Replating Differentiated Neurons Derived from Human Pluripotent Stem Cells
Transcription
Start with a large culture plate containing human pluripotent stem cell-derived differentiated neurons with a dense neuronal network.
Incubate these neurons with mild proteolytic enzymes to detach them from the plate, lifting them as a sheet.
With continued incubation, enzymes partially disrupt the cell connections, loosening the neuronal networks.
Add a culture medium to stop the enzyme activity.
Repeatedly pipette the detached neurons to separate them from each other.
Filter the neurons through a cell strainer to remove cell clumps and collect the individual neurons.
Centrifuge and remove the supernatant. Resuspend them in a growth medium.
For replating, seed these neurons onto a polymer and an adhesion protein-coated multi-well plate. Incubate to allow their attachment.
Neurons utilize nutrients from the medium to remain viable. Replace the culture medium regularly to maintain nutrient levels.
Incubate further to allow neuronal maturation and formation of connections between them.
