Isolation of renal tubular cells


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


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
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