Isolation and culture of kidney tumor organoids

Cells were isolated from the original tissue instantly after surgery
by dissociating the tissue into small pieces and treating it with
collagenase (40 units/mL) for 2–4 h. Benign and cancer cells
of the kidney of a clear cell renal cell carcinoma patient cells were
grown as organoids in F-medium [3:1 (v/v) of F-12 nutrient mixture
(Ham)–DMEM (Invitrogen), 5% FBS, 8.4 ng/mL cholera toxin (Sigma),
0.4 μg/mL hydrocortisone (Sigma), 10 ng/mL epidermal growth
factor (Corning), 24 μg/mL adenine (Sigma), 5 μg/mL insulin
(Sigma), 10 μM ROCK inhibitor (Y-27632, Enzo Life Sciences,
Lausen, Switzerland), and 1% penicillin-streptomycin with 10% Matrigel
(Corning). The bladder tumor-derived organoids were grown in hepatocyte
calcium medium (Corning)^{49 (link)} supplemented
with 5% CSFBS (Thermo Fisher Scientific), 10 μM Y-27632 RHO
inhibitor (Sigma), 10 ng/mL epidermal growth factor (Corning), 1%
GlutaMAX (Gibco), 1% penicillin-streptomycin, and 10% Matrigel. A
total of 6 × 10⁶ cells were collected by centrifugation,
washed in PBS to remove Matrigel, and snap frozen before analyses.