Brainphys medium

Manufactured by STEMCELL

Sourced in Canada

BrainPhys medium is a specialized cell culture medium designed to support the growth and maintenance of neural cells, including neurons and glial cells. It provides a balanced formulation of nutrients, growth factors, and other components essential for the optimal culture of various neural cell types.

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17 protocols using brainphys medium

Culturing Slice of Healthy Neocortical Tissue

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Healthy neocortical tissue was obtained with informed consent by resection of a small piece of the middle temporal gyrus from patients undergoing elective surgery for temporal lobe epilepsy (n = 7, both genders, median age 35 years) according to guidelines approved by the Regional Ethical Committee, Lund (Dnr. H15 642/2008). The tissue slices were derived and handled as previously described.¹⁵ Briefly, the surgically resected tissue was immediately kept in ice‐cold modified human artificial cerebrospinal fluid and sliced on a Vibratome (Leica VT1200S). Slices of 300‐μm thickness were transferred to inserts containing Alvetex scaffold membranes (Reinnervate) in six well plates filled with slice culture medium (BrainPhys medium, Stemcell) supplemented with B27, Glutamax (1:200), Gentamycin (50 μg/mL) (Life Technologies), and incubated in 5% CO2 at 37°C. The organotypic slices were kept in culture for at least 1 week before ex vivo transplantation of GFP+ lt‐NES cells.

Grønning Hansen M., Laterza C., Palma‐Tortosa S., Kvist G., Monni E., Tsupykov O., Tornero D., Uoshima N., Soriano J., Bengzon J., Martino G., Skibo G., Lindvall O, & Kokaia Z. (2020). Grafted human pluripotent stem cell‐derived cortical neurons integrate into adult human cortical neural circuitry. Stem Cells Translational Medicine, 9(11), 1365-1377.

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Postnatal Shank3 Hippocampal Neuron Culture

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Cultures were prepared from postnatal Shank3^Venus/Venus mice as previously described (Moutin et al., 2020 (link)). Briefly, hippocampi were mechanically and enzymatically dissociated with papain (Sigma-Aldrich, USA) and hippocampal cells were seeded in Neurobasal-A medium (Gibco, USA) supplemented with B-27, Glutamax, L-glutamine, antibiotics and Fetal Bovine Serum (all media from Gibco, ThermoFisher Scientific, USA). After 2 days in culture, cytosine β-D-arabinofuranoside hydrochloride (Sigma-Aldrich, USA) was added to curb glia proliferation. The day after, 75% of the medium was replaced by BrainPhys medium (Stemcell Technologies, Canada) supplemented with B-27, Glutamax and antibiotics.

Bouquier N., Sakkaki S., Raynaud F., Hemonnot-Girard A.L., Seube V., Compan V., Bertaso F., Perroy J, & Moutin E. (2022). The Shank3Venus/Venus knock in mouse enables isoform-specific functional studies of Shank3a. Frontiers in Neuroscience, 16, 1081010.

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Whole-cell recordings of hiPSC neurons

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For whole-cell recordings, hiPSC-derived neurons from one iPSC line (line 3) were visualized using an upright Olympus BX61 microscope equipped with a 40x objective and differential interference contrast optics. Neurons were constantly perfused with BrainPhys® medium (STEMCELL Technologies, Catalog #05790) preheated to 30-31°C. Patch electrodes were filled with internal solutions containing 130 mM K⁺gluconate, 6 mM KCl, 4 mM NaCl, 10mM Na⁺HEPES, 0.2 mM K⁺EGTA; 0.3mM GTP, 2mM Mg²⁺ATP, 0.2 mM cAMP, 10mM D-glucose. The pH and osmolarity of the internal solution were adjusted to resemble physiological conditions (pH 7.3, 290–300 mOsmol). Current- and voltage-clamp recordings were carried out using a Multiclamp 700B amplifier (Molecular Devices), digitized with Digidata 1440A digitizer and fed to pClamp 10.0 software package (Molecular Devices). For spontaneous EPSC recordings, neurons were held at chloride reversal potential of −75 mV. Data processing and analysis were performed using ClampFit 10.0 (Molecular Devices) and Prism software. CD49f⁺ astrocytes for co-cultures were from three iPSC lines. p-values to compare neurons alone to neurons with astrocytes or neurons with A0 astrocytes to neurons with A1 astrocytes were calculated using a two-tailed, unpaired t-test.

Barbar L., Jain T., Zimmer M., Kruglikov I., Sadick J., Wang M., Kalpana K., Rose I.V., Burstein S.R., Rusielewicz T., Nijsure M., Guttenplan K.A., di Domenico A., Croft G., Zhang B., Nobuta H., Hébert J.M., Liddelow S.A, & Fossati V. (2020). CD49f is a novel marker of functional and reactive human iPSC-derived astrocytes. Neuron, 107(3), 436-453.e12.

