SOP: Propagation and Quantification of Feline Calicivirus Using Cell Culture Methods

GIFSQ — Global Institute for Food Safety & Quality

1. Principle

Standardized stocks of feline calicivirus (FCV) are produced for use as positive controls in methods that extract and detect RNA viruses of interest from food commodities. Crandell-Reese Feline Kidney (CRFK) cells are grown and maintained, infected with FCV at a low multiplicity of infection, and the progeny virus is harvested, clarified and stored frozen. The infectivity of the harvested stock is quantified by a plaque assay on CRFK cell monolayers under an agarose overlay: each plaque (zone of clearing) is taken to originate from one infectious virus particle, giving a titre in plaque-forming units per millilitre (PFU/mL). PCR detection and confirmation of FCV can be done using the method OPFLP-06.

• Cytopathic effect (CPE): the destruction of cells caused by viral infection. In CRFK cells infected with FCV, CPE is characterized by cell rounding, lifting up from the monolayer, and eventual cell lysis.
• Plaque-forming unit (PFU): a measure of the concentration of infectious viruses in a sample. Each plaque, or zone of clearing, in the monolayer is assumed to originate from one infectious virus particle.
• Multiplicity of infection (MOI): the average number of virus particles present per cell. The MOI is found by dividing the number of infectious viral particles added to a well or flask (determined by the plaque assay in this procedure) by the number of cells (determined by the hemacytometer count in this procedure).

2. Apparatus required

• Biological safety cabinet providing a HEPA-filtered work zone
• Liquid nitrogen storage system or freezer capable of holding vials of cells (below −130°C)
• Water baths capable of maintaining 37°C and 45°C
• Incubator capable of maintaining a humidified environment (using a water tray) at 37°C with 5% CO₂
• Freezers capable of maintaining −20°C and −70°C
• Inverted phase-contrast microscope capable of visualizing tissue culture cells at 40× total magnification
• IEC DPR-6000 refrigerated centrifuge or equivalent, with rotors for 50 mL and 15 mL tubes or equivalent
• Hemacytometer with Neubauer rulings
• Microwave oven
• Autoclave
• Light box (for reading plaques)
• Vortex mixer
• Timer
• Cell freezing container to reduce temperature gradually while freezing the cells
• Pipetting aid with replaceable filter
• Adjustable micropipettors covering 0.5–10 µL, 10–100 µL and 100–1000 µL, with specific filtered pipette tips
• Laboratory protective goggles
• Protective padded gloves that reach past the elbows
• Sterile pipettes, 1 mL, 5 mL, 10 mL and 25 mL (glass reusable or plastic disposable)
• 0.22 µm bottle-top filters (Nalgene product number 292-3320 or equivalent)
• Nunc CryoTubes, 1.8 mL capacity (product number 375418 or equivalent)
• Tissue-culture-treated flasks, 75 cm², sterile with filter caps (Nunc product number 156499 or equivalent)
• 15 mL conical polypropylene sterile centrifuge tubes (Sarstedt product number 62-554-002 or equivalent)
• 50 mL polypropylene sterile centrifuge tubes (Sarstedt product number 62-547-004 or equivalent)
• 12-well tissue-culture-treated sterile microplates (Corning product number 3513 or equivalent)
• Microcentrifuge tubes, 1.5 mL capacity
• 0.22 µm syringe filters (Millipore product number SLGV033RB or equivalent)
• Sterile syringes (plastic disposable), 10 mL and 60 mL
• Vacuum pump for liquid aspiration, with a liquid trap that has a back-up to prevent chemical overflow

