Culture of human normal kidney and primary tumor tissues

Culture of human normal kidney and primary tumor tissues

Materials

The following materials were obtained from GIBCO™ Invitrogen unless stated otherwise.

Cell culture medium: Dulbecco's modified Eagle's medium with nutrient mixture F-12 (DMEM/F-12) and GlutaMAX-I™ supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin/streptomycin (50 U/mL/50 µg/mL), fungizone (2.5 µg/mL), and human transferrin (5 µg/mL).

Hank's buffered salt solution (HBSS) without CaCl2 and MgCl2.

Collagenase solution: dissolve 50 mg of collagenase type 2, 315 U/mg in 25 mL non-supplemented culture medium and filter sterilized with 0.22 µm filter. Used fresh.

0.05% Trypsin-EDTA solution.

Freezing solution: 90% FBS and 10% dimethylsulfoxide.

Incubation vessel: autoclaved 250 mL PYREX® glass-jacketed flask with a magnetic bar in it. The temperature of the collagenase solution in the reservoir is maintained at 37°C by circulating warm water through the jacket of the flask.

Collagen coated flasks: 75-cm2 plastic culture flasks coated overnight at 37°C with a 40 µg/mL solution of collagen G from bovine calf skin in PBS.

Sterile cell strainers with sieve sizes of 100, 70, and 40 µm (BD Falcon™, BD Biosciences, USA).

0.4% Trypan blue solution (Sigma Aldrich, St. Louis, MO).

1X PBS

Immunocytochemical staining: mouse monoclonal anti-pan cytokeratin antibody, goat anti-mouse IgG-FITC antibody, and Hoechst 33258 (Sigma Aldrich, St. Louis, MO).

RT-PCR analysis: RNeasy Mini RNA isolation kit (Qiagen, USA) and RevertAid™ H Minus First Stand Synthesis Kit。

Tissue Collection

Human kidney tissue samples were obtained from a total of 6 patients (3 males and 3 females) undergoing radical nephrectomy for renal cell carcinoma at the Portuguese Oncology Institute-Porto (IPO-Porto). Patient mean age was 62±5 years old. Tissue samples (about 10 g each) were collected from areas macroscopically identified as normal (in the cortex) or tumoral immediately after the specimen extraction, by an expert uropathologist. The nature of those areas was subsequently confirmed by histopathological evaluation of mirror samples. Tissues were placed in separated sterile 50-mL tubes with ice-cold culture medium and then transferred to a cell culture laboratory on ice.

All specimens underwent subsequent routine tissue processing (formalin fixation and paraffin embedding). Histopathologic analysis of sections to assess the type of renal cancer, grading and staging was performed at the Department of Pathology, IPO-Porto.

HPTEC and HRTC Isolation Protocol

Renal cell isolation took place within 30 minutes of renal tissue collection. All subsequent procedures were performed in a tissue-culture flow hood, under sterile conditions. To avoid cell cross-contamination, we recommend handling normal and tumor specimens in two individual tissue-culture hoods. If this is not possible, then, the following guidelines should be reinforced: only one surgical specimen should be used in a tissue-culture hood at any one time (during this time keep the other tissue on ice in a sterile container), the hood should be cleaned before the introduction of the other surgical specimen, and bottles or aliquots of medium should be dedicated for use with only normal or tumor cells.

In the laboratory culture cabinet, transfer the tissue to a 60-mm Petri dish. Using forceps and scissors, dissect off (i) the fibrous capsule and adjacent medulla from the cortical tissue and (ii) any fat, blood clots and connective tissues from tumor tissues.

Cut the tissue sample into small pieces with scalpels.

Transfer the tissue fragments to a sterile 50-mL centrifuge tube, rinse them vigorously with ice-cold HBSS (contains EGTA that loosen cell junctions via calcium chelating action) and decant the supernatant. Repeat this last step until the solution is clear of blood.

Pour off the supernatant, transfer the fragments to a clean 60-mm Petri dish and finely mince the tissue into approximately 1-mm3 pieces with scalpels.

Resuspend the small fragments in 25 mL pre-warmed non-supplemented culture medium and combine it with the collagenase solution (1 mg/mL final concentration) in the incubation vessel. Incubate for 20 minutes at 37°C with gentle stirring.

