Encyclopedia of Experiments: Cancer Research
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Transcript
After counting, resuspend the cells to a 1 times 10 to the fifth cells per milliliter of mesoderm induction medium concentration and aspirate the extracellular matrix solution from the basement membrane matrix 2-coated plates. Rinse the plates two times with warm medium and mix the human-induced pluripotent stem cell suspension with gentle pipetting.
Add 1 milliliter of cells to each well of the basement membrane matrix 2-coated 12-well plates and gently shake the plates to distribute the cells more evenly. Then, place the plate into the cell culture incubator. On days 2 to 15 of the differentiation, replace the mesoderm induction medium with 1 milliliter of intermediate mesoderm induction medium per well.
If substantial cell growth and a rapid depletion of nutrients is observed as indicated by yellowing of the medium, the volume of the intermediate mesoderm differentiation medium can be increased to 1.3 milliliters per well. On day 16 of culture, rinse the intermediate mesoderm cells with warm medium and incubate the cells with 500 microliters of 0.05% trypsin-EDTA per well for 3 minutes at 37 degrees Celsius.
When the cells begin to dissociate, scrape the cells with a cell lifter and gently mix the cells by pipetting. Stop the reaction with about 2 milliliters of trypsin neutralizing solution per well and transfer the cells to a 50-milliliter conical tube. Bring the volume up to 50 milliliters with medium and collect the cells by centrifugation.
Resuspend the pellet and podocyte induction medium at a 1 times 10 to the fifth cells per milliliter of medium concentration and add the cells to basement membrane matrix 2-coated plates. Then, gently shake the plate to help distribute the cells more evenly and place the cells into the incubator for up to 5 days.