3. Reagents and kits

• CRFK cells: American Type Culture Collection (ATCC) product number CCL-94, no substitute
• Feline calicivirus strain F9: American Type Culture Collection product number VR-782, no substitute
• Sterile double-distilled water, or other sterile water allowing cell growth such as Ultrapure Dnase/RNase-free distilled water
• 10X Minimal Essential Medium with Earle’s salts, powder (Invitrogen product number 11700-077 or equivalent) — see section 5.1
• Sodium bicarbonate (Sigma product number S-8875 or equivalent) — see section 5.2
• Non-essential amino acids (Invitrogen product number 11140-050 or equivalent)
• Penicillin/streptomycin (Invitrogen product number 15140-148 or equivalent)
• L-Glutamine, 200 mM liquid (Invitrogen product number 25030-081 or equivalent)
• Fetal bovine serum (Invitrogen product number 26140-079 or equivalent), heat inactivated
• Magnesium chloride (Sigma product number M-2670 or equivalent)
• Gentamycin, 50 mg/mL (Invitrogen product number 15750-060 or equivalent)
• Type II agarose (Sigma product number A-6877 or equivalent) — see section 5.5
• Filtered DMSO (Sigma product number D5879 or equivalent)
• Prepared 10X Minimal Essential Medium — see section 5.1
• 7.5% sodium bicarbonate solution — see section 5.2
• Growth medium — see section 5.3
• Maintenance medium — see section 5.4
• Overlay medium — see section 5.5
• Freezing medium — see section 5.6
• 70% ethanol
• Sodium chloride (ACS grade or better)
• 37% formaldehyde solution (Anachemia chemicals product number 41860 or equivalent)
• 3.7% formaldehyde solution — see section 5.8
• Phosphate-buffered saline (PBS), pH 7.2 (Invitrogen product number 20012-027 or equivalent)
• 0.05% Trypsin-EDTA (Invitrogen product number 25300-054 or equivalent)
• Trypan blue, 0.4% prepared in 0.85% saline (Invitrogen product number 15250-061 or equivalent)
• Earle’s balanced salt solution (EBSS) (HyClone product number SH3002902 or equivalent)
• Crystal violet (Sigma product number C6158 or equivalent)
• 0.1% crystal violet solution — see section 5.7

Reagent preparations

• 70% Ethanol
• To prepare: mix 70 parts ethanol with 30 parts distilled water (v/v)
• 37% Formaldehyde Solution
• Use as supplied
• Phosphate Buffered Saline (PBS)
• Stock Solution 1
• Sodium phosphate dibasic anhydrous reagent grade (Na₂HPO₄) — 28.4 g
• Sodium chloride reagent grade (NaCl) — 85.0 g
• Distilled water — 1.0 L
• pH approximately 8.2
• Stock Solution 2
• Sodium phosphate monobasic monohydrate reagent grade (NaH₂PO₄·H₂O) — 27.6 g
• Sodium chloride reagent grade (NaCl) — 85.0 g
• Distilled water — 1.0 L
• pH approximately 5.6
• Complete Medium: make 1 to 9 dilutions of aliquots of each stock solution to obtain a final volume of 500 mL of diluted solution 1, and 100 mL of diluted solution 2; by means of a pH meter, titrate the diluted solution 1 to a pH of 7.3–7.4 by adding approximately 65.0 mL of diluted solution 2; the resulting solution is 0.02 M phosphate saline buffer
• Note: do not titrate 0.2 M phosphate buffer to pH 7.3–7.4 and then dilute to 0.02 M strength — this results in a drop in pH of approximately 0.25. Addition of 0.85% salt after pH adjustment also results in a drop of approximately 0.2
• 0.05% Trypsin-EDTA
• Use as supplied
• Trypan Blue, 0.4% in 0.85% Saline
• To prepare: dissolve 0.4 g of trypan blue in 0.85% saline and make up to 100 mL

4. Procedure

Storage and handling of viral and cell stocks

No sampling is required: this procedure develops standard virus and cell stocks for use as controls.

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Store viral stocks at −70°C

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Store cells in a freezer or in the vapour phase of liquid nitrogen (below −130°C)

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Transport virus and cell stocks on dry ice and do not allow them to thaw

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Prepare all buffers and solutions as described in section 5

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Ensure that all reagents and equipment that will contact virus or cells are sterile and opened only in the biological safety cabinet

Reviving CRFK cells from liquid nitrogen

Warm growth medium to 37°C in the water bath

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Add 14 mL of warm growth medium to one 75 cm² culture flask

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Wearing a face shield and gauntlets, remove 1 cryovial of CRFK cells from the liquid nitrogen storage container or the −150°C freezer

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Transfer the vial to a 37°C water bath until just thawed

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Dry the cryovial with a paper towel and decontaminate it with 70% ethanol; unscrew the cap and pipette all the contents (1 mL) into the 14 mL of growth medium in the culture flask

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Mix by gently tilting the flask back and forth; identify the flask with the name of the cell line, the passage number, the date and the initials

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Incubate the flask at 37°C with humidity and 5% CO₂

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Examine the cells under the microscope after 5 hours; ensure the cells are adhered to the surface when the flask is gently tilted back and forth

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Remove and discard the growth medium; add 15 mL of fresh, warmed growth medium

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Incubate the flask at 37°C with humidity and 5% CO₂ for 1–2 days, until the cells form a confluent monolayer

Splitting CRFK cells for propagation

Cells die if they dry out — replace all media quickly.

