Work with iPS and ES cells was conducted under the approval and auspices of the University of Washington Embryonic Stem Cell Research Oversight Committee. Specific cell lines used in this study are described below and were sourced from commercially available iPS and ES cell lines obtained with informed consent. Stem cell stocks were maintained in mTeSR1 media with daily media changes and passaging using Accutase (STEMCELL Technologies). One thousand to six thousand cells per well were placed in each 24-well plate precoated with 300 μL of DMEM-F12 containing 0.2 mg/mL Matrigel and sandwiched the following day with 0.2 mg/mL Matrigel in mTeSR1 (STEMCELL Technologies) to produce scattered, isolated spheroid colonies. Forty-eight hours after sandwiching, spheroids were treated with 12 μM CHIR99021 (Tocris Bioscience) for 36 hours, then changed to RB (Advanced RPMI + 1× Glutamax + 1× B27 Supplement, all from Thermo Fisher Scientific) and replaced every 3 days thereafter. Organoids were differentiated for 21 days from the time of plating, at which time tubular structures had formed. Gene-edited PKD2−/− organoids were picked from the adherent plate at day 21, placed in suspension culture with RB media replaced every 3 days until day 30 when cyst growth was prominent (37 (link)).
Advanced rpmi 1 glutamax 1 b27 supplement
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Advanced RPMI + 1× Glutamax + 1× B27 Supplement is a cell culture medium designed to support the growth and maintenance of a variety of cell types. It contains the basal RPMI formulation supplemented with Glutamax and B27 Supplement, which provide essential nutrients and growth factors to cells.
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3 protocols using advanced rpmi 1 glutamax 1 b27 supplement
1
Kidney Organoid Differentiation Protocol
2
Generating PKD2-Deficient Organoids from iPSCs
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hPSC stocks were maintained in mTeSR1 medium with daily medium changes and weekly passaging using Accutase or ReLeSR (STEMCELL Technologies, Vancouver). For differentiation into organoids, iPSCs (WTC-11 cell line, Coriell # GM25256) bearing knockout mutations in PKD2 were plated at 2,000 cells/well in 24-well plates, or 200 cells/well in 384-well plates, pre-coated with 300 μl of DMEM-F12 containing 0.2 mg/ml Matrigel and sandwiched the following day with 0.2 mg/ml Matrigel in 500 μl of mTeSR1 (STEMCELL Technologies, Vancouver) to produce scattered, isolated spheroid colonies. Coating and plating of 384-well plates was performed using a Matrix Wellmate liquid handling robot. 48 h after sandwiching, hPSC-derived spheroids were treated with 12 μM CHIR99021 (Tocris Bioscience) for 36 h in 1,000 μl of Advanced RPMI + 1× Glutamax + Pen-strep (all from Thermo Fisher Scientific), then changed to RB (Advanced RPMI + 1× Glutamax + 1× B27 Supplement, all from Thermo Fisher Scientific). For full details see Supplementary Methods .
3
Generating PKD2-Deficient Organoids from iPSCs
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