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.

Abstract

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.