Pass the digested tissue onto the first sieve (100 µm) into a 50-mL centrifuge tube. The same procedure is then applied to the following sieves (70 and 40 µm). Sieving through the 100-µm sieve allows the removal of any undigested fibrous tissue, whereas the function of the 70 and 40-µm sieves is to remove contaminating tubular fragments and glomeruli, respectively.

Wash the sieved cells by centrifugation (400 g, 5 min at 4°C), and resuspend the pellet in HBSS. Repeat this process two more times, and then resuspend the cell pellet in culture medium with supplements. Determine cell number and viability in a Neubauer hemocytometer using the trypan blue solution.

Cell Culture Protocol

Seed the isolated cells on collagen-coated 75 cm2 culture flasks at a density of 5×104 cells/cm2 and incubate in a humidified atmosphere of 95% air/5% CO2 at 37°C. (Note: The RPMI 1640 medium with supplements can be used alternatively to grow HRTC)

Change the medium 24 h after initial seeding and at 48 h intervals thereafter.

Allow the cells to grow until ~80% confluence before they are subcultured or frozen.

When cultured as described above, the cells reach confluence in approximately 10–13 days after seeding.
Cell Subculture Protocol

Remove the cell culture medium and wash the cell monolayer with either pre-warmed HBSS or PBS and 1 mL of trypsin-EDTA to weaken cell adhesion to the flasks surface.

Add enough trypsin-EDTA solution to cover the flask surface and incubate at 37°for 3 min. Observe the cells under an inverted optical microscope to ensure adequate cell detachment.

Terminate trypsin action by adding 10 mL of supplemented growth medium and resuspend the detached cells by repeatedly pipeting over the surface of the culture flask.

Transfer the resuspended cells to a centrifuge tube, rinse the flask with medium, and add the rinsed cells to a centrifuge tube. Seed the cell suspension into new 75-cm2 flasks at a 1:3 subculture ratio.

Under these conditions, HPTEC beyond the first passage reach confluence in 3–5 days, and HRTC in 7 days.
Cell Cryopreservation, Thawing and Replating Protocol

After trypsinization, transfer the detached cells to a centrifuge tube and pellet the cells by centrifugation (400 g, 5 min at 4°C).

Resuspend the pellet from each flask in 3 mL of freezing solution.

Tranfer 1 mL cell suspension to 2 mL cryovials and freeze immediately at −80°C.

To reseed the suspensions, cells are rapidly defrosted by adding pre-warmed supplemented medium and transferred to a 15-mL centrifuge tube.

Pellet the cells by centrifugation at 400 g, for 5 minutes.

The pellet is resuspended in warmed culture medium, and transferred to 75 cm2 flasks, one vial per flask.

HPTEC and HRTC are successfully cryopreserved from both isolated cells and cell suspensions obtained after trypsinisation, maintaining a normal growth after defrosting.
Morphological Evaluation
Monolayer cultures derived from all 12 primary isolates (6 normal, 4 clear cell RCC and 2 chromophobe RCC) and the corresponding first three passages were examined by light microscopy under an inverted optical microscope. An immortalized proximal tubule epithelial cell line derived from normal adult human kidney, HK-2 cell line (ATCC, CRL-2190), was also examined for comparison.
Immunocytochemical Staining for Cytokeratin
To investigate the epithelial origin of obtained normal and tumor renal cells, the reactivity to anti-cytokeratin antibody was assessed in suspensions from passages 1 to 3. Two immortalized cell lines derived from normal (HK-2) and tumor (A-498) human kidney were used as positive controls.
Briefly, HPTEC or HRTC were grown to about semiconfluence on 24-well plates (initial density: 1.0×105 cells/well), washed with sterile PBS, fixed for 20 minutes in 4% p-formaldehyde, and permeabilized for 5 minutes in 1% Triton X-100 solution. After blocking with a 1% bovine serum albumin, 0.4% Triton X-100 and 4% sodium azide mixture, the cells were incubated overnight at 4°C with mouse monoclonal anti-pan cytokeratin antibody (1:100), and then with goat anti-mouse IgG-FITC antibody (1:100) for 2 hours at room temperature. After rinsing with PBS, nuclei were counterstained with 5 µg/mL Hoechst 33258 for 5 minutes and then washed with PBS again. Images were captured using a fluorescence microscope (Nikon, Eclipse E400). Appropriate negative controls were included to guarantee positive antibody reactivity.