The BrainSpheres were generated as described (Pamies et al., 2017 (link)). Briefly, at 90% confluency, NPC were detached mechanically and counted. A number of 2 × 106 cells per well were plated in uncoated 6 well-plates. Cells were grown in NPC media for two days and then medium was changed to a differentiation medium (Neurobasal® electro Medium (Gibco) supplemented with 5% B-27® Electrophysiology (Gibco), 1% Glutamax (Gibco), 0.01 μg/ml human recombinant GDNF (Gemini), 0.01 μg/ml human recombinant BDNF (Gemini). Cultures were kept at 37 °C in an atmosphere of 5% CO2 under constant gyratory shaking (88 rpm, 19 mm orbit) for up to 8 weeks.
Neurobasal electro medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Neurobasal® electro Medium is a basal medium designed for the maintenance and growth of primary neuronal cultures and embryonic stem cell-derived neuronal cells. It provides a defined environment to support the survival and differentiation of these cell types.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (9 mentions)
B 27 electrophysiology, by Thermo Fisher Scientific (8 mentions)
Maxq 2000 co, by Thermo Fisher Scientific (2 mentions)
Neurobasal medium without phenol red, by Thermo Fisher Scientific (2 mentions)
Human recombinant gdnf, by Gemini Bio (2 mentions)
Human recombinant bdnf, by Gemini Bio (2 mentions)
Maxq 2000 co2, by Thermo Fisher Scientific (2 mentions)
Cell scraper, by Sarstedt (1 mentions)
Countess automated cell counter, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (1 mentions)
Neural induction medium, by Thermo Fisher Scientific (1 mentions)
12 protocols using neurobasal electro medium
1
Generation of BrainSpheres from NPCs
2
Glioblastoma Cells Incorporation into Brain Spheroids
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BS were generated as previously described18 (link). In order to incorporate glioblastoma cells into the BS, the protocol was slightly modified as follows: NPCs were grown (as above) in 175 mm2 poly-l-ornithine and laminin-coated flasks. When NPCs were at 90% confluency, 7 × 105 glioblastoma cells were plated on top of the NPCs. After 24 hours the cells were detached mechanically using a cell scraper (Sarstedt). The mixture of cells was pipetted repeatedly to disaggregate cell clumps. A density of 2 × 106 cells per well were plated on a non-coated 6 plate-well. Cells were grown in differentiation medium (Neurobasal® electro Medium (Gibco) supplemented with 5% B-27® Electrophysiology (Gibco), 1% Glutamax (Gibco), 0.01 μg/ml human recombinant GDNF (Gemini), 0.01 μg/ml human recombinant BDNF (Gemini). Cultures were kept at 37 °C in an atmosphere of 5% CO2 under constant gyratory shaking (88 rpm, 19 mm orbit) for up to 7 weeks, Fig. 1 Supplementary Data .
3
Generation of Brain-like Organoids from iPSCs
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The CRL-2097 line was derived from CCD-1079Sk ATCC® CRL-2097™ fibroblasts purchased from ATCC and was kindly provided by Dr. Hongjun Song within our joint NIH NCATS funded project (Pamies et al., 2017 (link); #1U18TR000547-01). The iPS2C1 line was kindly provided by Dr. Herbert Lachman. All studies followed Institutional Review Board protocols approved by the Johns Hopkins University School of Medicine. Differentiation from iPSCs to NPCs has been previously described (Wen et al., 2014 (link)). The BrainSpheres were generated as described in Pamies et al. (2017 (link)). Briefly, at 90% confluency, NPCs were detached mechanically and counted. The 2 × 106 cells per well were plated in uncoated six well-plates. After 2 days, NPC medium was changed to differentiation medium (Neurobasal® electro Medium (Gibco) supplemented with 2% B-27® Electrophysiology (Gibco), 1% Glutamax (Gibco), 0.01 μg/ml human recombinant GDNF (Gemini), 0.01 μg/ml human recombinant BDNF (Gemini). Cultures were kept at 37°C in an atmosphere of 5% CO2 under constant gyratory shaking (88 rpm, 19 mm orbit) for up to 8 weeks. The medium was partly exchanged three times a week.
4
Neural Progenitor Cell Differentiation
5
Differentiation of Neuronal Progenitor Cells
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The production of BS was published previously (Pamies et al., 2017 (link)). Briefly, to produce BS, NPCs were detached mechanically with a cell scraper (Sarstedt, 2-position, Blade 25, 83.1830), re-pipetted for disaggregation, and counted using the Countess Automated Cell Counter (Invitrogen, Carlsbad, CA, United States). 2 × 106 cells per well were plated in non-treated FalconTM Polystyrene 6-well plates (Corning, Corning, NY, United States). Cells were grown in differentiation medium [Neurobasal® electro Medium (Gibco, Gaithersburg, MD, United States)] supplemented with 2% B-27® Electrophysiology (Gibco), 1% GlutaMAX (Gibco), 0.01 μg/mL human recombinant GDNF (Gemini, Woodland, CA, United States), and 0.01 μg/mL human recombinant BDNF (Gemini). Cultures were maintained at 37°C in an atmosphere of 5% CO2 under constant gyratory shaking (88 rpm) for 7 weeks. Differentiation medium was changed every 2 days.
