Murine Renal Cell Isolation: A Technique to Isolate and Generate Primary Culture of Renal Tubular Epithelial Cells

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Tissue Processing and Primary Tubular Cells Isolation

1. Remove the renal capsules and medulla, mince both kidneys into tiny pieces, and incubate them in 10 mL of a digestion buffer in a 37 °C oven with gentle rotation for 5 min.
2. Remove any undigested kidney tissues by passing the buffer through a 70 μm filter. Add 10 mL of culture media to stop the digestion.
3. To collect tubular cells, centrifuge the filtered cell suspension at 50 x g for 5 min to collect the first pellet. Transfer the supernatant to a new tube and add 5 mL of culture media, centrifuge it at 50 x g for 5 min to ensure all tubular cells are collected into the second pellet. The centrifugation is at a lower speed to primarily pellet heavy tubules. Later, after the cells recover from the isolation, the pure tubular culture is centrifuged at a higher speed during the sub-cultures.
4. Resuspend the first pellet in 20 mL of culture media and centrifuge it at 50 x g for 5 min to collect the third pellet.
5. Resuspend the second and third pellets in 1 mL of culture media. Mix 10 µL of the cell suspension with 10 µL of Trypan Blue, load the mixture into chamber A of a counting slide, and record the cell viability from the automatic cell counter.
6. Seed up to 10⁷ cells (a heterogeneous population) onto a single 60 mm dish pre-coated with collagen and let the tubular cells attach overnight.