Isolation of Fetal Kidney Cells

All research using human fetal tissue was approved by the University of Michigan institutional review board. Normal human fetal kidneys at 87, 105, 110, 115 and 132 days of gestation were obtained from the University of Washington Laboratory of Developmental Biology. Gestation age is estimated based on the date of the last period. All tissues were shipped overnight in Belzer's solution at 4°C and were processed immediately upon arrival to the laboratory. Single-cell dissociation was performed using a cold active protease, as described recently (Adam et al., 2017 (link)). The embryonic kidneys were decapsulated and cut in half. All tissue samples collected for digestion spanned from the cortical nephrogenic zone to the inner medulla, and were dissected in ice-cold PBS and finely minced in a petri dish on ice using razor blades. About 20 mg of tissue were added to 1 ml of ice-cold active protease solution [PBS, 10 mg of Bacillus Licheniformis protease (Sigma, #P5380), 5 mM CaCl₂, 20 U DNAse I (Roche, #4716728001)]. The tissue was incubated in a 2 ml reaction tube for 15-20 min on a slow-moving shaker (nutator) in a coldroom at 4°C with repeated trituration steps for 20 s every 5 min. Single-cell dissociation was confirmed with a microscope. The dissociation was stopped with 1 ml ice-cold PBS supplemented with 10% fetal bovine serum (FBS). Afterwards, the cells were immediately pelleted at 300 g for 5 min at 4°C. Subsequently, the supernatant was discarded and cells were suspended in 2 ml PBS/10% FBS and pelleted again at 300 g for 5 min at 4°C. Then cells were suspended in PBS/0.01% BSA and pelleted again (300 g for 5 min at 4°C), suspended in 1 ml PBS/0.01%BSA and passed through a 30 µM filter mesh (Miltenyi MACS smart strainer). Viability was then investigated with the Trypan-blue exclusion test and cell concentration was determined using a hemocytometer and adjusted to 200,000 cells/ml for the Drop-seq procedure.