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Warm growth medium, PBS and trypsin to 37°C in a water bath

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Remove the flask from the incubator and perform all work in the biological safety cabinet

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Remove and discard the growth medium

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Add 10 mL of PBS; tilt the flask to distribute the PBS over the surface of the cell monolayer

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Remove and discard the PBS

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Add 2 mL of trypsin to the flask

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Incubate the flask at room temperature for 30 seconds, or until the cells are easily detached

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Keeping the flask level and the cells covered with liquid, tap the side of the flask sharply to dislodge the cells

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Add 8 mL of growth medium to the flask

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Pipette up and down 10–15 times to break up cell clumps, avoiding foaming; direct the liquid onto the coated surface of the flask (where the cells were adhered)

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Transfer 0.5 mL of the cell suspension into a 1.5 mL microcentrifuge tube; add 0.5 mL of trypan blue solution and mix with a pipette (do not vortex); leave in contact for 2 minutes (but not more than 15 minutes)

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With a micropipettor, fill both sides of the hemacytometer with the diluted cell suspension

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Examine under the microscope with a 4× objective; count the cells in 5 squares from each side of the hemacytometer (there are 9 squares of equal size on the grid). A cell touching the top or right border of a square counts as “in”; a cell overlapping the bottom or left border is “out” and is not counted. Count only the viable (translucent) cells and not the non-viable (blue) cells. Counting may also be done under a 10× objective; in that case only one square (of 9) is visible at one time in the field, and the hemacytometer is moved around to complete the count of five squares

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Estimate the number of cells in 1 mL of the initial suspension with the formula below

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Clean the hemacytometer with water and dry it

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Dilute the cell suspension in growth medium to obtain 100,000 cells/mL in a volume of 15 mL and transfer to a new flask

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Mix gently by tilting the flask back and forth; label the flask with the name of the cell line, the passage number, the date and the initials

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Incubate the flask at 37°C with humidity and 5% CO₂ for 2–3 days, until the cells form a confluent monolayer

Do not use cells for viral propagation or titration after more than 35 consecutive passages or around three months. Note the cell viability from the trypan blue staining — if over 10% of the cells stain dark blue (non-viable), thaw a new vial before proceeding.

Cell count formula:

(Number of viable cells ÷ Number of squares) × Conversion factor of hemacytometer × Dilution factor = Number of cells/mL

Example: (200 ÷ 10) × 10,000 × 2 = 400,000 cells/mL

• Number of viable cells = the total count of viable (translucent) cells
• Number of squares = the number of hemacytometer squares counted
• Conversion factor of hemacytometer = 10,000
• Dilution factor = 2 (the 0.5 mL + 0.5 mL trypan blue mix)

Freezing CRFK cells

Keep the passage number low — freeze large batches at as low a passage number as possible (no more than 2 to 5 passages).

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Warm PBS buffer and trypsin to 37°C in a water bath; maintain the freezing medium at 4°C

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Remove the flask from the incubator and perform all work in the biological safety cabinet. Cells intended for freezing should be in exponential growth phase (actively dividing); the monolayer should have been split no more than 2 days previously

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Remove and discard the growth medium

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Add 10 mL of PBS; tilt the flask to distribute the PBS over the surface of the cell monolayer

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Remove and discard the PBS

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Add 2 mL of trypsin to the flask

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Incubate the flask at room temperature for 30 seconds, or until the cells are easily detached

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Keeping the flask level and the cells covered with liquid, tap the side of the flask sharply to dislodge the cells

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Add 8 mL of growth medium to the flask

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Pipette up and down 10–15 times to break up cell clumps, avoiding foaming; direct the liquid onto the coated surface of the flask (where the cells were adhered)

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Count the cells as described in the splitting procedure (trypan blue mix, hemacytometer count, cell count formula)

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Transfer the cell suspension to a sterile 50 mL centrifuge tube; if multiple flasks are being split, combine the cell suspensions at this point; centrifuge at 400 × g for 10 minutes

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Remove and discard the supernatant

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Add the appropriate volume of freezing medium to the cell pellet to obtain 4 × 10⁶ cells per mL

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Gently resuspend the cells and transfer 1 mL to cryovials labelled with the name and passage of the cell line, the date and the initials

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Tighten the cryovial caps and place them in a regulated freezing container or in a styrofoam box

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Freeze in the regulated container for 24 hours in a −70°C freezer; alternatively, keep the styrofoam box at −20°C for 5 to 6 hours and then at −70°C overnight

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Transfer the cryovials to a liquid nitrogen tank or a −150°C freezer for long-term storage

Thaw and check one cryovial of freezing cells per lot for its capacity to grow before using the cell lot for experiments.

