To begin, obtain the human embryonic stem cells in the stem cell medium with a cell density of 80 to 90%After removing the spent medium, rinse the cells in each well with one milliliter of 1X DPBS. Incubate the stem cell colonies in each well with one milliliter of 0.5 millimolar EDTA for approximately five minutes at 37 degrees Celsius. Check the colony morphology to confirm that the edges are slightly curled up with loose gaps.
Aspirate the EDTA, and if necessary, incubate cells at 37 degrees Celsius for another one to two minutes, but not exceeding eight minutes. Gently rinse the cells in each well with six milliliters of medium A containing six microliters of 10 millimolar ROCK inhibitor Y-27632 solution. Gently tap the bottom of the dish to help rinse the cells.
After 24 hours, observe the cells under the microscope to confirm the embryo body formation. Using a 10 milliliter pipette, collect the embryo bodies into a 15 milliliter centrifugal tube and wait for the embryo bodies to settle to the bottom for five minutes. Then carefully aspirate the supernatant and resuspend the embryo bodies in six milliliters of fresh medium A.Transfer them into a new low adhesion well of a six-well plate and culture in a 37 degrees Celsius incubator.
On day four, coat a 10 centimeter dish with three milliliters of 0.1%fish gelatin solution and incubate for at least one hour in a 5%carbon dioxide, 37 degrees Celsius incubator. Then remove the gelatin solution and rinse the dish once with five to six milliliters of 1X DPBS. Carefully collect the embryo bodies into a 15 milliliter centrifugal tube and wait for the embryo bodies to settle to the bottom In five minutes.
Gently resuspend the embryo bodies with 15 milliliters of medium B and transfer them to the coated 10 centimeter dish. Culture in a 37 degrees Celsius, 5%carbon dioxide incubator for one week after 49 days. Centrifuge the cells at 200 x g for five minutes at room temperature.
Carefully aspirate the supernatant and resuspend the cells with two milliliters of fresh medium C.Transfer into a new low adhesion well of a six-well plate. Incubate the plate in a 5%carbon dioxide, 37 degrees Celsius incubator. On day 56, replace the spent medium with two milliliters of medium C per well.
Examine the plate under a microscope to identify the branched microglia adhere to the bottom of the low adhesion well. To harvest microglia for co-culturing, gently replace the medium C in each well with one milliliter Accutase and incubate at 37 degrees Celsius for three minutes. Using a five milliliter pipette, collect the microglia cells into a 15 milliliter tube and centrifuge the cells at 200 x g for five minutes at room temperature.
After discarding the supernatant, suspend the microglia with one milliliter of medium E.
