Propagation and Culture of Human Renal Fibroblasts

Fig. 1

Phase contrast microscopic appearance of confluent human renal fibroblasts . Cells show characteristic “fingerprint” monolayers without forming hillocks

2 Materials

2.1 General Sterile Cell Culture Materials

Glass media bottles: 100, 200, and 500 mL.

60 mm² cell culture petri dishes.

50 mL centrifuge tubes.

Tissue culture flasks: 25 and 75 cm².

Six-well cell culture plates.

Graduated 5 and 10 mL pipettes.

Cell scrapers.

Disposable 50 mL syringes.

Disposable 0.2 μm pore syringe filters.

10.

0.2 μm 500 mL bottle-top vacuum filter unit.

11.

Sterile containers for tissue collection.

12.

Disposable basic sterile dressing pack for dissection.

13.

N° 22 scalpel blades.

14.

Pasteur transfer pipettes.

15.

Waste bottle.

16.

Laboratory grade double deionized water (ddH₂O).

2.2 Specialist Cell Culture Equipment

Biohazard hood.

Inverted light microscope.

Vacuum pump.

Tissue culture incubator at 37 °C with 95 % O₂/5 % CO₂.

Water bath.

Autoclave.

Pipet-Aid™ (BD Biosciences, San Jose, CA, USA) or equivalent pipet controller to aspirate and change media.

Hemocytometer.

Mechanical tally counter.

10.

Cryogenic dewar for storage of frozen cells.

11.

−70 °C freezer.

2.3 Reagents

0.01 M phosphate-buffered saline (PBS), pH 7.4: For each 1 L weigh 8.0 g NaCl, 0.2 g KCl, 2.9 g Na₂HPO₄⋅H₂O, and 0.2 g KH₂PO₄ and transfer to the bottle containing 980 mL of ddH₂O. Adjust to pH 7.4 with NaOH at 25 °C. Make up to 1 L with ddH₂O.

Coating solution: 2 % solution of Type B gelatin from bovine skin (Sigma-Aldrich, St Louis, MO, USA).

Tissue collection buffer: Combine 100 mL Hank’s balanced salt solution with 2.5 μL gentamicin (50 mg/mL gentamicin sulfate). Filter through a 0.2 μm pore syringe filter using a 50 mL syringe (see Note 1 ).

Enriched medium (Dulbecco’s Modified Eagle Medium [DMEM] with 20 % serum): Combine 80 mL fetal calf serum (FCS), 10 mL 1 M HEPES, 4 mL of 200 mM glutamine, and 8 mL 5000 U/mL penicillin-5000 μg/mL streptomycin, and make up to 400 mL with 1× DMEM. This solution is sterilized by passing through a sterile 0.2 μm pore cellulose acetate filter unit into a sterile 500 mL glass bottle by vacuum extraction. The bottle is capped and stored at 4 °C until use (see Note 2 ).

Maintenance medium (DMEM with 10 % serum): as above for enriched medium but containing 40 mL FCS per 400 mL medium.

Ethylenediaminetetraacetic acid (EDTA): 0.02 % solution.

EDTA/trypsin solution: 1:10 dilution of 1× trypsin in 0.02 % EDTA.

Freezing medium: 1 mL dimethyl sulfoxide (DMSO) added to 9 mL of maintenance medium.

Liquid nitrogen (N₂).

2.4 Immunoperoxidase Cytochemistry

2.4.1 General Materials

Established primary human renal cells.

Sterile glass cover slips (22 × 40 mm).

Sterile 0.01 M PBS, pH 7.4.

Borosilicate glass test tubes.

Filter paper.

Fixative, e.g., ice-cold methanol, acetone, or 4 % paraformaldehyde in 0.01 M PBS (see Note 3 ).

Glass microscope slides.

Hydrophobic wax pen.

2.4.2 Immunoperoxidase Cytochemistry

Primary antibody (Table 1).

Table 1

Immunocytochemical antiserum specifications

Antibody	Clone	Raised in	Dilution	Specificity	Manufacturer
α-SMA	1A4	Mouse	1:50	Smooth muscle isoform of actin	Dako, Glostrup, Denmark
Collagen I	–	Rabbit	1:1000	Collagen type I	Southern Biotech, Birmingham, AL, USA
Cytokeratin	MNF116	Mouse	1:100	Pan-keratin	Dako
Desmin	D33	Mouse	1:100	Desmin	Dako
E-cadherin	36/E-cadherin	Mouse	1:100	E-cadherin	BD Biosciences, San Jose, CA, USA
Vimentin	V9	Mouse	1:50	Vimentin	Dako

Antibody diluent (Dako, Glostrup, Denmark).

0.035 % hydrogen peroxide in methanol: Dilute 10 μL of 35 % hydrogen peroxide in 990 μL of 100 % methanol. Make as required.

Peroxidase IgG kits containing normal serum and appropriate biotinylated secondary antibody (2°Ab) (Table 1) (Vectastain™ Kit; Vector Laboratories, Burlingame, CA, USA).

Avidin-biotin complex (ABC) Elite kit (Vector Laboratories).

Chromogen substrate (e.g., SIGMAFAST™, Sigma-Aldrich: dissolve 1× 3′3-diaminobenzidine (DAB) tablet and 1× UreaH₂O₂ tablet in 1 mL 0.01 M PBS).

Harris hematoxylin.

Aqueous mounting media.

Humidified chamber (e.g., plastic container lined with blotting paper and soaked with 0.01 M PBS).

3 Methods

The methodology described in Subheadings 3.1–3.3 provides techniques to establish a primary human fibroblast cell line originating from explanted human renal cortical tissue. The procedures outline (a) the preparation of cell culture reagents, (b) explanting of human kidney tissue, and (c) subculturing of the established primary cell line. Subheading 3.4 offers methods in characterizing a primary human fibroblast cell line. Subheadings 3.5 and 3.6 describe the methods for cryogenic storage and subsequent experimental use once the primary human fibroblast cell line has been established.

All cell culture work described in this protocol should be performed in a biohazard cabinet. All reagents for use with cell culture work should be prewarmed to 37 °C prior to use in order to maintain cellular viability and integrity, unless otherwise specified.

3.1 Gelatin Coating of Petri Dishes

Petri dishes are coated with 1 % gelatin solution prior to explanting of kidney tissue. This will enable minced tissue to secure to the petri dish and provide a substrate for the initial cultivation of cell populations.

Dilute 2 % gelatin solution with 0.01 M PBS (1:1 dilution) and filter sterilize into a centrifuge tube using a disposable syringe and a 0.2 μm pore syringe filter.

Coat entire surface of the petri dish with 1 mL of 1 % gelatin solution and incubate for ≥30 min at 37 °C.

Remove excess gelatin solution and rinse the petri dish with 0.01 M PBS prior to explanting.

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