Propagating FCV

Use a separate flask of cells (not infected) to count the cells and calculate the MOI.

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Begin with CRFK cultures seeded at 100,000 cells/mL and grown for 24–48 hours

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Warm growth medium, maintenance medium, PBS, EBSS and trypsin to 37°C in a water bath

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Observe the cell monolayers under the microscope; choose one representative flask, trypsinize and count its cells as described in the splitting procedure

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Calculate the amount of virus to infect the remaining flasks — the target MOI is 0.1 PFU of virus per cell. For example, if the representative flask has 2 × 10⁷ cells, infect each flask with 2 × 10⁶ PFU of virus in a total volume of 1 mL

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Immerse the frozen FCV stock in the 37°C water bath until thawed

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Dilute the virus to the required concentration in EBSS; prepare enough virus suspension to infect all flasks

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Remove the flasks to be infected from the incubator and perform all work in a biological safety cabinet

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Remove and discard the growth medium

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Add 10 mL of PBS; tilt the flask to distribute the PBS over the surface of the cell monolayer

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Remove and discard the PBS

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Add 1 mL of diluted virus suspension to each flask

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Gently tilt the flask back and forth to cover the monolayer with the virus suspension

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Incubate the flask at 37°C with humidity and 5% CO₂ for 60 minutes, rocking the flask gently every 10 minutes to spread the virus evenly

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Gently add 9 mL of maintenance medium to the flask without disturbing the cells

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Incubate the flask at 37°C with humidity and 5% CO₂ for 24 hours

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Examine the cells under the microscope to determine if CPE is present — CPE is indicated by cells rounding and floating in the medium

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When 80% of the cells show CPE (this may take up to 48 hours), freeze the flask at −70°C for 45 minutes

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Thaw the flask at room temperature for 15 minutes

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Repeat the freeze and thaw once more, for a total of two freeze/thaw cycles

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Vigorously pipette the cells up and down about 25 times

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Pool the contents of all infected flasks and transfer to sterile 50 mL centrifuge tubes

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Centrifuge at 400 × g for 20 minutes

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Gently transfer the supernatant to a new tube and discard the pellet; filter the supernatant through a 0.22 µm filter

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Dispense the cell-free viral stock into cryogenic vials in 1 mL aliquots; label each vial with the name of the virus, the lot number, the date and the initials; store at −70°C

Preparing plates for the plaque assay

Warm growth medium, PBS and trypsin to 37°C in a water bath

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Remove the flask from the incubator and perform all work in the biological safety cabinet

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Remove and discard the growth medium

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Add 10 mL of PBS; tilt the flask to distribute the PBS over the surface of the cell monolayer

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Remove and discard the PBS

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Add 2 mL of trypsin to the flask

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Incubate the flask at room temperature for 30 seconds, or until the cells are easily detached

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Keeping the flask level and the cells covered with liquid, tap the side of the flask sharply to dislodge the cells

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Add 8 mL of growth medium to the flask

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Pipette up and down 10–15 times to break up cell clumps, avoiding foaming; direct the liquid onto the coated surface of the flask (where the cells were adhered)

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Transfer the cell suspension to a sterile 50 mL centrifuge tube; if multiple flasks are being split, combine the cell suspensions at this point

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Pipette 0.5 mL of cell suspension into a microcentrifuge tube; add 0.5 mL of trypan blue solution and mix with a pipette; leave in contact for 2 minutes (but not more than 15 minutes)

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Count the cells as described in the splitting procedure (hemacytometer count, cell count formula)

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Dispense 5 × 10⁵ cells per well of a 12-well plate

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Add additional growth medium to each well to make a total volume of 1 mL (the cells could alternatively be diluted to 5 × 10⁵ cells per mL and 1 mL added to each well)

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Mix gently by moving the plate in a circular motion without splashing

