Donald J. Merchant

The relation between glucose utilization, lactic acid production and utilization and the growth cycle of L strain fibroblasts☆

Abstract 1. 1. Glucose and lactate metabolism of L strain fibroblasts growing in an agitated suspension was correlated with the growth phase of the culture and with changes in the average cell volume and pH changes in the medium. 2. 2. In agreement with previous work, it was found that the rate of glucose uptake and lactate production was most rapid during the first part of the logarithmic growth period. It was found, however, that lactate was taken up by the cells when glucose was still present in relatively high concentrations. During the last half of the logarithmic growth period, lactate was rapidly taken up and the rate of glucose uptake was low. Lactate was found to accumulate in the medium for the second time as the cultures entered the plateau phase of growth. Later in the plateau phase, lactate was reduced to very low values and glucose was exhausted from the medium after 192 hrs. 3. 3. From the observation that the pH decreased while lactate decreased in concentration it was concluded that other factors causing pH depression must have been functioning at that time. 4. 4. A calculation of the rate of cell carbohydrate metabolism indicated that there was no constant relation between the rate of carbohydrate uptake per cell and the rate of mitosis during progressive intervals of the growth cycle. It is hypothesized that the rate of cell carbohydrate metabolism is directly related to average cell volume.

Growth of L-M strain mouse cells in a chemically defined medium.

Summary Using the criteria of generation time, final population density, and chromosome analysis, it has been demonstrated that Eagle basal medium is adequate for continued propagation of L-M strain mouse cells both as monolayers and in suspension. Three distinct advantages to this system are: (1) the relative simplicity of the medium in terms of number of components as compared to other defined media currently available, (2) the 3- to 5-fold increase in cell population achieved in 7 to 10 days, in contrast with results using most defined media, (3) the growth of these cells in suspension culture, which is an ideal system for both nutritional and parasite-host cell relationship studies. The effectiveness of methylcellulose as a physical protective agent for cells grown in a simple, chemically defined medium has been demonstrated.

The mechanism of cell binding and cell-sheet formation in L strain fibroblasts

Abstract L strain mouse fibroblasts grow as single, rounded, independent units in suspension cultures containing horse serum. Serum is apparently not essential as a growth factor, since analogous and sustained growth can be obtained when methyl cellulose is substituted for serum in suspension culture. In the absence of either serum or methyl cellulose, binding occurs between adjacent cells and in suspension cultures cell sheets form. In this situation there is little or no net synthesis of DNA, RNA or protein. The protein involved in cell sheet formation is present on the surface of the cells at all times even when they are growing as rounded units in suspensions containing horse serum or methyl cellulose, since elaboration of fibers by such cells can be observed in a very short interval when they are transferred to saline. Binding between cells is dependent upon pH and these studies suggest that the phenomenon is due to cross linkage by polyvalent cations.

Amino acid utilization by L-M strain mouse cells in a chemically defined medium

SummaryThe amino acid requirements of strain L-M mouse cells grown in a chemically defined medium (2×Eagle) containing only the 13 essential amino acids (EAA) were investigated. Medium and acid hydrolysate samples were analyzed for amino acid content by the method of ion exchange chromatography. The extent of utilization of the EAA differed;e.g. after 120 hr of cell growth without medium change, glutamine was exhausted from the medium; methionine, leucine, isoleucine, cystine, arginine, and valine were depleted 60 to 80%; other EAA were used to lesser extents. Although the EAA were used in excess of their requirements for protein synthesis, a correlation could generally be made between utilization and protein amino acid composition.Glutamine appeared to be, a growth-limiting factor. Use of U-14C-labeled glutamine indicated that over one-half of the metabolized glutamine was converted to carbon dioxide, 17% to cell material, and 15% was extracted from the amino acid pools.Nonessential amino acids (NEAA), viz. alanine, aspartic acid, glutamic acid, glycine, proline, and serine, were released into the medium during growth, and some were reutilized. Exogenous provision of these did not improve cell growth. In contrast to the other NEAA, only serine showed net utilization when provided exogenously. When glutamic acid largely replaced the glutamine in the medium, it exerted a sparing effect on the glutamine requirement for protein synthesis. Suggestions are given for the improvement of Eagle medium for cell growth.

