In the indicated experiments, organoids were cultured in medium with or without vitamin A. To prepare the medium, 1x B27 minus vitamin A (Thermo Scientific) was used instead of 1x B27 (Thermo Scientific) to prepare both ENR and WENRC media. To allow proper comparison, in these experiments, B27 minus vitamin A (Thermo Scientific) was supplemented with retinyl acetate (Merck, catalogue number 46958) corresponding to the standard B27 supplement in order to produce medium for control conditions.
B27 minus vitamin a
Manufactured by Thermo Fisher Scientific
Sourced in United States
B27 minus vitamin A is a cell culture supplement developed by Thermo Fisher Scientific. It is designed to support the growth and maintenance of various cell types in vitro, with the exception of vitamin A.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (12 mentions)
Dmem f12, by Thermo Fisher Scientific (11 mentions)
Neurobasal medium, by Thermo Fisher Scientific (9 mentions)
N2 supplement, by Thermo Fisher Scientific (8 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (6 mentions)
L glutamine, by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Matrigel, by Corning (4 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (4 mentions)
Pen strep, by Thermo Fisher Scientific (4 mentions)
Recombinant human egf, by Thermo Fisher Scientific (3 mentions)
Neurobasal a, by Thermo Fisher Scientific (3 mentions)
Accutase, by STEMCELL (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Heparin, by Merck Group (3 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (3 mentions)
Epidermal growth factor (egf), by Merck Group (3 mentions)
Chir99021, by Bio-Techne (3 mentions)
Retinyl acetate, by Merck Group (2 mentions)
46 protocols using b27 minus vitamin a
1
Organoid Culture with Vitamin A
2
Organoid Culture with Vitamin A
3
Induced Astrocyte Differentiation Protocol
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We differentiated HiAs as previously reported (Perriot et al., 2018 (link)). Lt-NES cells were dissociated with trypsin-EDTA, plated on Matrigel (Corning, Corning, NY)-coated T25-flask at 1 × 106 cells in Astrocyte Induction Medium (DMEM/F12; Sigma-Aldrich, St Louis, USA, GlutaMax; Thermo Fisher Scientific, Waltham, MA, 1/100, N2 supplement; Thermo Fisher Scientific, 1/100, 10 ng/ml LIF; Sigma-Aldrich, 10 ng/ml EGF; PeproTech, B27 minus vitamin A; Thermo Fischer Scientific, 1/50) and medium was changed every 3–4 days for 2 weeks. After 2 weeks, cells were trypsinized, and a half volume of cells was plated on Matrigel-coated T25-flasks in Astrocyte Maturation Medium (DMEM/F12, GlutaMax 1/100, B27 minus vitamin A 1/50, 20 ng/ml CNTF; PeproTech) and incubated for 4 weeks, changing the medium every 3 to 4 days. Four weeks later, the cells were removed by trypsin and plated in Matrigel-coated T75 flasks in 10% FBS/DMEM (DMEM, FBS 1/10, GlutaMax 1/100, MITO+ Serum Extender; Corning, 1/1000) and incubated for a week. Finally, the cells were reacted with mitomycin C (Nacalai Tesque, Kyoto, Japan) in 10 μg/ml for 2 hours to inhibit cell proliferation. For cryopreservation, cells were trypsinized, and around 1–2 × 106 cells were spun down and resuspended in CELLBANKER 2 before freezing at −80°C.
4
Generating Human Cortical Spheroids from hiPSCs
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HiPSCs were differentiated into human cortical spheroids (hCS) using methods published by Pasca et al. In this method, dual SMAD inhibition was performed using KOSR media supplemented with 10 µM ROCKi and SMAD inhibitors: 5 µM dorsomorphin (Tocris) and 10 µM SB431542 (Tocris) to direct hiPSCs towards a cortical lineage. Cultures were maintained in 5% CO2 at 37 °C for 48-h period to promote formation of spheroids. From day 2 until day 4, media changes were performed daily with fresh KOSR media supplemented with 5 µM dorsomorphin and 10 µM SB431542. From day 5 until day 25 (or when hCSs were harvested), culture media used was B27 minus Vitamin A (Life Technologies) media plus 20 ng/mL of recombinant human EGF and recombinant human bFGF (both Preprotech). From day 25, B27 minus Vitamin A media with 20 ng/mL of recombinant human BDNF (Preprotech) and recombinant human NT3 (LifeTechnologies) was used. Media was changed every 24 h during the first 15 days and once every 48 h thereafter. Spheroids were harvested at days 7, 17 or 30.