6
Generation and Differentiation of 3D Neuronal Cultures
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NPCs were thawed and expanded for 2–3 weeks. BrainSpheres were then prepared as previously reported [61 (link)]. Briefly, two million NPCs per well were plated in non-coated 6-well plates in 2 mL of NPC medium and incubated at 37 °C, 5% CO2 as free-floating cultures under constant gyratory shaking at 88 rpm (Figure 2 A). After two days, medium was exchanged to differentiation medium (Neurobasal® electro medium (GIBCO) supplemented with 1% Penicillin Streptomycin Glutamine (GIBCO), 1% Glutamax (GIBCO), 1% B-27® Electrophysiology supplement (GIBCO), 0.02 μg/mL human recombinant glial-cell derived neurotrophic factor (GDNF, Gemini Bio, West Sacramento, CA, USA), and 0.02 μg/mL human recombinant brain-derived neurotrophic factor (BDNF, Gemini Bio, West Sacramento, CA, USA)). Cultures were maintained for eight weeks at 37 °C, 5% CO2 under constant gyratory shaking at 88 rpm, and differentiation medium was changed every second day (Figure 1 B).
7
Neural Progenitor Cell Differentiation
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At 100% confluence, NPCs were detached by scraping and counted. 2×106 cells per well were plated in 2 ml medium in non-treated 6 well-plates. Cells were grown in NPC medium for two days under constant gyratory shaking (88 rpm), allowing aggregation by using a MaxQ™ 2000 CO2 (ThermoFisher Scientific) plate shaker. Subsequently, medium was changed to differentiation medium (Neurobasal® Electro Medium (Gib- co) supplemented with 5% B-27® Electrophysiology (Gibco), 1% glutamax (Gibco), 0.02 μg/ml human recombinant GDNF (Gemini), 0.02 μg/ml human recombinant BDNF (Gemini)). Cultures were maintained at 37°C, 5% CO2 under constant gyratory shaking (88 rpm) for up to eight weeks. Differentiation medium was routinely changed every two days.
8
iPSC-Derived Brain Organoid Differentiation
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We differentiated brain organoids from the iPSC NIBSC8 cell line [U.K. National Institute for Biological Standards and Control (NIBSC)], following our in-house two-step protocol (8 (link)). The NIBSC-8 iPSC cell line is mycoplasma free, with a normal female karyotype. Briefly, we differentiated iPSCs in a monolayer to NPCs using serum-free, chemically defined neural induction medium (Gibco, Thermo Fisher Scientific). We expanded the NPCs, and a single-cell suspension was distributed into uncoated six-well plates and cultured under constant gyratory shaking (80 rpm, 19-mm orbit) to form 3D aggregates. After 48 hours, we induced differentiation with serum-free, chemically defined differentiation medium [Neurobasal electro medium (Gibco, Thermo Fisher Scientific) supplemented with 1× B27-electro (Gibco, Thermo Fisher Scientific), 2× glutamax, glial cell line–derived neurotrophic factor (10 ng/ml; Gemini), brain-derived neurotrophic factor (10 ng/ml; Gemini), and 5% penicillin-streptomycin]. We differentiated brain organoids for 8 to 10 weeks before recording. By this time, the brain organoids consist of different types of neurons, astrocytes, and oligodendrocytes (8 (link)).
9
Oxidative Stress Response in BrainSpheres
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At 4 weeks of differentiation, the medium was replaced with 2 ml of fresh differentiation medium (Fig. 1A ). Subsequently, BrainSpheres were exposed for 24 h to compounds known to induce ROS production (6-OHDA, MPTP, or MPP+) at a wide range of concentrations (10 μM – 5000 μM) (Fig. 1A ). For the ROS assay, differentiation medium Neurobasal® electro medium [Gibco] was replaced with Neurobasal medium without phenol red [Gibco]. The 6-wells plates were returned to the incubator with gyratory shaking (5.0% CO2, 37 °C) for 24 h. After exposure, multiple assays were performed as described below. In addition, in order to study the recovery of the cells, different treatments were removed from the media, BrainSpheres were washed 3 times with PBS, and then cultured for 5 days before collection.
10
BrainSpheres Differentiation Protocol
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BrainSpheres were generated as previously described (Pamies et al., 2016 (link)). Briefly, at 90% confluence, NPCs were detached mechanically. 2 × 106 cells per well were plated in 2 ml of medium in non-coated 6 wellplates. Cells were kept under constant gyratory shaking (88 rpm, 19 mm orbit) by using a MaxQ™ 2000 CO2 [ThermoFisher] plate shaker in NPC expanding medium for two days, then the medium was changed to differentiation medium (Neurobasal® electro Medium [Gibco] supplemented with 2% B-27® Electrophysiology [Gibco], 1% glutamax [Gibco], 10 ng/ml human recombinant GDNF [Gemini Bio Products], 10 ng/ml human recombinant BDNF [Gemini Bio Products]. Cultures were maintained further under gyratory shaking (5.0% CO2, 37 °C, 88 rpm) for up to 4 weeks. Differentiation medium was exchanged every 2–3 days.
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