Isolation of human renal tubule 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