This article outlines a method for differentiating neuroepithelial progenitor cells into mature brain organoids using neurospheres and an extracellular matrix. The process involves embedding neurospheres in a matrix, incubating them, and transferring them to a spinner flask for enhanced growth.
Begin with a culture of neurospheres, free-floating clusters of neuroepithelial progenitor cells.
Transfer these neurospheres onto an embedding sheet.
Remove media. Add a drop of extracellular membrane matrix containing proteins and growth factors to each neurosphere.
Incubate for the matrix to solidify, embedding the neurospheres within it.
Now, transfer the matrix-embedded neurospheres using neurosphere media to a culture plate containing neurosphere media.
Incubate. The extracellular membrane matrix and its constituents facilitate neuroepithelial progenitor cell differentiation into neuroepithelial cells which proliferate and form fluid-filled lumens.
Post-incubation, transfer neurospheres into a spinner flask containing pre-warmed brain organoid media. Incubate with stirring for enhanced oxygen and nutrient diffusion, promoting cellular growth.
Growth factors and small molecule inhibitors in media promote neural differentiation and develop into cells of specific brain regions, forming mature brain organoids.
To begin this procedure, collect the neurospheres with a 200-microliter micropipette, using a 2-millimeter tip previously cut with sterile scissors. Next, place the neurospheres approximately 5 millimeters away from each other on a paraffin film in a 100-millimeter dish, and carefully remove as much of the remaining medium as possible. Then, add a drop of EHS matrix onto each single neurosphere.
Incubate the EHS matrix drops with the neurospheres for 15 minutes at 37 degrees Celsius. After that, wash the neurospheres carefully from the paraffin film by flushing them with neurosphere medium. Subsequently, incubate the neurospheres for the next four days, and add 2 milliliters of fresh neurosphere medium on day two.
In this procedure, add 100 milliliters of brain organoid medium to each spinner flask through its side arms and pre-warm the medium in the incubator at 37 degrees Celsius for at least 20 minutes. Then make sure the neurospheres are all separated. If two or more are connected through EHS matrix, separate them by cutting the connecting matrix with a scalpel.
Set up a stirring program at 25 RPM. Carefully transfer the EHS matrix-embedded neurospheres into the spinner flasks containing 100 milliliters of organoid medium. Following that, place the spinner flasks on a magnetic stirring platform in the incubator for 14 days at 37 degrees Celsius. This is day zero of the organoid culture. Change the medium once a week by removing half of the medium and adding the same amount of fresh medium.
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