ATCC® Number:  CRL-1442™       
Designations:  BRL 3A 
Depositors:   SP Nissley, MM Rechler 
Biosafety Level: 1 
Shipped:  frozen 
Medium & Serum:  See Propagation 
Growth Properties: adherent
Organism: Rattus norvegicus （rat） 
Morphology:
 
Source: Organ: liver
Strain: Buffalo
Cellular Products: somatomedin like multiplication stimulating activity （MSA） 
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 CRL-1442 BRL 3A 大鼠肝细胞
Comments: The serum-free conditioned medium from this cell line is a source of MSA. MSA is a family of polypeptides that can partially satisfy the serum reguirement of chick embryo fibroblasts. [1060]
Propagation:  ATCC complete growth medium: Minimum essential medium （Eagle） with 2 mM L-glutamine ａnd Earle＇s BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, ａnd 1.0 mM sodium pyruvate, 90%; fetal bovine serum, 10%
Atmosphere: air, 95%; carbon dioxide （CO2）, 5%
Temperature: 37.0°C
Growth Conditions: For 24 hrs after initiating culture from a thawed ampule or after subculturing, grow the cells in culture medium with 5% fetal bovine serum. After 24 hrs, the cells may be transferred to serum-free medium.
Subculturing:  Protocol:
Remove ａnd discard culture medium.
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.
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.
Add 6.0 to 8.0 ml of complete growth medium ａnd aspirate cells by gently pipetting.
To remove trypsin-EDTA solution, transfer cell suspension to centrifuge tube ａnd spin at approximately 125 xg for 5 to 10 minutes.Discard supernatant ａnd resuspend cells in fresh growth medium. Add appropriate aliquots of cell suspension to new culture vessels.
Incubate cultures at 37°C.

Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:6 is recommended
Medium Renewal: 2 times per week
Preservation:  Freeze medium: Complete growth medium supplemented with 10% fetal bovine serum ａnd 5% DMSO
Storage temperature: liquid nitrogen vapor phase
Related Products: Recommended medium （without the additional supplements or serum described under ATCC Medium）:ATCC 30-2003
References: 1060: Nissley SP, et al. Proliferation of buffalo rat liver cells in serum-free medium does not depend upon multiplication-stimulating activity （MSA）. Cell 11: 441-446, 1977. PubMed: 302146
22582: Coon HG, Weiss MC. A quantitative comparison of formation of spontaneous ａnd virus- produced viable hybrids. Proc. Natl. Acad. Sci. USA 62: 852-859, 1969. PubMed: 4308097
22711: Dulak NC, Temin HM. A partially purified polypeptide fraction from rat liver cell conditioned medium with multiplication-stimulating activity for embryo fibroblasts. J. Cell. Physiol. 81: 153-170, 1973. PubMed: 4735141
22712: Dulak NC, Shing YW. Large scale purification ａnd further characterization of a rat liver cell conditioned medium multiplication stimulating activity. J. Cell. Physiol. 90: 127-130, 1977. PubMed: 833209
22762: Schalch DS, et al. Nonsuppressible insulin-like activity （NSILA）. I. Development of a new sensitive competitive protein-binding assay for determination of serum levels. J. Clin. Endocrinol. Metab. 46: 664-671, 1978. PubMed: 755052 
 
 CRL-1442 BRL 3A 大鼠肝细胞