Neurobasal

Organotypic hippocampal slices were prepared from 6- to 7-day-old Wistar rats derived from the Animal House of the Mossakowski Medical Research Centre. All experiments were approved by the local bioethical committee. Six- to seven-day-old rats were anesthetized and decapitated, and the brains were carefully removed and placed into the HBSS buffer (Gibco). After that, the hippocampi were isolated and cut into 400-μm slices using McIlwan tissue chopper. The healthy, presenting proper anatomy slices were chosen under the binocular and transposed onto the Millicell-CM membranes, with four hippocampal slices on each membrane. All procedures were performed on ice. Afterwards, the membranes with hippocampal slices were placed into the six-well dishes containing hippocampal slices medium with serum (50% Neurobasal (Gibco), 25% horse serum (Gibco), 22% HBSS (Gibco), 1 M HEPES (Sigma), glucose, and penicillin/streptomycin). Next day, the medium was changed and then replaced gradually by hippocampal slices medium free of serum (73% Neurobasal (Gibco), 22% HBSS (Gibco), 1 M HEPES (Sigma), B-27 supplements (Gibco), glucose, and penicillin/streptomycin). The organotypic hippocampal slices were incubated at 35 °C, 21% O₂, and 5% CO₂ for 7 days.

Dissociated hippocampal cultures were prepared from E17-E18 Wistar rats as previously described (Martin and Henley, 2004 (link)). Glass coverslips were coated in poly-D-lysine or poly-L-lysine (1 mg/mL, Sigma) in borate buffer (10 mM borax, 50 mM boric acid) overnight and washed in water. Dissociated hippocampal cells were plated at different densities in plating medium (Neurobasal, Gibco supplemented with 10% horse serum, Sigma; 2 mM GlutaMAX, Gibco; and either GS21, GlobalStem, or B27, Thermo Fisher) which was changed to feeding medium (Neurobasal supplemented with 1.2 mM GlutaMAX and GS21 or B27) after 24 hr. For RUSH experiments, cells were plated and fed in media containing GS21 instead of B27 because it does not contain biotin. Cells were incubated at 37°C and 5% CO₂ for up to 2 weeks. Animal care and procedures were carried out in accordance with UK Home Office and University of Bristol guidelines.
Transfection of neuronal cultures was carried out at DIV 12 using Lipofectamine2000 (Invitrogen) according to the manufacturer’s instructions with minor modifications. Cells were left for 20–48 hr before fixation.
HEK293T cells (ECACC) were passaged and maintained in complete DMEM (DMEM + 10% FBS + 2 mM L-Glutamine). HEK293T cells were regularly treated with ciprofloxacin (10 µg/mL) to prevent mycoplasma contamination.

iPSCs were differentiated into fbNPCs as previously described (46 (link), 63 (link)). Upon dissociation at 70 to 90% confluency, the cells were plated on LN111 (1.14 μg/cm²; BioLamina)–coated Nunc multidishes at a density of 10,000 cells/cm² and grown in N2 medium [1:1 Dulbecco’s modified Eagle’s medium (DMEM)/F12 (21331020, Gibco) and Neurobasal (21103049, Gibco) supplemented with 1% N2 (Gibco), 2 mM l-glutamine (Gibco), and 0.2% penicillin/streptomycin]. SB431542 (10 μM; Axon) and noggin (100 ng/ml; Miltenyi) were given for dual SMAD inhibition. The medium was changed every 2 to 3 days. On day 9, N2 medium without dual SMAD inhibitors was used. On day 11, cells were dissociated and replated on LN111-coated Nunc multidishes at a density of 800,000 cells/cm² in B27 medium [Neurobasal supplemented with 1% B27 without vitamin A (Gibco), 2 mM l-glutamine, and 0.2% penicillin/streptomycin Y27632 (10 μM), brain-derived neurotrophic factor (BDNF; 20 ng/ml; R&D), and l-ascorbic acid (0.2 mM; Sigma-Aldrich)]. Cells were kept in the same medium until day 14 when cells were harvested for downstream analysis.