Propagation of strain L (Earle) cells in agitated fluid suspension cultures.

Summary The agitated culture has been used in studying the growth cycle of strain L (Earle) mouse fibroblasts. From data available, it is seen that mammalian cells growing in vitro display a growth pattern quite similar to that of other microbiological populations. Some points which have been studied and can now be evaluated are the following: 1) the lag period for these cells appeared to be eliminated completely when log-phase cells were used as inoculum in media equilibrated to pH 7,2) growth rate appeared to be independent of the inoculum size and also independent of serum concentration, 3) generation time for these connective tissue cells. under conditions studied, was approximately 40 hours: 2 generations could usually be observed in these cultures and saturation concentration in some instances reached 2,000.000 cells per ml, 4) steady-state cultivation of tissue cells has been approached. The results indicated that populations could be maintained in such a manner, and this problem will be investigated.

Inhibition of the Phagocytic Action of Leucocytes by Mumps and Influenza Viruses.

Summary Mumps virus and influenza A virus (PR8) have been shown to reduce the phagocytosis of bacteria by the polymorphonuclear leucocytes of the guinea pig in vitro. This inhibitory effect is destroyed by mild heating of the viruses which leaves their hemagglutinating capacity intact. Mumps antiserum prevents the inhibition of phagocytosis by mumps virus but has no effect on the inhibitory action of influenza virus.

Fiber formation in suspension cultures of L strain fibroblasts.

Summary The in vitro formation of fibrous membranes by the L and LLC-Ml strains of mouse fibroblasts in a protein-free medium has been demonstrated. When cells of either of these strains are suspended in medium 199 plus 0.5-1.0% peptone and incubated at 35°C on a rotary action shaker at 100 rpm, delicate membranes form at the liquid-air interface. Enzymatic digestion, staining reactions and electron micrographs suggest that the fibers are fibrous protein in nature and belong to the collagen class of protein. The possible relationship of this fibrous protein to the cell surface is discussed.

The plateau phase of growth of the L-M strain mouse cell in a protein-free medium : III. The effect of added glucose on protein and nucleic acid synthesis and on carborhydrate utilization

Abstract The addition of radioactive glucose to suspension cultures of L-M strain mouse cells during the plateau phase has been shown to extend the duration of this period from the normal 48–72 hr to 144–240 hr. Supplemental carbohydrate serves both as an energy source, as detected by the production of radioactive lactic acid and carbon dioxide, and as a precursor for nucleic acid and protein synthesis during this period. On the basis of the specific activities of the nucleic acids and proteins recovered from both the cells and the medium, possibilities of synthesis of multiple proteins and of extracellular RNA are discussed. The addition of radioactive homologous nucleic acidprotein fractions to culture in logarithmic growth is shown to reduce the mean generation time by 50 per cent. The recovery of 12 to 13 per cent of the radioactivity in these same fractions isolated from the cells at the end of logarithmic growth suggests that cells have the potential for re-utilization of cellular material release into the medium from other cells in the population.

Serologic and karyologic evidence of incorrect identity of an animal cell line (guinea pig spleen).

SummaryA cell line labeled GPS and presumed to be guinea pig spleen has been shown to be mouse and to carry the H-2k allele. Identification of characteristic chromosome markers suggests that the proper identity may be L-M mouse cells. The use of immunologic and karyologic identification procedures for routine checking of cell lines is discussed.

The plateau phase of growth of the L-M strain mouse cell in a protein-free medium: II. Prolongation of the plateau phase by supplemental glucose☆

Abstract Supplementation of glucose at the beginning of the plateau phase has been shown to extend the duration of this phase by as much as fourfold in suspension cultures of L-M strain mouse cells in a protein-free medium. The role of glucose seems to be, at least in part, one of sparing action on the cells by providing a readily available energy source. Continued cell synthetic activity also may be promoted. No net increase in cell numbers occurred. Decrease in the average cell size and loss of viability were significantly delayed while a cyclic pattern of cell size changes suggested a low level of population turnover and renewal.