ZFL [ZF-L] (ATCC® CRL-2643)

Organism: Danio rerio, zebrafish  /  Tissue: liver  /  Disease: ­normal

Permits and Restrictions

View Permits

Organism Danio rerio, zebrafish
Tissue liver
Product Format frozen
Morphology epithelial
Culture Properties adherent
Biosafety Level 1
Disease ­normal
Age adult
Applications
The cells are useful for studies of liver cell metabolism and xenobiotic formation. They may be used for in vitro toxicology studies.
Storage Conditions liquid nitrogen vapor phase
Karyotype hypodiploid; modal number = 47
Images
Derivation The ZFL cell line was established in 1992 from a pool of approximately 10 normal adult zebrafish livers. It exhibits some properties characteristic of liver parenchymal cells.
Comments
The cells synthesize and release several proteins into the culture medium, including a 70 kDa protein recognized by anti-bovine serum albumin IgG.

A culture submitted to the ATCC in August of 2001 was found to be contaminated with mycoplasma. Progeny were cured by a 21-day treatment with BM Cycline.

The cells were assayed for mycoplasma, by the Hoechst stain, PCR and the standard culture test, after a six-week period following treatment. All tests were negative.

The dioxin, TCDD, induces one or possibly two forms of cytochrome P450 immunochemically and functionally related to rainbow trout P4501A1.

ZFL cell homogenates exhibit alanine and aspartate aminotransferase, glucose-6-phosphatase and alkaline phosphatase enzyme activities.

Complete Growth Medium These cells are grown in
  • 50 % L-15 (ATCC 30-2008)
  • 35 % DMEM HG (GIBCO 12100)
  • 15 % Ham's F12 (GIBCO 21700)
All without sodium bicarbonate Supplemented with:
  • 0.15 g/L sodium bicarbonate
  • 15 mM HEPES
  • 0.01mg/ml bovine insulin
  • 50 ng/ml mouse EGF
  • 5% heat-inactivated fetal bovine serum
  • 0.5% Trout Serum
Do not filter complete medium.
Subculturing Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.

  1. Remove and discard culture medium.
  2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
  3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
    Note: To avoid clumping, do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
  4. Add 6.0 to 8.0 mL of complete growth medium containing 10% heat-inactivated FBS and aspirate cells by pipetting gently.
  5. To remove trypsin-EDTA solution, transfer cell suspension to centrifuge tube and spin at approximately  125 x g for 5 to 10 minutes.
  6. Discard supernatant and resuspend cells in fresh serum-free growth medium.  Add appropriate aliquots of cell suspension to new culture vessels. 
  7. Place culture vessels in incubators at 28°C for 30 minutes.
  8. Examine to ensure attachment, and then add heat-inactivated FBS for a final concentration of 5% and Trout serum for a final concentration of 0.5%.

Subcultivation Ratio: 1:2 to 1:3
Medium Renewal: Every 2 to 3 days.
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells: a Manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.

Cryopreservation
Freeze medium: Culture medium supplemented with an additional 10% heat-inactivated fetal bovine serum and 5% DMSO
Storage temperature: liquid nitrogen vapor phase
Culture Conditions
Atmosphere: air, 100%
Temperature: 28°C; (Max. 29 °C, Min. 26°C)
Population Doubling Time 72 hours
Name of Depositor DW Barnes
Year of Origin 1992
References

Ghosh C, et al. Derivation and characterization of a zebrafish liver cell line. Cell Biol. Toxicol. 10: 167-176, 1994. PubMed: 7994634

Miranda CL, et al. Regulation of cytochrome P450 expression in a novel liver cell line from zebrafish (Brachydanio rerio). Arch. Biochem. Biophys. 305: 320-327, 1993. PubMed: 8373170

Collodi P, et al. Induction of zebrafish (Brachydanio rerio) P450 in vivo and in cell culture. Xenobiotica 24: 487-493, 1994. PubMed: 7975714

Notice: Necessary PermitsPermits

These permits may be required for shipping this product:

  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.
Basic Documentation
Other Documentation
References

Ghosh C, et al. Derivation and characterization of a zebrafish liver cell line. Cell Biol. Toxicol. 10: 167-176, 1994. PubMed: 7994634

Miranda CL, et al. Regulation of cytochrome P450 expression in a novel liver cell line from zebrafish (Brachydanio rerio). Arch. Biochem. Biophys. 305: 320-327, 1993. PubMed: 8373170

Collodi P, et al. Induction of zebrafish (Brachydanio rerio) P450 in vivo and in cell culture. Xenobiotica 24: 487-493, 1994. PubMed: 7975714