Human recombinant insulin-like growth factor I. I. Development of a serum-free medium for clonal density assay of growth factors using BALB/c 3T3 mouse embryo fibroblasts.
A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on poly-D-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1:1 (vol/vol) mixture of Ham's F12 and Dulbecco's modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 micrograms/ml human transferrin, 100 micrograms/ml ovalbumin, and 1.0 microM dexamethasone. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-D-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold more cells than control cultures without IGF-I after 4 d. Deletion of BFGF resulted in no IGF-I stimulation of growth. The concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED50) ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin, ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED50 of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in culture.
Riss
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TL
University of Texas Medical School, Department of Biochemistry and Molecular Biology, Houston 77225.
Karey
K P
KP
Burleigh
B D
BD
Parker
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D
Sirbasku
D A
DA
D000818:Animals; D001681:Biological Assay; D002455:Cell Division / Q000187:drug effects; D003470:Culture Media; D003907:Dexamethasone / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005346:Fibroblast Growth Factors / Q000032:analysis*; D007328:Insulin / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000032:analysis* / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C / Q000196:embryology; D010047:Ovalbumin / Q000494:pharmacology; D011994:Recombinant Proteins; D013002:Somatomedins / Q000032:analysis*; D014168:Transferrin / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D003470:Culture Media; D007328:Insulin; D011994:Recombinant Proteins; D013002:Somatomedins; D014168:Transferrin; D005346:Fibroblast Growth Factors; D004815:Epidermal Growth Factor; D007334:Insulin-Like Growth Factor I; D003907:Dexamethasone; D010047:Ovalbumin; D014357:Trypsin