Isolation of papillary cells

实验概要

This protocols provides a general protocol for isolation of papillary cells.

实验步骤

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 NaHCO₃, 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% CO₂and 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.