Cellular Products: somatomedin like multiplication stimulating activity （MSA）
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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 approximay 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.
Subc*tion Ratio: A subc*tion 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
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