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Primary Hippocampal Neuron Culture

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All procedures involving animals were in accordance with the US National Institutes of Health Guide for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee at Harvard University.
Before the plating of primary hippocampal neurons, 14 mm glass-bottom dishes were first incubated with 40 μg/ml poly-D-lysine (PDL) in PBS at room temperature for 1 h and subsequentially with 20 μg/ml laminin (Fisher Scientific; 23-017-015) at 4 °C overnight, followed by thorough wash with PBS. Hippocampi (BrainBits; SKU: SDEHP) from embryonic day 18 (E18) rats were dissected and resuspended in Brainphys^™ medium (BPNM, Stemcell Technologies; 05790) supplemented with 2% SM1 (Stemcell Technologies; 05792), 5 mM L-Glutamine (Stemcell Technologies; 07100), and 35 μg/ml L-Glutamic Acid (Sigma Aldrich; 49449), to a final concentration of 3.0 ×10⁶ cells/ml. The neurons were then plated at a density of 30,000 cells/cm² on the pretreated glass-bottom dishes, with subsequent addition of 2 ml BPNM with 2% SM1 (BPNM/SM1). Neuronal health was monitored daily from DIV1 to DIV 7. Every 3-4 days, 1 ml of the medium in each dish was replaced with 37 °C fresh BPNM/SM1 medium.

Sievert S.M., Kuever J, & Muyzer G. (2000). Identification of 16S Ribosomal DNA-Defined Bacterial Populations at a Shallow Submarine Hydrothermal Vent near Milos Island (Greece). Applied and Environmental Microbiology, 66(7), 3102-3109.

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Hippocampal Neuron Isolation and Culture

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Hippocampal neurons were prepared from E18 mouse embryos as described (Sun et al, 2015 (link)). Briefly, hippocampi were dissected and digested with papain (2 mg/ml, Sigma) for 30 min at 37°C. Dissociated cells were plated onto poly‐L‐lysine‐coated 6‐well plates at a density of 6–10 × 10⁴ cells/cm² or confocal dishes/coverslips in 24‐well plates at a density of 6–10 × 10³ cells/cm² in Neurobasal medium (Gibco) supplemented with 2% SM1 (STEMCELL) and 2 mM glutamine and kept at 37°C under 5% CO₂. Half of the culture medium was replaced with BrainPhys medium (STEMCELL) supplemented with SM1 at DIV5 and then every 7 days.

Sun J., Liu Y., Hao X., Lin W., Su W., Chiang E., Baudry M, & Bi X. (2022). LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function. The EMBO Journal, 41(5), e108119.

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Huntington's iPSC-Derived 2D Neuronal Cultures

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2D neuronal cultures were differentiated from ND42229∗B iPSCs as described by Keskin et al.⁴¹ (link) ND42229∗B is an iPSC line derived from Huntington’s patient fibroblasts. iPSCs were cultured in poly(D) and laminin-coated 24-well plates. Neural differentiation was performed by dual SMAD inhibition according to the STEMCELL Technologies kit and yielded a mixture of neuronal and astrocytic cells. The cells were cultured in BrainPhys medium (STEMCELL Technologies, Vancouver, BC, Canada) before transduction.

Depla J.A., Sogorb-Gonzalez M., Mulder L.A., Heine V.M., Konstantinova P., van Deventer S.J., Wolthers K.C., Pajkrt D., Sridhar A, & Evers M.M. (2020). Cerebral Organoids: A Human Model for AAV Capsid Selection and Therapeutic Transgene Efficacy in the Brain. Molecular Therapy. Methods & Clinical Development, 18, 167-175.

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Isolation and Characterization of Retinal Ganglion Cells

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Single cells were harvested from 60-day normal and OPA1 c.2807_2811delTTAG mutant retinal organoids. They were resuspended with 1–2 mL Dulbecco’s PBS containing 2% fetal bovine serum and 1 mM EDTA. GFP⁺ RGCs were then enriched by FACS using the FACSAria Fusion cell sorter (BD Biosciences). Approximately 30,000 cells were plated on a poly-ornithine/laminin-coated coverslip in a well of 24-well plate, cultured in BrainPhys medium (Stem Cell Technology) for 2 weeks, and then fixed in 4% paraformaldehyde for immunocytochemistry. Images were captured with a laser scanning confocal microscope (Carl Zeiss, LSM700) and analyzed using ImageJ software.

