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.

 

For primary neuron cultures, CultureOne Supplement reduces the number of proliferating glial progenitors which can overgrow post-mitotic neurons. It can also be used to control the expression of glia through modifying timing of addition.

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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.

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.

B. mRNA expression at 2 weeks by qPCR.

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.

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.

Figure 1. CultureOne Supplement controls astrocyte outgrowth and proliferation in neuronal cultures. (A) Primary rat cortical neurons and (B) mouse cortical neurons were cultured for 3 weeks In B-27 Plus Neuronal Culture System complete medium. Cells were treated with CultureOne Supplement at the time of plating (DO) or treatment was delayed to day 2 (D2), day 4 (D4), day 6 (D6), and day a (DB). Cells were fixed at day 21 and stained (images, A and B) with astrocyte-specific GFAP antibody (1:100, red). Nuclei were counterstained with DAPI (blue). Images were captured on the Thermo Scientific CellInsight CX5 HCS Platform. Thermo Scientific HCS Studio Cell Analysis Software was used to quantify the relative fluorescence from GFAP-labeled cells (graphs, A and B). The data represent the average GFAP fluorescence per field from 3 wells; 15 fields were captured per well. Data are reported as the mean ±SEM. Similar results were observed with mouse hippocampal neurons (data not shown).

No effect on number or morphology of neurons.

Figure 2. No effect on number or morphology of neurons. Rat cortical neurons or mouse cortical neurons were plated on poly-d-lysine-coated plates and cultured in the B-27 Plus Neuronal Culture System. (A) Cells were treated with CultureOne Supplement at the time of plating, DO, or the onset of treatment was delayed to D2, D4, D6, and D8. (B) Rat cortical neurons were fixed at day 21 and neurons were stained with MAP2 (green) and HuC/HuD (red) antibodies. HCS Studio Cell Analysis Software was used to quantify the number of neurons and number of HuC/HuD-positive cells. Nuclei were labeled with DAPI (blue). The data represent the average number of HuC/HuD-positive cells per field from 3 wells; 6 fields were captured per well. Data are reported as the mean ±SD. Similar results were observed with mouse cortical and hippocampal neurons (data not shown).

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.

B. mRNA expression at 2 weeks by qPCR.

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.

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.

Figure 1. CultureOne Supplement controls astrocyte outgrowth and proliferation in neuronal cultures. (A) Primary rat cortical neurons and (B) mouse cortical neurons were cultured for 3 weeks In B-27 Plus Neuronal Culture System complete medium. Cells were treated with CultureOne Supplement at the time of plating (DO) or treatment was delayed to day 2 (D2), day 4 (D4), day 6 (D6), and day a (DB). Cells were fixed at day 21 and stained (images, A and B) with astrocyte-specific GFAP antibody (1:100, red). Nuclei were counterstained with DAPI (blue). Images were captured on the Thermo Scientific CellInsight CX5 HCS Platform. Thermo Scientific HCS Studio Cell Analysis Software was used to quantify the relative fluorescence from GFAP-labeled cells (graphs, A and B). The data represent the average GFAP fluorescence per field from 3 wells; 15 fields were captured per well. Data are reported as the mean ±SEM. Similar results were observed with mouse hippocampal neurons (data not shown).

No effect on number or morphology of neurons.

Figure 2. No effect on number or morphology of neurons. Rat cortical neurons or mouse cortical neurons were plated on poly-d-lysine-coated plates and cultured in the B-27 Plus Neuronal Culture System. (A) Cells were treated with CultureOne Supplement at the time of plating, DO, or the onset of treatment was delayed to D2, D4, D6, and D8. (B) Rat cortical neurons were fixed at day 21 and neurons were stained with MAP2 (green) and HuC/HuD (red) antibodies. HCS Studio Cell Analysis Software was used to quantify the number of neurons and number of HuC/HuD-positive cells. Nuclei were labeled with DAPI (blue). The data represent the average number of HuC/HuD-positive cells per field from 3 wells; 6 fields were captured per well. Data are reported as the mean ±SD. Similar results were observed with mouse cortical and hippocampal neurons (data not shown).

Mechanism of action of CultureOne Supplement

Simple, scalable, neuronal cell culture protocol

Typical preparation for neuronal differentiation medium (based on 100 mL final volume)

Reagent Cat. No. Volume
Gibco Neurobasal Plus Medium A3582901 96 mL
Gibco B-27 Plus Supplement (50x)
A3582801
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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