Generation of Spheroids from Human Pluripotent Stem Cells

To begin, plate the hPSC colonies on an hESC-qualified basement membrane matrix from one well of a 6-well plate at 60% confluency into three wells to achieve 20% to 30% density, and then, proceed with induction of 2D neuroectodermal colonies.

Add fresh N2 media supplemented with dual SMAD inhibitors daily to each well for the next 3 days. For generating 3D neuroectodermal spheroids from induced 2D neuroectodermal colonies, remove 2 milliliters of N2 medium from the 6-well plate, and wash one time with HBSS to ensure that all of the N2 medium is removed.

Add 1 milliliter of dispase to each colony-containing well. Incubate the plate for 20 to 25 minutes at 37 degrees Celsius, and check for colony detachment regularly. At the end of the incubation, to stop the activity of the dispase enzyme, add 1 milliliter of N2 medium to the well. Using a wide-bore P1000 pipette tip or a modified P1000 pipette tip cup with sterile scissors, transfer the colonies into a 15-milliliter tube, and allow the colony clumps to sink to the bottom of the tube with gravity.

Then, with a standard P1000 pipette tip, carefully remove the supernatant. Replace it with 1 milliliter of fresh N2 medium, and repeat washing thrice to ensure complete removal of dispase. After resuspending the cell clumps and N2 medium, transfer the cell suspension to one well of a 6-well plate, and add 40 nanograms per milliliter of bFGF.