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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
- 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.
- Remove any undigested kidney tissues by passing the buffer through a 70 μm filter. Add 10 mL of culture media to stop the digestion.
- 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.
- 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.
- 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.
- 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.