Abstract
In earlier papers (1, 2) we reported chemically defined medium CMRL-1415 1 and described the preparation and use of certain macromolecular supplements, namely, α1-acid glycoprotein (orosomucoid) from the supernatant solution of Cohn's fraction V (method 6), sometimes referred to as fraction VI, of human plasma together with α2-macroglobulin from horse serum or with commercial dextran (mol wt 100,000–200,000). The latter combination (CMRL-1415-DSCV 1 ) yielded a population doubling time of 2.5 days for newly-isolated mouse embryo cells, whereas in replicate cultures prepared with unsupplemented CMRL-1415 it was 4.33 days.
Although many established cell lines have been adapted to serum-free, chemically defined media, newly-isolated mammalian cells in continuous culture require certain proteins that are usually supplied in the form of a native or dialyzed serum supplement in a suitable defined medium. The nature of these proteins and their nutritional role have not been fully elucidated. It is generally agreed that normal sera contain several proteins that greatly influence cell growth; and these proteins include both activators (3, 4, 6–10) and inhibitors (3–6) of growth.
In view of the multiplicity of these factors in serum, no single technique such as column chromatography or gel filtration will resolve them without certain preliminary separations. It is the purpose of this report to present a method for the separation of growth-active globulins from fetal calf serum. All operations were performed at room temperature unless otherwise specified. A few crystals of chlorobutanol were added as a preservative to all solutions during fractionation (except during the Rivanol 1 treatment). The method of separation is outlined in Table I.
Bentonite adsorption removes certain lipoproteins and clotting components (11, 12). The potassium phosphate precipitates the globulins, leaving most of the albumin in solution (13).
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