Superior neuronal cell cultures

Gibco™ CultureOne™ Supplement may be added to any conventional neuronal differentiation medium to eliminate more than 75% of contaminating neural progenitor cells—resulting in superior neuronal cell cultures of evenly-distributed, differentiated neurons, enabling improved downstream assays, accelerated neuronal maturation, and seamless maintenance for 5 weeks or more.

Example dataSimple protocolOrdering information

See how CultureOne Supplement improves your neuronal cultures

Get reduced neural progenitor cell contamination, accelerated neuronal maturation, and weeks of reliable maintenance. This video compares the clear difference with and without the use of CultureOne Supplement over 5 weeks in culture.

Neuronal cultures can be maintained for 5+ weeks

CultureOne Supplement was developed to help ensure differentiated neurons from human pluripotent stem cell (hPSC)–derived neural stem cells (NSCs) have maximum utility in downstream applications.

Neuronal cell cultures 5+ weeks
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Example data

CultureOne Supplement enables improved imaging, RNA expression, and electrophysiology assays. After 2 weeks of differentiation with CultureOne Supplement, images show evenly distributed differentiated neurons (MAP2+) with greater than 75% reduction in NSCs (SOX1+) and cell clumps compared to conventional differentiation methods (A). These cells had increased neuronal mRNA expression, reduced NSC mRNA expression (B), and exhibited higher spike rates as measured by multi-electrode array (MEA).

A. Imaging of differentiation at 2 weeks.

2 week conventional differentiation chevron diagram
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Conventional differentiation
2 week CultureOne differentiation chevron diagram
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CultureOne Supplement

B. mRNA expression at 2 weeks by qPCR.

B. mRNA expression at 2 weeks by qPCR
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Neurons differentiated from NSCs with CultureOne Supplement showed an increase in cytosolic calcium when depolarized with KCl, meaning they express significantly higher numbers of voltage-gated calcium ion channels, an important marker for neural maturity and excitability. This, along with longer neurite outgrowth after 2 weeks of differentiation, demonstrates that CultureOne Supplement accelerates neuronal maturation.

Fluo-4 calcium response to 30 mK KCI
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Neuronal cell cultures differentiated with CultureOne Supplement were compared to anti-mitotic enrichment methods including Ara-C and mitomycin C. CultureOne Supplement–treated cultures exhibited improved neurite outgrowth and reduced cell clumping and death.

Neuronal cell cultures differentiated with CultureOne Supplement reduced cell clumping and death
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Mechanism of action of CultureOne Supplement

How CultureOne Supplement Works
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Simple, scalable, neuronal cell culture protocol

Neuronal cell culture workflow days 0 to 14
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Typical preparation for neuronal differentiation medium (based on 100 mL final volume)

Reagent Cat. No. Volume
Gibco™ Neurobasal™ Medium 21103049 96 mL
Gibco™ B-27™ Supplement (50x) 17504044 2 mL
Gibco™ GlutaMAX™ Supplement (100x) 35050061 1 mL
Gibco™ CultureOne™ Supplement (100x) A3320201 1 mL
Ascorbic acid (200 mM, e.g., Sigma-Aldrich™ Cat. No. A8960) NA 100 µL
Glial cell-derived neurotrophic factor (GDNF) at 10-20 ng/mL and brain-derived neurotrophic factor (BDNF) at 10-20 ng/mL can be added into NDMC to improve neuron survival depending on NSC lines

Volumes of NDMC (neuronal differentiation medium with CultureOne Supplement) required for a typical two-week NSC differentiation regimen.

Poly-D-lysine– and laminin-coated culture plates (# of wells) Well surface area (cm2) Recommended
NSCs per well (5 x 104 cells/cm2)
Concentration of NSCs in NDMC (cells/mL) Plating volume of NSC/NDMC suspension per well (mL) NDMC feed volume per well (mL) Total NDMC feeds per well (feed every 2–3 days) Total volume of NDMC per plate (mL) Plates per 500 mL of NDMC (equal to 1 unit CultureOne Supplement)
96 0.32 16,000 160 0.1 0.1 5 57.6 8
48 0.95 47,500 238 0.2 0.2 5 57.6 8
24 1.90 95,000 190 0.5 0.5 5 72.0 6
12 3.80 190,000 190 1.0 1.0 5 72.0 6
6 9.60 480,000 240 2.0 2.0 5 72.0 6

Resulting cell yields with CultureOne Supplement

In our labs, 16,000 H9 ESC-derived NSCs plated in each well of a 96-well plate and differentiated with CultureOne Supplement for 2 weeks typically yield an enriched culture of approximately 9,000 differentiated neurons, or ~60% of the NSC starting numbers. Note that the yield may vary depending on the specific NSC line.

We offer CultureOne Supplement as a stand-alone reagent which can be easily paired with the Gibco™ Neural Cell Culture Starter Kit for simple, scalable, and superior neuronal cell cultures.

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