Generating Postganglionic Sympathetic Neurons from Human Pluripotent Stem Cells
Trascrizione
One day before differentiation induction, coat the appropriate number of six-well plates with two milliliters of basement membrane matrix per well, and store the plates at four degrees Celsius overnight. The next morning, warm the plates to room temperature, and wash the ready-to-split human pluripotent stem cell culture two times with fresh PBS per wash.
After the second wash, treat the cells with seven milliliters of 0.5 millimolar EDTA for 15 minutes in the cell culture incubator, before aspirating the floating human pluripotent stem cells and transferring the harvested cells into a 50-milliliter tube.
Add an equal volume of PBS to the tube to dilute the EDTA, and collect the cells by centrifugation. Resuspend the pellet in one milliliter of day 01 differentiation medium. Before adding an appropriate volume of medium for counting, dilute the cells to a 1.25 x 105 cells per square centimeter concentration in two milliliters of differentiation medium per well.
Aspirate all of the basement membrane matrix solution from each well of the previously prepared six-well plate. Then, seed 2 milliliters of cells into each well, and place the plate in the cell culture incubator. The next morning, feed the cells with 3 milliliters of day 01 differentiation medium per well.
On day two of differentiation, feed the cells with three milliliters of day two to ten differentiation medium per well. Then, feed the cells every other day until day 10. To aggregate the neural crest cells into spheroids, on day 10, wash the cells with PBS, and add two milliliters of dissociation medium to each well. After 20 minutes in the cell culture incubator, transfer the floating cells into a 50-milliliter conical tube, and bring the volume of suspension in the tube to 50 milliliters with fresh PBS.
Collect the cells by centrifugation, and resuspend the pellet in a sufficient volume of day 10 to 14 spheroid medium for counting. After counting, dilute the cells to a 5 x 105 cells per 500 microliters concentration using day 10 to 14 spheroid medium, and plate 500 microliters of cells into each well of an ultra-low attachment 24-well plate. Then return the cells to the cell culture incubator.
To induce a minimal spheroid culture, on day 11 of culture, feed the neural crest spheroids with an additional 500 microliters of day 10 to 14 spheroid medium per well. On day 12, tilt the plate to accumulate the spheroids on one side of the plate, and carefully aspirate as much medium as possible from each well without aspirating spheroids. Then, feed the spheroids with one milliliter of day 10 to 14 spheroid medium per well.
To induce an expanded spheroid culture on day 15, feed the spheroids with 1.5 milliliters of day 10 to 14 spheroid medium supplemented with 0.5 micromolar retinoic acid per well. Then, return the spheroids to the cell culture incubator.
For sympathetic neuron differentiation on day 14 or 28, tilt the plate to accumulate the spheroids on one side of the plate, and carefully aspirate as much medium as possible. Feed the cells with one milliliter of sympathetic neuron medium.
To break up big spheroid aggregates into smaller spheroids, add no more than one milliliter of medium per well, and pipette the spheroids five to 10 times before splitting.
And remove the excess laminin fibronectin from previously prepared 24-well plates. Split the plate one milliliter of spheroids from each well on the 24-well spheroid culture plate between four separate wells of the newly coated 24-well plate. Add 250 microliters of fresh sympathetic neuron medium to each well, and place the plate in the cell culture incubator. The next morning, replace the medium in each well with one milliliter of sympathetic neuron medium supplemented with 0.125 micromolar retinoic acid, and return the plate to the cell culture incubator. After day 35, feed the neurons by carefully replacing only half of the existing medium in each well with fresh medium.
