Cultivation of mammalian cells in defined media with protein and nonprotein supplements.

An improved chemically defined basal medium (CMRL-1415) has been used to advantage in studying the effects on trypsinized, newly explanted mouse embryo cells of certain glycoproteins from plasma and serum, certain nonprotein macromolecules, and various combinations of these, in stationary cultures. When protein and nonprotein fractions were separated from fetal calf serum, the entire growth activity was found to be associated with the protein. When 100 mg % of dialyzed, freeze-dried, supernatant solution of Cohn's fraction V (method 6) from human plasma was used as a supplement for CMRL-1415, there was considerable improvement in the cultures; and seromucoid prepared from calf serum had a similar effect. Supernatant V was further fractionated by gel filtration to give a threefold concentration of growth activity in a single, highly purified alpha(1)-acid glycoprotein (orosomucoid). Starch gel electrophoresis of horse serum that was used to supplement the basal medium revealed a decrease of both alpha(1)-acid glycoprotein and alpha(2)-macroglobulin during the cultivation of mouse embryo cells. When horse serum was fractionated on DEAE-cellulose columns, the only fraction that showed growth activity was a slow alpha(2)-globulin. When the alpha(2)-macroglobulin of Schultze was prepared from horse serum by salt precipitation, it was equally effective. When the alpha(2)-macroglobulin from horse serum was tested (at 100 mg %) in combination with alpha(1)-acid glycoprotein from Supernatant V, seromucoid from calf serum, or unfractionated Supernatant V, the growth response was greatly in excess of that produced by any of these supplements tested separately. The alpha(2)-macroglobulin from horse serum could be replaced by certain nonprotein macromolecules (e.g., dextran or Ficoll). Thus, dextran (mol. wt. 100,000 to 200,000) had no visible effect on the cells when used alone at 0.1 or 1%. But when these levels of dextran were used in combination with low molecular weight glycoproteins (e.g., unfractionated Supernatant V at 100 mg %), the cultures remained active and healthy for unusually long periods.