief, we first removed the meninges and blood vessels of the brain. The brain tissues were then digested with 0.25% trypsin for 5 min, and the digestion was terminated by washing the tissue three times with PBS. The brain tissue suspension was centrifuged at 500×g for 5 min, and the pellet was then resuspended in neural basal medium (all from GIBCO, Carlsbad, CA, USA). Finally, cells were seeded in 12-well plates and 6-well plates in fresh medium. Afterward, half of the medium was changed every 2 d.

Zhang M., Lu H., Xie X., Shen H., Li X., Zhang Y., Wu J., Ni J., Li H, & Chen G. (2020). TMEM175 mediates Lysosomal function and participates in neuronal injury induced by cerebral ischemia-reperfusion. Molecular Brain, 13, 113.

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Neural Induction of iPSCs

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iPSCs were differentiated into neural lineage by following a published protocol^{35 (link)}. In brief, iPSCs were dissociated into small clumps with Accutase and seeded on polyornithine (0.1 mg/ml, P4957, Sigma) and laminin (4 μg/ml) pre-coated 4-well-plates (179820, Nunc) and cultured in mTSER supplemented with Y-27632 (10 μM). The day after, cell culture media were changed to N2B27 medium (1:1 mix of DMEM-F12 (31331093, Gibco) and Neural Basal Medium (1103049, Gibco)) supplemented with N2 (17502048, Gibco), B27 (17504044, Gibco), Noggin (100 ng/ml, 78060.1, STEMCELL), Y-27632 (10 μM), and 1% PS. The media were replaced every 3 days.

Ucuncu E., Rajamani K., Wilson M.S., Medina-Cano D., Altin N., David P., Barcia G., Lefort N., Banal C., Vasilache-Dangles M.T., Pitelet G., Lorino E., Rabasse N., Bieth E., Zaki M.S., Topcu M., Sonmez F.M., Musaev D., Stanley V., Bole-Feysot C., Nitschké P., Munnich A., Bahi-Buisson N., Fossoud C., Giuliano F., Colleaux L., Burglen L., Gleeson J.G., Boddaert N., Saiardi A, & Cantagrel V. (2020). MINPP1 prevents intracellular accumulation of the chelator inositol hexakisphosphate and is mutated in Pontocerebellar Hypoplasia. Nature Communications, 11, 6087.

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Tumorsphere Formation Assay Protocol

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Tumorsphere formation assays were measured by in vitro limiting dilution assay, as previously reported [31 (link), 32 (link)]. Briefly, decreasing numbers of cells per well (50, 20, 10, 5 and 1) were plated into Ultra-Low Attachment 96-wells plates (Costar®) in Neural basal medium (Gibco) supplemented with 100 × penicillin and streptomycin (Gibco Life Technologies), 100 × Glutamine (Gibco), 100 × D-( +)-Glucose solution (Sigma), 100 × Insulin-Transferrin-Selenium (Gibco), 50 × B27 Supplement (Gibco), N-Acetyl Cysteine (16ug/ml), EGF (20 ng/ml) and FGF (20 ng/ml). Spheres with a diameter equal or higher than 40 μm were deemed tumourspheres. The presence and number of tumorspheres in each well were recorded seven days after plating. Extreme limiting dilution analysis was performed using the software available at [31 (link), 33 (link)].

Cai J., Wang Y., Wang X., Ai Z., Li T., Pu X., Yang X., Yao Y., He J., Cheng S.Y., Yu T., Liu C, & Yue S. (2023). AMPK attenuates SHH subgroup medulloblastoma growth and metastasis by inhibiting NF-κB activation. Cell & Bioscience, 13, 15.

