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Amino acid analyses of two biologically efficacious fractions from a peptone dialysate (PD) revealed high concentrations of glycine, presumably present in PD as glycine-rich peptides. Before undertaking further analyses of PD the growth-promoting abilities of several commercially available glycine peptides were tested with strain L (NCTC 2071) cells continuously subcultured in chemically defined medium NCTC 135, and with RH-PD cells which require peptone or peptone fractions for continued survival and proliferation. NCTC 2071 populations 5 to 10% greater than control cultures were obtained with 0.18 to 0.72mm peptide-supplemented medium NCTC 135; slightly better results were obtained with peptides containing an even number of glycine residues. No amplification of this response was observed after 28 to 29 weekly culture transfers in 0.72mm supplemented NCTC 135, and evidence suggested that the peptides were poorly hydrolyzed to monomeric glycine. Addition of the glycine peptides may permit use of a reduced minimum inoculum for NCTC 2071. Marked growth enhancement of peptone-dependent RH-PD cells was obtained only when glycine peptides were tested in the presence of 0.5 mg of PD per ml.

SummaryA non-dialyzable substance was recovered in crude form from growth fluid of mouse lung fibroblasts adapted to a serum-free medium. This substance was capable of replacing the serum protein normally required for human liver cell growth. The substance in the material from mouse lung cell growth fluid is heat labile. The crude material was heat coagulable with loss of growth promoting activity for human liver cells. A graded dose response of growth of liver cells was obtained.

SummaryA biological titration of whole peptone dialysate (PD) demonstrated a broad concentration range over which large cell populations were obtained. Sephadex G-10 chromatography of PD resulted in six UV-absorbing fractions, three of which were recovered in sufficient quantities for biological analysis. Fractions G-10(I) and G-10(II) supported proliferation and survival, whereas fraction G-10(III) maintained cell populations at their seed inoculum level. Greater cell populations were observed in cultures supplemented with fraction G-10(II) than in those to which fraction G-10(I) was added.

SummaryBy means of an electrometric method, surface charge density was measured in rabbit, myocardial cells and mouse lung cells. As the pH was varied from 7.4 to 2.2, changes in the surface charge yielded a titration curve of the surface colloids. The isoelectric point of each surface is close to pH 2.2, and most of the titratable groups occur between pH 2.2 and 3.7. Net negative charges of 0.066 equivalents (myocardial cells) and 0.060 equivalents (lung fibroblasts) are found at pH 7.4 Bivalent cations, Ca and Mg, lower the negative charge through binding and electrostatic interactions. Incubation of myocardial cells with trypsin also diminishes the density of titratable groups. Interactions of cells with each other and with other organisms, macromolecules, and electrolytes are determined by the physicochemical properties of the amphoteric surface colloids.

Two mammalian cell lines which multiply in vitro in culture medium devoid of serum were investigated for sensitivity to twice crystallized trypsin. The minimum trypsin concentration which showed a concomitant increase in cell numbers and detachment from the surface of the culture vessel in one cell line was 0.01 μg per ml or approximately 10−8 μg per cell. These effects could be neutralized by normal human or calf serum at a dilution of 1∶500. The second cell line, which normally grows in suspension, was unaffected by these concentrations of trypsin.

Electron micrographs of acinar cells of the submandibular glands of CFW mice infected with murine cytomegalovirus, Smith strain, were studied. The resulting viral intracytoplasmic inclusion bodies were classified into two groups termed "light" (relatively electron-lucent) and "dark" (relatively electron-dense). It appeared that the "dark" type was the late stage of inclusion body maturation.

A low passage rat liver cell line demonstrated in vitro growth stimulation when cultured in the presence of serum of homologous, partially hepatectomized rats. After 4-day incubation a 3.25-fold increase in the cell population was observed in cultures supplemented with posthepatectomy serum at a dilution of 1∶10. No response was observed with sham-operated animal serum. Continous cultures of Chang human liver and Don hamster lung cells were not responsive to the posthepatectomy serum. The limitations of tetraphenylboron as a dispersing agent for primary rat liver cells are discussed.

The biological efficacy of several commercially available peptones was evaluated for three continuous mammalian cell lines. Peptic peptone, a peptic digest of beef lung, and Bacto-peptone were found to be most suitable.

THE haemadsorption test1,2 has been found to be a simple and useful means of detecting foci of infection by myxoviruses in tissue culture monolayers. The same response may be observed in cell cultures whether these viruses are non-cytopathic3 or cytopathic. Briefly, the technique consists of decanting the growth fluid and covering the monolayer with a 0.4 per cent saline suspension of guinea pig or chicken red blood cells and allowing haemadsorption to proceed for 10 min. at 4° C. After two saline washes, the areas of haemadsorption are examined under low magnification.

AT the present time two well-defined systems of viral interference by cell products have been demonstrated in vitro. They are interferon1 and viral inhibitory factor2, the latter possibly being a variety of interferon. Both at least have in common the fact that they are produced in cell cultures infected with virus, active virus in the case of the latter, or inactivated where interferon is concerned. Other differences and similarities are discussed in a recent report by Ho and Enders3. It is well known that virus inhibitors are present in a variety of mammalian tissues and fluids4 and may be demonstrated either in vivo or in vitro.

THE use of tissue culture cells for the production of virus preparations and vaccines is increasing and will gain prominence in the future. A report has recently appeared concerning the possible use of cell cultures for the standardization of smallpox vaccines1. At present the cell lines used for the production of virus vaccines are grown in media which contain of necessity serum of one species or another2. Several cell lines which grow in media free of serum have been reported3, and the advantages that these cells might have for vaccine production are obvious. Their use, however, could well be limited by their ability to support virus growth to a high titre. This report describes a comparison of vaccinia growth in serum-free and serum-containing mouse lung cells.

NEARLY all media routinely used for cell culture contain from 5 to 40 per cent serum of a particular species of animal. As one might expect, there has been much research into the nature of the fraction or fractions of serum which form the active part of this material1–6. Despite this the exact nature of the activity of serum or its fractions is unknown.