5
Differentiation of hIPSCs into Cortical Spheroids
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HiPSCs were differentiated into human cortical spheroids (hCS) using methods published by Pasca et al. [2] . In this method dual SMAD inhibition was performed using KOSR media supplemented with 10 µM ROCKi and SMAD inhibitors: 5 µM dorsomorphin (Tocris) and 10 µM SB431542 (Tocris) to direct hiPSCs towards a cortical lineage. Cultures were maintained in 5% CO2 at 37°C for 48-hours period to promote formation of spheroids. From day 2 until day 4, media changes were performed daily with fresh KOSR media supplemented with 5 µM dorsomorphin and 10 µM SB431542. From day 5 until day 25 (or when hCSs were harvested), culture media used was B27 minus Vitamin A (Life Technologies) media plus 20 ng/mL of recombinant human EGF and recombinant human bFGF (both Preprotech). From day 25, B27 minus Vitamin A media with 20 ng/mL of recombinant human BDNF (Preprotech) and recombinant human NT3 (LifeTechnologies) was used. Media was changed every 24 hours during the first 15 days and once every 48 hours thereafter.
Spheroids were harvested at days 7, 17 or 30.
Spheroids were harvested at days 7, 17 or 30.
6
Culturing Diverse DMG Cell Lines
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All DMG cell lines used in the study (except for SF7761) were cultured in ultra-low attachment flasks in culture medium with 1:1 ratio of DMEM/F-12 (Invitrogen, 11330-032) and Neurobasal A (Invitrogen, 10888-022) and ten percent each of HEPES Buffer Solution 1 M (Thermo Fisher, 15630080), Sodium Pyruvate solution 100 nM (Life Technologies, 11360070), MEM non-essential amino acids solution 10 mM (Thermo Fisher, 11140050), Glutamax-I Supplement (Thermo Fisher, 35050061), and Penicillin/Streptomycin solution (Life Technologies, 15140122). The media was supplemented with B27 Minus Vitamin A (Invitrogen, 12587-010), epidermal growth factor (EGF; StemCell Tech. Inc., 78006), fibroblast growth factor (FGF; GF003, StemCell Tech., Inc., 78003) and heparin solution, 0.2% (StemCell Tech. Inc., 07980), as well as PDGF-AA (Shenandoah Biotech, 100-16) and PDGF-BB (Shenandoah Biotech, 100-18). SF7761 cell line was cultured in medium with Neurobasal A (Invitrogen, 10888-022) and N-2 Supplement (Invitrogen, 17502), further supplemented with B27 Minus Vitamin A (Invitrogen, 12587-010), epidermal growth factor (EGF; StemCell Tech. Inc., 78006), fibroblast growth factor (FGF; GF003, StemCell Tech. Inc., 78003) and heparin solution, 0.2% (StemCell Tech. Inc., 07980). Cells were dissociated using Accutase (StemCell Tech. Inc., 07922) and passaged every 3 or 4 days.
7
Directed Differentiation of iPSCs to NSCs
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Directed differentiation of patient-derived iPSCs into NSCs was performed according to a previous report [24 (link)] with modifications: Briefly, patient-derived iPSCs were seeded onto Geltrex-coated dishes and were maintained in mTeSR1 medium at 37 °C under humidified atmosphere of 5 % CO2. After 3 days in adherent culture, culture medium was switched to NSC induction medium [1:1 mixture of DMEM/F12 (Gibco) and Neurobasal medium (Gibco) supplemented with 1 × N2 (Gibco), 1 × B27 minus vitamin A (Gibco), 1 × GlutaMAX (Gibco), 100 units/mL penicillin (Gibco), 100 μg/mL streptomycin (Gibco), 10 μM SB431542 (Wako), 100 nM LDN193189 (Wako), 20 ng/mL EGF (Peprotech), 20 ng/mL bFGF (Peprotech)], and patient-derived iPSCs were maintained at 37 °C under humidified atmosphere of 5 % CO2. Emergent NSCs were expanded in NSC induction medium and were characterized by immunocytochemical staining. Fluorophore-conjugated primary antibody used was 5 μg/mL AlexaFluor 488-conjugated anti-Nestin (Millipore).
