Renal epithelial cell cultures can be used: (1) to study the Na+-dependent inorganic phosphorus transport system inhibited, for example, by parathyroid hormone; (2) to study the specificity of phosphonoformic acid and inhibitors of Na+ and P cotransport. (3) As a model for oxidant damage in renal tubules.
Operation method
Primary renal epithelial cell culture experiment
Principle
The autocortically excised tissue was quick chopped into pieces, rinsed and put into a mixture of collagenase and trypsin and shaken for digestion. The tissue pieces were ground with a pipette at regular intervals, and then the separated cells were collected. The cell suspension was filtered through a filter of a certain pore size to remove the undigested tissue mass. Then, the cells were washed to remove digestive enzymes. Suspend the cells with serum-added culture medium and inoculate the cells in plastic culture vessels.
Materials and Instruments
Kidney Tissue Slices Move I. Materials Caveat 1. at the beginning of primary culture, the level of culture solution is less than 3 ML, and the optimum is 1-1.5 ML, just enough to keep the plant mass moist. 2. leftover tissues and used test tubes, pipettes, and petri dishes should be treated with hypochlorite before being thrown in the trash. 3. cultures need to be opened only in a sterile environment to avoid contamination. Common Problems Identification of renal tubular epithelial cells: Cultured renal tubular epithelial cells were placed on a 24-well culture plate with coverslips, and when the cells grew to occupy about 80% of the culture area, the coverslips were taken out, and the slices were washed with PBS. Fixed in methanol at -20℃ for 5min, PBS washed, 10% sheep serum was added and closed at 37℃ for 30min, PBS washed, rabbit anti-human keratin 18 antibody was added, negative control PBS primary antibody was added and reacted at 37℃ for 1h, FTTC-labeled sheep anti-rabbit lgG (1:200) was added, and reacted at 37℃ and protected from light for 45min, PBS washed, 90% glycerol was added and sealed, and the results of staining were observed with fluorescence microscope. Staining results were observed. For more product details, please visit Aladdin Scientific website.
DMEM FBS Collagenase Trypsin
Scalpel Tissue forceps Orbital oscillator Petri dishes Culture flasks Test tubes Strainers
aseptic
1. Base culture solution: 50:50 mixture of DMEM and F12.
2. FBS (Sterile Systems, Hyclone)
3. complete culture medium: DMEM and F12 mixture with 10% FBS
4. BSS
5. 0.1% collagenase (type IV, Worthington) and 0.1% trypsin (1:250, Sigma) mixture prepared in 0.15 mol/L NaCl
6. 0.5 mg/ml DNase in saline.
7. trypsin and EDTA mixture: prepared with 0.1% trypsin (1:250) and 1 mm/L EDTA (culture medium, Sigma)
8. scalpel and tissue forceps
9. 160um pore size Nitex filter (Tetko)
10. Petri dishes and culture flasks
11. 50 ml test tubes
Non-sterile
1. Orbital oscillator (Bellco)
II. Operating steps
Digested tissue
1. Cut the kidney into 5-10 mm thick tissue slices in the coronal plane. Wash the tissue slices with ice-cold basal culture solution.
2. Cut the tissue from the outer layer of the cortex and then cut it into 1-2 mm pieces with a cross blade.
3. Place approximately 5 ml of tissue into a 50 ml test tube containing 20 ml of pre-warmed collagenase and trypsin mixture.
4. Incubate the block for 1 h with gentle shaking.
5. Aspirate the digestive solution and add fresh digestive solution.
6. Collect the cell suspension every 20 min, then add 20 ml of culture base containing 0.5 mg/ml DNase and gently grind for 10 revolutions with a 10 ml pipette.
7. Dilute the collected cell suspension with an equal amount of complete culture medium. Place the diluted cell suspension on ice.
8. Repeat the collection step 5 or more times until the tissue mass is completely dispersed.
9. Mix the collected cell suspension and dispense into 50 ml test tubes.
10. After centrifugation (100 g, 15 min), suspend the cells with 45 ml of complete culture medium containing DNase.
11. Filter the cell suspension through a Nitex filter with a pore size of 160um.
12. This isolation method isolates both lineage clusters and individual cells, so cell counting is unreliable. Place 15 ml of cell suspension in each 75 cm2 culture flask, and passage culture or freeze storage after cell expansion.
NHK-C cells significantly showed functional features of proximal tubules, such as the Na+-dependent inorganic phosphorus transport system inhibited by parathyroid hormone (PTH). In addition, NHK-C cells transport hexose, which is sensitive and Na+-dependent to rhizopyranoside. cAMP in NHK-C cells is stimulated by the kinin in the same manner as in the proximal tubule, i.e., it is responsive to PTH and insensitive to pressin. These cells expressed enzymes from the brush border of the proximal tubule (epimerase, leucine aminophthaleinase, and 7-glutamyltransphthalase). In addition, NHK-C cells were used to study the specificity of phosphonoformic acid and inhibitors of Na+ and P isotropic transport and as a model of oxidant injury in renal tubules.
Source: Animal Cell Culture: A Guide to Basic Techniques (5th Edition)