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Neural Differentiation of Human iPSCs

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Embryonic bodies were produced after treating iPSC colonies with dispase and placing them in human neural stem cell differentiate medium (50 % DMEM/F12 plus 50 % Neural basal medium (Gibco) containing 1 % N2 supplement (Gibco), 2 % B27 supplement (Gibco), 1 μM Dorsomorphin (Sigma-Aldrich), 10 μM SB431542 (Sigma-Aldrich)) in a 100 mm petri dish. Medium was changed every two days. After 2 weeks, spheres were seeded on polyornithine (Sigma-Aldrich) and laminin (Sigma-Aldrich) coated dishes and kept culturing in the same medium for another 7-14 days for neural rosette formation. Neural rosettes were collected with Stem Diff neural rosette selection reagent and re-plated onto polyornithine/laminin coated dishes as a single cell suspension in neural stem cell culture medium (ReNcell NSC maintenance media (Millipore) containing, 1 % N2 supplement, 1 % NEAA, 20 ng/ml bFGF and 20 ng/ml EGF). For three germ layers differentiation, iPSC colonies were digested with dispase and culture in DMEM with 10 % FBS for 4 days in 10 cm petri dish. After 4 days in suspension culture, floating embryonic bodies were re-seeded onto gelatin-coated dishes in the same culture medium for 10 days. The medium was changed every other day.

Wren M.C., Zhao J., Liu C.C., Murray M.E., Atagi Y., Davis M.D., Fu Y., Okano H.J., Ogaki K., Strongosky A.J., Tacik P., Rademakers R., Ross O.A., Dickson D.W., Wszolek Z.K., Kanekiyo T, & Bu G. (2015). Frontotemporal dementia-associated N279K tau mutant disrupts subcellular vesicle trafficking and induces cellular stress in iPSC-derived neural stem cells. Molecular Neurodegeneration, 10, 46.

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Neural Stem Cell Culture Conditions

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Growth factors: bFGF (recombinant human fibroblast growth factor-basic; PeproTech) and EGF (recombinant human epidermal growth factor; PeproTech), DMEM/F-12 medium (1 : 1) and Neural Basal medium (Gibco/Life Technologies), glucose at 30% (w/v) in distilled water (ThermoFisher Scientific) and stored at 4°C (d-(+)-glucose, Sigma-Aldrich, Australia), GlutaMAX™-1 (Gibco/Life Technologies), phosphate-buffered saline solution (PBS) (Sigma-Aldrich, Australia) made according to the manufacturer's instructions, poly-d-lysine hydrobromide (Sigma-Aldrich, Australia) reconstituted in double distilled water at 1 mg ml⁻¹ and stored at 20°C, laminin supplied reconstituted at 1 mg ml⁻¹ Tris–HCl (50 mM, pH 7.4), NaCl (0.15 M) and stored at 20°C (Invitrogen/Life Technologies), ITS solution (insulin/transferrin/selenium-A solution, Gibco/Life Technologies), N-2 supplement (Gibco/Life Technologies), B27 retinol-free supplement (Gibco/Life Technologies) and Pen/Strep solution (penicillin/streptomycin solution with 10 000 U ml⁻¹ penicillin and 10 000 mg ml⁻¹ streptomycin, Gibco/Life Technologies).

D'Abaco G.M., Mattei C., Nasr B., Hudson E.J., Alshawaf A.J., Chana G., Everall I.P., Nayagam B., Dottori M, & Skafidas E. (2018). Graphene foam as a biocompatible scaffold for culturing human neurons. Royal Society Open Science, 5(3), 171364.

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Molecular Mechanisms of Neuronal Differentiation

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Retinoic acid (RA), dimethylsulfoxide (DMSO), FM 1-43FX, MTT, β-mercaptoethanol (β-ME), 4,6-Diamidino-2-phenylindole (DAPI), inositol 1,4,5-triphosphate (IP3), L165041 and GSK0660 were purchased from Sigma-Aldrich (St. Louis, MO, USA). DMEM medium, fetal bovine serum (FBS), B27 supplement, neuralbasal medium was obtained from Gibco BRL (Burlington, Ontario, Canada). Non-essential amino acids (NEAA) stock solution was purchased from Hyclon (Logan, USA). Recombinant mouse leukemia inhibitory factor (LIF) was obtained from Millipore (CA, USA). Antibodies were used as follows: β-tubulin III, neuronal nuclei (NeuN), and neurofilament 160 (NEFM) were purchased from Sigma-Aldrich (St. Louis, MO, USA); synaptophysin and GAPDH were products of Cell Signaling; Nestin was from Millipore (CA, USA); PPAR-α, -β, -γ, Mfn1 and Mfn2 were ordered from Abcam; PGC-1α, Fis1, Drp1 and OPA1 were purchased from Santa Cruz Biotechnology. Secondary antibodies were purchased from Multisciences. Rhod-2AM was purchased from Dojindo Molecular Technologies. MitoTracker Green was a product of Life technologies.

