Isolation of papillary cells

                                   Isolation of papillary cells    

Isolation of renal papillary cells
1. For isolation of papillary cells, kidneys were harvested and kept in HBSS containing 15 mM HEPES, penicillin/streptomycin, and 0.35 g/l NaHCO^₃, pH 7.4, at 4°C.
2. After removal of the perinephric fat, the kidneys were sectioned along the anterior-posterior axis, ensuring that the papilla was left intact and attached to one of the two half-kidneys.
3. After isolation of the papilla, the tissue was minced and digested with 2 mg/ml collagenase I while being shaken at 175 rpm in a 37°C shaker.
4. Thereafter, with a spatula, the tissue was forced through sequential 106-μm and 20-μm steel filters.
5. The dispersed cells were then collected by centrifugation.
Cell culture.
1. For tissue culture, the collected cells were dispersed with DMEM/Ham F12 containing 10% FCS, 5% chicken embryo extract, and 5% rat serum, as well as 2 mM glutamine, penicillin, and streptomycin.
2. Except where indicated otherwise, all cultures were maintained at 37°C in an atmosphere of 5% CO^₂ and 95% room air.
3. When cells were grown in serum-free media, the culture media consisted of DMEM/Ham F12 containing glutamine and antibiotics plus 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml sodium selenite, 20 ng/ml dexamethasone, 20 ng/ml l-thyroxine, 50 ng/ml bFGF, 100 ng/ml PDGF, and 20 ng/ml EGF.
4. Cells were grown either in plastic dishes or in dishes coated with 5 μg/cm2 fibronectin.
5. When used, LIF was added at a concentration of 100 ng/ml.
6. Individual cell clones were obtained.