Lei Q., Xiang K., Cheng L, & Xiang M. (2023). Human retinal organoids with an OPA1 mutation are defective in retinal ganglion cell differentiation and function. Stem Cell Reports, 19(1), 68-83.

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Culturing Hippocampal Neurons from Mice

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Cultures were prepared as recently described [30 ]. Briefly, hippocampi from P0–P3 mice were dissected and grown in neurobasal-A medium supplemented with B27 2% (Gibco), Glutamax 0.25% (Gibco), l-glutamine 0.5 mM (Gibco), fetal bovine serum 10% (Gibco), and antibiotics (penicillin and streptomycin) 1% (Gibco). After 2 days, culture was supplemented overnight with Cytosine β-D-arabinofuranoside hydrochloride (Sigma-Aldrich) 1 μM to curb glia proliferation. Then, approximately 75% of the culture medium was replaced by BrainPhys medium (Stemcell Technologies) supplemented with B27 2% (Gibco), Glutamax 0.25% (Gibco), and antibiotics (penicillin and streptomycin) 1% (Gibco).

Bouquier N., Moutin E., Tintignac L.A., Reverbel A., Jublanc E., Sinnreich M., Chastagnier Y., Averous J., Fafournoux P., Verpelli C., Boeckers T., Carnac G., Perroy J, & Ollendorff V. (2020). AIMTOR, a BRET biosensor for live imaging, reveals subcellular mTOR signaling and dysfunctions. BMC Biology, 18, 81.

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Calcium Imaging of Differentiated Neural Progenitors

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5 × 10⁵ cells were seeded in 35 mm MatTek Dishes (MatTek corporation) in the StemDiff Neural progenitor medium and differentiated for 10, 15, or 21 days in BrainPhys medium (STEMCELL Technologies) supplemented with N2, B27 supplement, BDNF, and GDNF(10 ng/μL). At the day of the acquisition, the Fluo4 calcium indicator (ThermoFisher Scientific) was incubated for 30 minutes at a final concentration of 1 μM. Cells were then washed twice for 5 minutes with the differentiation medium before acquisition.

Jefri M., Bell S., Peng H., Hettige N., Maussion G., Soubannier V., Wu H., Silveira H., Theroux J., Moquin L., Zhang X., Aouabed Z., Krishnan J., O'Leary L.A., Antonyan L., Zhang Y., McCarty V., Mechawar N., Gratton A., Schuppert A., Durcan T.M., Fon E.A, & Ernst C. (2020). Stimulation of L‐type calcium channels increases tyrosine hydroxylase and dopamine in ventral midbrain cells induced from somatic cells. Stem Cells Translational Medicine, 9(6), 697-712.

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Electrophysiological Analysis of Parkinson's Disease Neurons

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Control iCell DopaNeurons (Fujifilm Cellular Dynamics Inc.) and MyCell SNCA A53T DopaNeurons were thawed and dotted (~80k cells) onto a 48-well MEA plate (Axion Biosystems) using the iCell DopaNeuron MEA application protocol. Cultures were treated with BrainPhys medium (Stem Cell Technologies), with 50% medium changes performed every two to three days for 20 days. Five-minute recordings were made on DIV20 post-plating (N = 24). Raw voltage recordings were processed via a Butterworth (200–4 kHz) filter; action potentials were then detected by a 5.5 standard deviation detection threshold, and network-level bursting behaviors were analyzed off-line using the Axion’s NeuralMetric toolbox via the ‘Envelope’ algorithm (threshold factor 3, minimum inter-burst-interval 10 s, burst inclusion percentage 75, minimum number of electrodes percentage 25) and synchrony index (20 ms)^{48 (link)}. The activity and network-level bursting behaviors assessed included mean firing rate, network bursting rate (bursts per minute: BPM), intensity (Hz), and duration (seconds). A two-tailed student t-test was used to assess statistical differences on each measure independently between the two groups of neurons.

Stern S., Lau S., Manole A., Rosh I., Percia M.M., Ben Ezer R., Shokhirev M.N., Qiu F., Schafer S., Mansour A.A., Mangan K.P., Stern T., Ofer P., Stern Y., Diniz Mendes A.P., Djamus J., Moore L.R., Nayak R., Laufer S.H., Aicher A., Rhee A., Wong T.L., Nguyen T., Linker S.B., Winner B., Freitas B.C., Jones E., Sagi I., Bardy C., Brice A., Winkler J., Marchetto M.C, & Gage F.H. (2022). Reduced synaptic activity and dysregulated extracellular matrix pathways in midbrain neurons from Parkinson’s disease patients. NPJ Parkinson's Disease, 8, 103.

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