Mei Y.Q., Pan Z.F., Chen W.T., Xu M.H., Zhu D.Y., Yu Y.P, & Lou Y.J. (2016). A Flavonoid Compound Promotes Neuronal Differentiation of Embryonic Stem Cells via PPAR-β Modulating Mitochondrial Energy Metabolism. PLoS ONE, 11(6), e0157747.

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Induced Pluripotent Stem Cell Derivation

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Human skin fibroblasts NB1RGB [20–22 (link)] and human iPSCs 201B7 [23 (link)] were obtained from RIKEN Bio Resource Center. NB1RGB was maintained with Dulbecco’s Modified Eagle Medium and supplemented with 10% FBS and penicillin/streptomycin (Nacalai Tesque, Japan). iPSCs C2 were derived from NB1RGB by messenger RNA (mRNA) integration-free methods using the Stemgent® StemRNATM-NM Reprogramming Kit for Reprogramming Adult and Neonatal Human Fibroblasts (Stemgent, USA) as previously reported [11 (link), 24 (link)]. iPSCs were maintained as feeder-free culture with the NutriStem™ XF/FF medium (ReproCELL, Japan) and iMatrix511 silk (Nippi, Japan). NPCs were derived from iPSCs C2 using the PSC neural induction medium (Gibco, Thermo Fisher Scientific, USA) and maintained with the neural basal medium (Gibco, Thermo Fisher Scientific) and iMatrix 511 silk according to the manufacturer’s protocols with small modifications. iPSCs and NPCs culture media were supplemented with the Y27632 ROCK inhibitor (WAKO Pure Chemical Industries, Japan) during passage; on the subsequent day, the media were replaced with fresh culture media without the ROCK inhibitor.

Shimada M., Tokumiya T., Miyake T., Tsukada K., Kanzaki N., Yanagihara H., Kobayashi J, & Matsumoto Y. (2023). Implication of E3 ligase RAD18 in UV-induced mutagenesis in human induced pluripotent stem cells and neuronal progenitor cells. Journal of Radiation Research, 64(2), 345-351.

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Astrocyte-Neuron Interactions in Inflammatory Conditions

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To examine the direct effects of RUX pre-treated astrocytes on neurons after exposure to 3 ng/mL of IL-1α, 30 ng/mL of TNF-α, and 400 ng/mL of C1q (A1-like astrocyte induction medium, A1IM), a co-culture model was established using Transwell inserts (Corning, One Riverfront Plaza Corning, NY, USA). Neurons were cultured in 500 µL of neural basal medium (Gibco) in the lower chamber for 24 hours. In the upper chamber, treated astrocytes were cultured in 500 µL of 10% Dulbecco’s modified Eagle medium. Excitotoxicity was induced with 100 µM glutamate solution. For neuronal calcium imaging, the neurons were cultured in confocal petri dishes (Biosharp, Nanjing, China). Astrocyte-conditioned medium with or without RUX (1 µM) and/or A1IM pretreatment was added, followed by 8 hours of incubation. Neurons were then stimulated with astrocyte-conditioned medium to evaluate their response to glutamate exposure.

Cao J., Yu X., Liu J., Fu J., Wang B., Wu C., Zhang S., Chen H., Wang Z., Xu Y., Sui T., Chang J, & Cao X. (2024). Ruxolitinib improves the inflammatory microenvironment, restores glutamate homeostasis, and promotes functional recovery after spinal cord injury. Neural Regeneration Research, 19(11), 2499-2512.

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