8
Differentiation of hiPSCs into NPCs
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One clone from each subject (n = 13) was differentiated into hiPSC–NPC using AggreWell™ methodology (StemCell Technologies), that involves the formation of embryoid bodies (EB) using STEMdiff™ Neural Induction Medium, according to manufacture instructions. After 5 days, EBs were plated in six-well plates coated with Membrane Matrix. Rosette selection was performed after 7 days using STEMdiff Rosette Selection Reagent (StemCell Technologies) and plated on Membrane Matrix-coated wells. STEMdiff™ Neural Induction Medium was changed every day. When confluent, hiPSC–NPC were lifted using Accutase (Innovative Cell Technologies, Inc.) and expanded on Membrane Matrix-coated plates in Neurobasal Medium 50/50 (50% DMEM/F12 (Corning), 50% Neurobasal Medium (Gibco), 1x GlutaMAX (Gibco), 1x NEAA (Gibco), 1x PenStrep (Gibco), 1x N-2 Supplement (Gibco), 1x B27 minus vitamin A (Gibco)) supplemented with 20 ng/ml FGF-Basic (AA 1–155) Recombinant Human Protein (Gibco) until passage 3. hiPSC–NPCs were immunocytochemically characterized using markers for proteins highly expressed by NPCs (Nestin and SOX-1).
9
Cerebral Organoid Differentiation from hESCs
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Differentiation of hESCs into cerebral organoids was based on a previously described protocol26 (link),29 (link). Briefly, hESC cells were inoculated into a spinner flask, and to enable embryoid body formation, after six days the medium was changed to neural induction media (DMEM/F12, 1X N2 supplement (Gibco), 2 mM Glutamax (Invitrogen), 1% MEM-NEAA, and 1 μg/mL heparin (Sigma)) and the aggregates were cultured for five more days. After being embedded in matrigel, differentiation media composed of 1:1 DMEM/F12:Neurobasal (Gibco), 0.5X N2, 1X B27 minus vitamin A (Gibco), 2 mM Glutamax, 0.5% MEM-NEAA, 0.2 μM 2-mercaptoethanol and 2.5 μg/mL insulin was used. After 4 days, cell aggregates were grown in neuronal differentiation media, composed as aforementioned except by replacing with 1X B27 containing vitamin A (Gibco). The medium was changed once per week. Cerebral organoids were grown for 45 days (30 days in neuronal differentiation media).
10
Neuronal Differentiation from Patient-Derived Stem Cells
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Terminal differentiation of patient-derived NSCs and NCCs into neurons was performed as follows: Briefly, patient-derived NSCs and NCCs were seeded at high cell density onto Geltrex-coated 6-well culture plates and were maintained at 37 °C under humidified atmosphere of 5 % CO2. After 3 days in culture, culture medium was switched to neuron induction medium [Neurobasal medium (Gibco) supplemented with 1 × N2 (Gibco), 1 × B27 minus vitamin A (Gibco), 1 × GlutaMAX (Gibco), 100 units/mL penicillin (Gibco), 100 μg/mL streptomycin (Gibco), 10 ng/mL BDNF (Peprotech), 10 ng/mL GDNF (Peprotech), 10 ng/mL NGF (Peprotech), 500 μM dbcAMP (Sigma), 200 μM ascorbic acid (Wako)], and patient-derived NSCs and NCCs were maintained at 37 °C under humidified atmosphere of 5 % CO2 for more than 2 weeks. For immunocytochemical detection of emergent NSC-derived neurons, 5 μg/mL anti-TUJ1 (Abcam) and 2.5 μg/mL Alexa Fluor 568 (Molecular Probes) were used. For immunocytochemical detection of emergent NCC-derived neurons, 5 μg/mL PE-conjugated anti-Peripherin (Santa Cruz) was used.
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