Robert E. Neuman

Serumless medium for cultivation of cells of normal and malignant origin.

Summary A serumless medium suitable for prolonged growth and repeated subculture of Walker carcinosarcoma 256, sarcoma 180, KB and human heart cells in vitro was described. In addition to amino acids, vitamins, minerals and glucose, it contained insulin, salmine sulfate, pyruvate, mucate, methyl oleate, folinic acid and lactalysate. The latter group of materials with exception of mucate was required for maximal growth rates, comparable to those on media containing dialyzed serum. Suspension cultures of Walker carcinosarcoma 256 were grown with continuous passage in the serumless medium.

A medium free of agar, serum and peptone for plaque assay of herpes simplex virus.

Summary Methods have been described for quantitative plaque assay of herpes simplex virus either in bottle culture or in Leighton tubes. The overlay employed was devoid of agar, peptones and serum, which are viewed as undesirable constituents. Use of agar overlay results in production of plaques from only a small fraction of the potential plaque-forming population. The inhibition of plaque formation by agar can be reversed by addition of protamine sulfate at a level of approximately 800 μg/ml. Possible uncertainties associated with the use of mixtures of agar and protamine can be avoided by substitution of methyl cellulose with which a further 10-fold increase in sensitivity of the assay was obtained.

Iron replacement of lactalysate and embryo extract in growth of cell cultures.

Summary Iron salts and iron-bearing materials replaced lactalysate in stimulation of growth of Walker carcinosarcoma 256 cells in serumless medium and replaced lactalysate and embryo extract in stimulation of chick embryo lung cultures in a similar medium with or without further addition of serum. Presumptive evidence is presented that a stimulatory activity of lactalysate is due to iron and iron-bearing materials. A specific function of lactalysate and embryo extract in overcoming toxicity of serum for embryo cell cultures was served equally well by iron or iron-bearing materials. The avian cells revealed in their incapacity to respond to transferrin a characteristic distinguishing them from the Walker tumor cells. We wish to acknowledge excellent technical assistance of Miss Joan L. Molowski.

Amino acid requirements for growth of avian lung cultures.

Summary Primary chick embryo and maturing cockerel lung cultures were found to grow in a test medium containing dialyzed chick embryo extract, bovine serum, and a variety of small molecular constituents. The avian cultures required the 13 amino acids usually found to be essential for mammalian cells. They also were markedly stimulated by glycine or L-serine and by small quantities of L-alanine. Folinic acid and higher levels of folic acid partially replaced the glycine requirement for optimal growth. Pyruvate, oxalacetate, or alpha-ketoglutarate, replaced the requirement for L-alanine. Pyridoxal phosphate partially replaced the L-alanine.

Inhibitory Effects of Hadacidin on KB Cell Cultures.

Summary The cytotoxicity of hadacidin in KB cell cultures was found to be potentiated by phenethylbiguanide as has been observed previously with known inhibitors of glycolysis. Glucose utilization and lactic acid production were inhibited in proportion to the cytotoxicity. The effects of hadacidin were antagonized by pyruvate, α-ketobutyrate, oxalacetate, and L-aspartate, but not by asparagine or pyrimidines. A variety of other metabolites, notably those in the Krebs tricarboxylic cycle, possessed some activity. Alpha-ketobutyrate and aspartate were additive in their capacity to overcome the cytotoxicity of hadacidin. Adenine at low concentrations effectively antagonized the cytotoxicity of hadacidin while adenosine, inosine, and hypoxanthine were ineffective. The data support interference with purine metabolism as a mechanism for cytotoxicity of hadacidin in tissue culture. In addition, interference with energy metabolism appears likely.

Partial Replacement of Serum and Embryo Extract Requirements for Growth of Avian Cell Cultures.

Summary Salmine, pyruvate, insulin and lactalysate or its derivatives have been shown to replace partially requirements of avian cell cultures for serum and embryo extract. In addition to these factors, however, serum was required for growth comparable to that attained in the basal medium containing both serum and embryo extract. A function of lactalysate and embryo extract was inhibition of a toxic effect of serum. The stimulatory and“anti-toxic” effects of lactalysate were confined largely to the non-dialyzable portion. Nevertheless, the activity of acid hydrolysates of the lactalysate residue demonstrated that these properties were not dependent on a proteose or peptone.

Stimulated Glycolysis of KB Cell Cultures by Guanidine Derivatives and Other Compounds Affecting Respiration.

Summary A variety of respiratory inhibitors, phosphorylation uncouplers, amidine and guanidine derivatives, including the hypoglycemic agents, synthalin and phenethyl-biguanide, appreciably stimulated lactic acid production and glucose utilization of cell cultures. The simplicity and effectiveness of the procedures described suggest their applicability to further studies of respiratory inhibitors, uncouplers of phosphorylation, and the evaluation of certain types of hypoglycemic compounds.

Stimulatory Effects of Glycine, L-Serine, Folic Acid and Related Compounds on Growth of Cell Cultures.

Summary Growth of the majority of a wide variety of primary and stable line cultures was stimulated significantly by inclusion of glycine or L-serine in the medium. Folinic acid or high levels (5-20 μg/ml) of folic acid also accomplished this effect for most cultures, although generally not to the same extent as did amino acids. The requirement of Walker carcinosarcoma 256 and of primary green monkey kidney cells for folic acid was fulfilled by glycine, thymidine, and adenine or several related compounds. L-serine could not replace glycine under similar conditions. The implications of these findings are discussed.

Potentiated Cytotoxicity of Glycolytic Inhibitors by Phenethylbiguanide in Cell Culture.

Summary Cultured cells exposed to phenethylbiguanide and other respiratory inhibitors or uncouplers of phosphorylation exhibited an increased susceptibility to cytotoxicity of certain inhibitors of glycolysis.

ANOMALOUS HEMADSORPTION BY CELL CULTURES.

Summary Chicken RBCs in the presence of calcium ion or after treatment with metaperiodate were absorbed by cultured primary and stable line monkey kidney cells and to a lesser extent by rabbit kidney cells. An extensive variety of other primary or stable cell lines of human or animal origin did not adsorb the cells. Treatment of responsive cells with crystallized trypsin or chymotrypsin inhibited the hemadsorption. Apparently the anomalous hemadsorption was unrelated to viral infection. The morphology of attachment varied with cell strain. BSC-1 cells which displayed few cytoplasmic projections adsorbed RBCs through extensive contact in contrast to adsorption by MK-2 cells by microvillae which characterized the surface of these cells.