For those having difficulties with primary neuronal cultures

Neuron Culture Medium

The product is a serum-free medium designed for primary culture of rat and murine neurons, and is suited for culturing cells of the central nervous system. The product contains culture supernatant of rat glial cells.

Features

Rapid production of mature neurons

Approximately 1/2 of conventional culture method

Neuron culture medium: 14 days

Conventional culture medium: approximately 1 month

Low density culture

1/5 - 1/10 the number of cells compared with conventional methods

Neuron culture medium: 0.1 ×106 cells/mL

Conventional culture medium: 0.5~1.0×106 cells/mL

Ready-to-Use

Cells can be cultured with the culture medium alone, with no need for additional supplements.

Confirming dendrite outgrowth

MAP2 immunostaining

Neuron culture medium alone

00821_img01.png

Competitor’s medium + supplements

00821_img02.png

Experimental conditions

Number of cells: 0.1×106 cells/mL(isolated from the hippocampus of a mouse at embryonic days 18.5)
Seeding density: 500 μL/well(polylysine-coated glass bottom dish)
Culture condition: Change half of the culture medium at days 3 and 7 (without the addition of Ara-C)

Data source

Dr. Hirotaka James Okano and Mr. Yuki Ogawa, Division of Regenerative Medicine, Jikei University School of Medicine

▶The data suggested that neurons grown in our culture medium had a faster rate of dendrite outgrowth compared with neurons cultured in the competitor’s medium.

Confirming dendrite outgrowth and cell viability

MAP2 and Hoechst immunostainings

Day 14

00821_img03.png

Experimental conditions

Number of cells: 0.1×106 cells/mL(isolated from the hippocampus of a mouse at embryonic days 18.5)

Seeding density: 500 μL/well (polylysine-coated glass bottom dish)

Culture condition: Change half of the culture medium at days 3 and 7 (without the addition of Ara-C)

Data source

Dr. Hirotaka James Okano and Mr. Yuki Ogawa, Division of Regenerative Medicine, Jikei University School of Medicine

▶The data suggested that neurons grown in our culture medium had a faster rate of dendrite outgrowth and were more viable compared with neurons cultured in the competitor’s medium.

Evaluating the maturity of neurons

Examination of dendritic spines

Day 14

00821_img04.png

Experimental conditions

Number of cells: 0.1×106 cells/mL (isolated from the hippocampus of a mouse at embryonic days 18.5)

Seeding density: 500 μL/well (polylysine-coated glass bottom dish)

Culture condition: Change half of the culture medium at days 3 and 7 (without the addition of Ara-C)

Data source

Dr. Hirotaka James Okano and Mr. Yuki Ogawa, Division of Regenerative Medicine, Jikei University School of Medicine

▶The data revealed the presence of dendritic spines, which indicate maturity of neurons, on neurons cultured in our medium on day 14.

Confirming the maturity of neurons

Examination of dendrites and axons

Day 14

00821_img05.png

MAP2:Marker for dendrites
AnkyrinG:Marker for axon initial segment

Experimental conditions

Number of cells: 0.1×106 cells/mL(isolated from the hippocampus of a mouse at embryonic days 18.5)

Seeding density: 500 μL/well (polylysine-coated glass bottom dish)

Culture condition: Change half of the culture medium at days 3 and 7 (without the addition of Ara-C)

Data source

Dr. Hirotaka James Okano and Mr. Yuki Ogawa, Division of Regenerative Medicine, Jikei University School of Medicine

▶The data demonstrated that neurons cultured in our medium had multiple dendrites and one axon that are extended from the cell body, suggesting that they have properly matured.

Culturing murine Purkinje cells

Calbindin immunostaining

Day 14

00821_img06.png

Experimental conditions

Number of cells: 0.1×106 cells/well(isolated from the hippocampus of a mouse at embryonic days 18.5)

Seeding density: 500 μL/well (polylysine-coated glass bottom dish)

Culture condition: Final concentration at 1nM, supplemented with triiodothyronine

Data source

Dr. Hirotaka James Okano and Mr. Yuki Ogawa, Division of Regenerative Medicine, Jikei University School of Medicine

▶Purkinje cells cultured in 1 nM of our culture medium supplemented with triiodothyronine stained positive for calbindin and had dendritic development.

Confirming the maturity of human iPS-derived neurons

Immunostaining with MAP2, NeuN, and Hoechst

Suspension culture of cells after directed dopaminergic neuron differentiation at day 14 (42 days from directed differentiation)

00821_img07.png

Data source

Dr. Hirotaka James Okano and Ms. Keiko Bouno, Division of Regenerative Medicine, Jikei University School of Medicine

▶The data demonstrated that iPS-derived neurons cultured in our medium stained positive for NeuN, suggesting that they are mature neurons.

Immunostaining with MAP2 and TH

Suspension culture of cells after directed dopaminergic neuron differentiation at day 14 (42 days from directed differentiation)

00821_img08.png

Data source

Dr. Hirotaka James Okano and Ms. Keiko Bouno, Division of Regenerative Medicine, Jikei University School of Medicine

▶The data demonstrated improved viability of TH-positive cells derived from directed dopaminergic neuron differentiation when maintained in our culture medium.

Examining the physiological function of human iPS-derived neurons

Ca2+ imaging

Suspension culture of cells after directed dopaminergic neuron differentiation at day 14 (42 days from directed differentiation)

Endogenous fluorescence

Human iPS-derived neurons

00821_img09.png

Fluorescence with the addition of ionomycin

Human iPS-derived neurons

00821_img10.png

Data source

Dr. Hirotaka James Okano and Ms. Keiko Bouno, Division of Regenerative Medicine, Jikei University School of Medicine

▶Ca2+ imaging revealed that some of the human iPS-derived neurons cultured in our medium had independent physiological functions.

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