This article describes a method for differentiating mouse embryonic carcinoma cells into neuronal progenitor cells and subsequently into mature neurons. The process involves the use of retinoic acid and hydrophobic culture surfaces to promote cell aggregation and differentiation.
Take a mouse embryonic carcinoma cell suspension capable of differentiating into various cell types.
Seed cells on a hydrophobic culture dish containing media and retinoic acid.
Incubate. Hydrophobic surfaces prevent cell adhesion, causing cells to form aggregates.
Retinoic acid enters cell aggregates, triggering signaling pathways and causing differentiation into neuronal progenitor cells.
Post-incubation, collect the cell aggregates.
Discard the media.
Add media containing retinoic acid and seed the aggregates onto a hydrophobic culture dish.
Incubate. Retinoic acid facilitates sustained cellular differentiation into neurons.
Collect the aggregates and remove the media.
Add a proteolytic enzyme to disrupt cell-cell adhesions within the aggregates.
Add media containing serum to stop the enzymatic activity.
Mechanically dissociate the aggregates, forming a neuronal suspension.
Centrifuge and discard the enzyme-rich supernatant.
Resuspend the neurons in media and seed them on an adherent multi-well plate containing media.
Incubate. Neurons adhere to the plate and mature, extending long projections.
For aggregate generation, start by adding 10 milliliters of differentiation medium supplemented with 5 microliters of RA to a 100-milliliter non-treated culture dish. Seed 1 million cells in the culture dish, and incubate at 37 degrees Celsius and 5% carbon dioxide for two days in order to promote aggregate formation.
After the incubation, with a 10-milliliter pipette, aspirate the medium containing the aggregates and transfer it to a 15-milliliter tube at room temperature. Wait for 1.5 minutes for the aggregates to settle at the bottom, and then, discard the supernatant.
Add 10 milliliters of fresh differentiation medium supplemented with 0.5 micromolar RA. Gently seed the aggregates in a new 100-millimeter non-treated culture dish, and incubate at 37 degrees Celsius and 5% carbon dioxide for two days.
To begin, use a 10-milliliter pipette to transfer the cell aggregates to a 15-milliliter tube. Wait for 1.5 minutes at room temperature for the aggregates to settle at the bottom, and then discard the supernatant. Wash the aggregates with serum and antibiotic-free DMEM. Wait for cell aggregates to settle at the bottom. Discard the supernatant, and add 2 milliliters of 0.25% trypsin EDTA.
Incubate the tube in a water bath at 37 degrees Celsius for 10 minutes, tapping every two minutes to keep the cells in suspension. Then, add 4 milliliters of maintenance medium in order to stop the trypsin activity, and pipette up and down 20 times using a 1-milliliter pipette.
Finally, centrifuge the cells at 200 times g and room temperature for five minutes. Remove the supernatant and re-suspend the cell pellet in 5 milliliters of maintenance medium. Use a cell counter to determine the cell number and proceed with plating the cells. To plate cells, first, add 3 milliliters per well of maintenance medium to a six-well culture plate. Then, seed the cells at a density of 500,000 per well. Incubate the plate at 37 degrees Celsius and 5% carbon dioxide.
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