Human induced pluripotent stem cell (iPSC)-derived NSCs (IxCell Biotechnology, Ltd.) were maintained as adherent culture in the medium, containing 50% Dulbecco’s modified Eagle medium: nutrient mixture F-12 (DMEM/F12, Gibco), 50% neurobasal (Gibco), 1*N2 supplement (Gibco), 1*B27 supplement (Gibco), 1*MEM non-essential amino acids solution, 1*GlutaMAX (Gibco), 10 ng/μl bFGF, 10 ng/μl hlif, 3 μM CHIR99021 (Selleckchem), 5 μM SB431542 (Selleckchem), and 200 μM ascorbic acid (Sigma) and cultured in a humidified incubator with 5% CO2/95% air (v/v) at 37°C. For differentiation, the human NSCs were seeded in 24-well plates coated by poly-D-lysine and laminin, at the density of 5*10⁴ cells per well. On the second day, medium was changed to neuron differentiation medium, neurobasal with 1*B27, 1*CultureOne supplement (ThermoFisher Scientific), 1*GlutaMAX, and 200 μM ascorbic acid. The differential medium was refreshed every other day (Lu et al., 2019 (link)). At the day 12 of differentiation, cells were treated by Aβ for 48 h.

Commissural neurons were prepared from the dorsal fifth of E13 rat neural tubes as described previously [15 (link),35 (link)]. Cells were re-suspended in plating media composed of Neurobasal (Gibco) supplemented with 10% heat-inactivated FBS and 2 mM GlutaMAX (Life Tech). 50 μl of plating media was added to both inlet reservoirs and 50 μl of cell suspension (3,160,000–5,630,000 cells/ml) was added to the outlet. One of the inlet reservoirs was removed and a reverse flow induced by connecting a syringe to the inlet hole via a short rubber hose and pulling on the plunger. While observing with an inverted microscope, neurons were drawn into the gradient chamber, after which the flow was stopped by releasing the plunger, disconnecting the syringe, and then returning the reservoir to the hole. After 4–6 h, inlet reservoirs were filled with 200 μl of plating media, again inducing a forward flow. Approximately 15 h later, the plating media was replaced with serum-free growth media composed of Neurobasal (Gibco) supplemented with 2% B27 (Gibco), 2mM GlutaMAX (Gibco) and penicillin/streptomycin (Gibco).

iPSCs were differentiated into fbNPCs as previously described (46 (link), 63 (link)). Upon dissociation at 70 to 90% confluency, the cells were plated on LN111 (1.14 μg/cm²; BioLamina)–coated Nunc multidishes at a density of 10,000 cells/cm² and grown in N2 medium [1:1 Dulbecco’s modified Eagle’s medium (DMEM)/F12 (21331020, Gibco) and Neurobasal (21103049, Gibco) supplemented with 1% N2 (Gibco), 2 mM l-glutamine (Gibco), and 0.2% penicillin/streptomycin]. SB431542 (10 μM; Axon) and noggin (100 ng/ml; Miltenyi) were given for dual SMAD inhibition. The medium was changed every 2 to 3 days. On day 9, N2 medium without dual SMAD inhibitors was used. On day 11, cells were dissociated and replated on LN111-coated Nunc multidishes at a density of 800,000 cells/cm² in B27 medium [Neurobasal supplemented with 1% B27 without vitamin A (Gibco), 2 mM l-glutamine, and 0.2% penicillin/streptomycin Y27632 (10 μM), brain-derived neurotrophic factor (BDNF; 20 ng/ml; R&D), and l-ascorbic acid (0.2 mM; Sigma-Aldrich)]. Cells were kept in the same medium until day 14 when cells were harvested for downstream analysis.

Cortices were micro-dissected from 5 SAS Sprague Dawley Rat embryos or 7 to 10 E14 embryos of PS19 mice. All steps of the dissection were performed in cold Gey’s balanced salt solution (GBSS) supplemented with 0.1% glucose. Dissected structures were digested with papain (20U/ML in Neurobasal, Sigma). After papain inactivation with FBS, structures were mechanically dissociated with a pipette in presence of DNAse. After several rounds of rinsing, cells were re-suspended in Neurobasal + B27 in a final density of 35 million cells/mL. For 24 well plate, 150 000 to 200 000 of cortical precursors are plated. For microfluidic cultures, WT cortical neurons were then seeded in the left and the right chamber: 2 µl of the cell suspension was introduced into the upper reservoir and cells flowed into the chamber and adhered within 20 min. For 24 well plate and for microfluidic devices, cortical neurons are plated in plating media for 24 h: Neurobasal + B27 (Gibco) + streptomycin/penicillin (Gibco) + 10% FBS. The next day, a media change is performed to remove the FBS, Neurobasal + B27 (Gibco) + streptomycin/penicillin (Gibco). Microfluidic chips were placed in plastic Petri dishes containing H2O-EDTA to prevent evaporation and incubated at 37 °C in a humid 5% C02 atmosphere. The culture medium was renewed every 6 days.