Encyclopedia of Experiments: Immunology
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To begin the differentiation process on day zero, add 2.25 milliliters of stage 1 media into two wells of an ultra-low attachment 6-well plate. Then, for 180% confluent well of iPSCs in a 6-well plate, replace the maintenance media with 1.5 milliliters of stage 1 media. Cut colonies using a new cell passaging tool. Using a pipette, transfer the cut colonies into the two wells of the ultra-low attachment 6-well plate.
On day 2, adjust the cytokine concentrations by adding 0.5 milliliters of hESC-SFM media to the 6-well well plate containing the day two embryoid bodies.
On day 4, coat four wells of a 6-well tissue culture plate with 0.1% gelatin. After incubating for at least 10 minutes, remove the gelatin, and add 2.5 milliliters of stage 2 media.
Collect formed embryoid bodies from the two wells of a 6-well plate, and place them in a 50-milliliter centrifuge tube. Allow them to settle at the bottom of the tube by gravity. Then, carefully aspirate the media from the tube, and resuspend the embryoid bodies in 2 milliliters of stage 2 media.
Transfer 10 to 15 embryoid bodies to a gelatin-coated well, containing 2.5 milliliters of stage 2 media, and incubate at 37 degrees Celsius with 5% carbon dioxide. For two to three weeks, change the media every three to four days.
Plating of the EBs is crucial. Having fewer than 10 or more than 15 EBs, or having them unevenly distributed, can lead to low yields of macrophages.
After two to three weeks, the embryoid bodies start releasing non-adherent hematopoietic cells into suspension. Using a 40-micrometer strainer, collect these cells into a 50-milliliter tube, and then, replenish the media. Centrifuge the suspension of hematopoietic cells at 200 x g for three minutes.
Resuspend the hematopoietic cells in stage 3 media. Then, plate the cells at a density of 0.2 x 106 per milliliter. Change the stage 3 media every five days.
After nine to 11 days the macrophages will be fully differentiated and fully functional.