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Incubate the plates at 37°C with humidity and 5% CO₂ for 24 hours

FCV titre by plaque assay

Warm EBSS, PBS and 2X overlay medium to 37°C in a water bath

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Autoclave the 1.2% type II agarose solution for 5 minutes and equilibrate to 45°C in a water bath

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Immerse one vial of FCV in the 37°C water bath until just thawed

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Make 10-fold serial dilutions of the virus in EBSS: combine 900 µL EBSS and 100 µL of FCV in a microfuge tube; cap tightly, vortex and spin briefly to remove viral solution from the underside of the cap; combine 900 µL of EBSS and 100 µL of the previous FCV dilution in a microfuge tube; cap tightly, vortex and spin briefly; repeat until you have made dilutions one order of magnitude past the expected titre of the virus (for example, if you expect 10⁷ PFU, dilute to 1/10⁸)

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Remove the plates from the incubator and perform all work in the biological safety cabinet

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Remove and discard the medium from each well

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Add 1 mL PBS to each well

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Remove and discard the PBS from each well

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Inoculate each well with 100 µL of a diluted virus solution — keep 3 wells uninfected (EBSS only) and 3 wells of each dilution to test. For a new virus stock, try 3 wells with 1/10⁵, 3 wells with 1/10⁶ and 3 wells with 1/10⁷ to make up one plate

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Rock the plate gently to spread the virus evenly; watch carefully to ensure that the virus-containing medium covers the cell monolayer instead of staying around the edges of the wells

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Put the plates in the 37°C incubator with 5% CO₂ for one hour, rocking to ensure even viral distribution every 10 minutes

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Mix the agarose and the 2X overlay medium 1:1 to make the overlay medium; gently dispense 2 mL of overlay medium to each well (use the prepared overlay medium within 10 minutes to prevent premature setting)

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Allow the overlay to solidify at room temperature for 15–20 minutes

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Incubate the plates at 37°C with humidity and 5% CO₂ for 2 days

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Remove the plates from the incubator; fill the wells with 3.7% formaldehyde solution in a fume hood and leave at room temperature overnight in the fume hood

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Remove the overlay and formaldehyde from the wells by breaking through the agarose plug using an electric pump aspiration system; plugs that do not dislodge may be loosened by tapping the plate upside down on a paper towel

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Dispense enough 0.1% crystal violet solution into each well to cover the bottom surface

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Leave at room temperature for 20 minutes

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Remove and discard the stain; rinse off excess stain with tap water; allow the plates to dry and count the zones of clearing in each well

Calculation of the virus titre

PFU/mL = plaque count × 0.1 × dilution factor

• plaque count = the number of zones of clearing counted in the well
• 0.1 = the constant given in the method
• dilution factor = the dilution of the virus suspension inoculated into the well

5. Reagent preparations (per the source method)

5.1 10X Minimal Essential Medium (MEM)

• Dissolve the entire contents of the 10X Minimal Essential Medium with Earle’s salts powder (Invitrogen product number 11700-077 or equivalent) in 1 L of sterile double-distilled water or equivalent.
• Filter-sterilize through a 0.22 µm filter into a sterile container.

5.2 7.5% sodium bicarbonate solution

• Dissolve 7.5 g of sodium bicarbonate in 100 mL of double-distilled water or equivalent.
• Filter-sterilize through a 0.22 µm filter into a sterile container.

5.3 Growth medium

• Mix: 100 mL of 10X MEM; 755 mL of double-distilled water or equivalent; 15 mL of 7.5% sodium bicarbonate solution; 10 mL of non-essential amino acids (100 mM); 10 mL of penicillin/streptomycin solution (100X); 10 mL of L-glutamine (200 mM).
• Filter-sterilize through a 0.22 µm filter into a sterile container.
• Prior to use, add 100 mL of sterile fetal bovine serum.
• The growth medium expires one month after preparation. Write the date of preparation, the date of expiration and the initials on the bottle.

5.4 Maintenance medium

• Mix: 100 mL of 10X MEM; 835 mL of double-distilled water or equivalent; 15 mL of 7.5% sodium bicarbonate solution; 10 mL of non-essential amino acids (100 mM); 10 mL of penicillin/streptomycin solution (100X); 10 mL of L-glutamine (200 mM).
• Filter-sterilize through a 0.22 µm filter into a sterile container.
• Prior to use, add 20 mL of sterile fetal bovine serum.
• The maintenance medium expires one month after preparation. Write the date of preparation, the date of expiration and the initials on the bottle.

