An In Vitro Method to Generate Human Brain Organoids
Transcrição
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.
