Isolation of renal tubular cells
Procedures
Tissue Homogenization
- Use kidney nephrectomy as fresh as possible –> put on ice cold PBS immediately after surgery
- Carry out all subsequent steps on ice under sterile conditions
- Remove cortical areas from portions not involved in renal cell carcinoma
- Mechanically dissociate kidney sections by mincing the tissue using clean scalpels
- Transfer tissue homogenate into a 50 ml tube using 20-30 ml HBSS
- Incubate chopped kidney with 40 ml Collagenase IV (0.5 units/ml) for 60 min at 37 °C
- Shake vigorously every 20 min
- Stop Collagenase digestion by adding 4 ml FCS
- Squeeze kidney pieces through a tea sieve using the sterile plunger of a 10 ml syringe
- Wash sieve extensively with cold HBSS (~ 200 ml)
- Divide cell suspension in four 50 ml tubes
- Pellet cells at 1 600 rpm for 10 min at 4 °C
- Pool pellets using HBSS and pellet cells again at 1 600 rpm for 10 min at 4 °C
- Carefully remove supernatant
Density Centrifugation
- Precool Sorvall centrifuge to 4 °C
- Prepare 50 ml of a 41.4 % Percoll solution by diluting 21.2 ml Percoll with 28.8 ml DMEM
- Resuspend cell pellet in 40ml of Percoll solution and transfer 20ml of the suspension into two clean ultracentrifuge tubes
- Balance tubes carefully by adding residual Percoll solution
- Centrifuge at 15 000 rpm for 30 minutes at 4 °C
- Carefully transfer the tubular band located at the water/Percoll interphase using a sterile pasteur pipette into a fresh 50 ml tube
- Wash cells with 10ml cold HBSS at 1 600 rpm for 10 min at 4 °C
Immunomagnetic separation
- Wash cells twice in chilled PBS
- Resuspend cells in cold PBS
- Add mAbs against CD13 (proximal) / Uromodulin (distal) (use IgG1 isotypes at 1 µg/ml)
- Incubate for 30 min on ice
- Wash cells twice in chilled PBS
- Resuspend pellet in MACS buffer (80 µl/107 cells)
- Add rat anti-mouse IgG1 immunomagnetic beads (20 µl beads/107 cells)
- Incubate for 15 min on ice
- Wash cells in cold MACS buffer
- Resuspend cells in 0.5 ml MACS buffer
- Choose column size depending on the total cell number following the instructions of the manufacturer
- Prepare columns by rinsing with appropriate amounts of MACS buffer
- Apply cell suspension onto the column and collect non-retained cells
- Flush column three times with MACS buffer
- Remove column from the separator and place it on a suitable collection tube
- Pipette appropriate amount of HRTC-medium onto the column
- Immediately flush out fraction with the magnetically labelled cells by firmly applying the plunger supplied with the column
- Analyze cell purity and cell number by FACS
Cell Culture
- Coat 6-well plates with fibronectin dissolved in PBS (1 µg/ml) for 30 min at 37 °C
- Dilute cells to a concentration of 50 000 cells/ml with HRTC-medium
- Seed ~100 000 cells per well
- Propagate cells at 37 °C and 5 % CO2 in a humidified atmosphere
- Exchange growth medium after 24 hours after isolation and subsequently each 3rd day
- Split cells when reaching confluency by treatment with trypsin / EDTA for 2 minutes
Materials
HRTC-medium | ||
DMEM Hams F12 | without Hepes or NaHCO3 | Sigma D0547 |
Glutamax | 2 mM | Invitrogen 35050-038 |
Pen/Strep | 100 U/ml | Invitrogen 15070-022 |
FCS | 10 % | Invitrogen 16170-078 |
D-valin | 46 mg/ml | Sigma V1255 |
Linoleic acid | 42 mg/ml | Sigma L1012 |
?-Lipoic acid | 100 mg/ml | Sigma T1395 |
MACS-buffer | ||
PBS | ad 500 ml | Invitrogen 10010-056 |
BSA | 0.5 % | Sigma A3803 |
EDTA | 5 mM | Sigma E7889 |
Trypsin-EDTA 0,25 % Trypsin, 1mM EDTA | Invitrogen 25200-056 | |
Collagenase, Type 4, 1 g | Worthington LS004188 | |
Fibronectin (human plasma) | Invitrogen 33016-015 | |
Percoll | GE Healthcare 17-0891-02 | |
6-well-plates, flat bottom 50 Stk | BD Biosciences 353046 | |
PBS (phosphate buffered saline) | Invitrogen 10010-056 | |
HBSS (Hanks’ balanced salt solution) | Sigma H1641 | |
Purified mouse anti human Uromodulin | CedarLane CL1032A | |
Purified mouse anti-human CD13 | BD Biosciences 347830 | |
Rat Anti-Mouse IgG1 MicroBeads | Miltenyi 130-047-102 |