5.5 Overlay medium

• For 2X overlay medium, mix: 100 mL of 10X MEM; 344 mL of double-distilled water or equivalent; 15 mL of 7.5% sodium bicarbonate solution; 10 mL of non-essential amino acids (10 mM); 10 mL of L-glutamine (200 mM); 3.7 g of magnesium chloride; 1 mL of gentamycin solution.
• Filter-sterilize through a 0.22 µm filter into a sterile container.
• Prior to use, add 20 mL of sterile fetal bovine serum.
• (The source carries the expiry note here as “The maintenance medium expires one month after preparation. The date of preparation, the date of expiration and the initials must be written on the bottle.”)
• For 1.2% agarose, mix: 6 g of type II agarose; 500 mL of double-distilled water or equivalent. Autoclave for 5 minutes and equilibrate to 45°C. Do not re-autoclave or re-dissolve agarose that has solidified.
• For the overlay medium, mix: 25 mL of 2X overlay medium; 25 mL of 1.2% agarose.
• Use the prepared overlay medium within 10 minutes to prevent premature setting.

5.6 Freezing medium

• Mix: 8.5 mL of 10X MEM; 61.5 mL of double-distilled water or equivalent.
• Filter-sterilize through a 0.22 µm filter into a sterile container.
• Prior to use, add 20 mL of sterile fetal bovine serum and 10 mL of sterile DMSO.
• The freezing medium expires one month after preparation. Write the date of preparation, the date of expiration and the initials on the bottle.

5.7 0.1% crystal violet solution

• Mix: 0.1 g of crystal violet; 100 mL of double-distilled water or equivalent.
• Filter-sterilize through a 0.22 µm filter into a sterile container.

5.8 3.7% formaldehyde solution

• Mix: 50 mL of 37% formaldehyde; 450 mL of double-distilled water or equivalent.
• Formaldehyde is very toxic and corrosive — handle in a fume hood with nitrile gloves (see section 6).

6. Precautions

• The laboratory supervisor must ensure the analysis is done in accordance with the International Standard ISO/IEC 17025:2005 (or latest version): General requirements for the competence of testing and calibration laboratories.
• It is each laboratory’s responsibility to ensure incubators, block heaters and water baths are maintained at the recommended temperatures: where 37°C is recommended the water bath must be at 37°C ± 1°C; for all other temperatures it must be ± 2°C.
• Use of cryotubes in the liquid phase of liquid nitrogen may cause entrapment of liquefied nitrogen inside the vial and lead to pressure build-up, resulting in possible explosion. Wear gauntlets and eye protection at all times when working with liquid nitrogen or materials stored in liquid nitrogen.
• Liquid nitrogen is extremely cold (−196°C): contact with skin causes chilblains and burn injuries. Use in a well-ventilated area to prevent asphyxiation with vapours.
• Formaldehyde is very toxic (inhalation hazard, skin contact, skin sensitizer, possible mutagen and a cancer hazard) and corrosive (eyes, skin and some metals). It evaporates readily and is explosive. Handle in a fume hood with nitrile gloves. Ensure that all formaldehyde has vaporized from biohazardous waste before autoclaving.
• Procedures involving cells and virus are carried out in a containment laboratory (level II) under a biological safety cabinet with sleeve protectors and gloves.
• All procedures involving uninfected cell culture must be carried out in a dedicated non-infectious tissue culture section in the laboratory.
• To avoid cross-contamination, maintain separate areas for the growth and manipulation of infected and uninfected cells; if a separate room is not available, distinct biological safety cabinets and incubators are recommended.

References

• Health Canada, Compendium of Analytical Methods, Volume 5 — Laboratory Procedure OPFLP-08, March 2010: “Propagation and quantification of feline calicivirus using cell culture methods.”
• Bidawid, S., et al. 2003. A feline kidney cell line-based plaque assay for feline calicivirus, a surrogate for Norwalk virus. Journal of Virological Methods, 107: 163–167.
• Crandell, R. A., et al. 1973. Development, characterization, and viral susceptibility of a feline (Felis catus) renal cell line (CRFK). In Vitro, 9: 176–185.
• Master, W., et al. 1999. UKCCCR Guidelines for the Use of Cell Lines in Cancer Research (First Edition), London.