Studies of fluid exchanges between rat liver slices and simple media.
(25/1955)
1. The exchange of fluid between slices of rat liver and solutions of monosaccharides, disaccharides, and sodium chloride has been studied in relation to the temperature of incubation. The point of apparent isotonicity (P.A.I.) of the tissue was defined as the concentration of the solution in which the slices neither gained nor lost weight after immersion for a period of 10 minutes. 2. In solutions of glucose, the P.A.I. of the slices was significantly lower at 4 degrees C. than at 20 degrees C. but similar at 20 degrees C. and 37 degrees C. Upon immersion for 15 to 60 minutes in 0.66 molar glucose the slices always swelled more at 37 degrees C. than at 20 degrees C. In solutions of sucrose change in the temperature of incubation was without effect on the hydration of the tissues. 3. In solutions of sodium chloride, the P.A.I. and the content of water, chloride, and sodium plus potassium were lower at 37 degrees C. than at 20 degrees C. 4. These findings emphasize the role of translocation of solute in providing an osmotic gradient for the movement of water between the tissue slices and the media. (+info)
The nutrition of animal tissues cultivated in vitro. IV. Amino acid requirements of chick embryonic heart fibroblasts.
(26/1955)
1. The amino acid requirements of freshly explanted chick embryonic heart tissues cultivated in completely synthetic media have been determined, employing a nutritional depletion technique. Arginine, histidine, lysine, tyrosine, tryptophan, phenylalanine, cystine, methionine, threonine, leucine, and valine were found to be essential. Serine, isoleucine, glycine, and glutamine were found to be non-essential. Glutamic acid, aspartic acid, alpha-alanine, proline, and hydroxyproline were found to be inhibitory in this test system. 2. A total amino acid level of approximately 100 mg. per cent was found to be optimal and DL-amino acids were found to be non-toxic, unless used in high concentrations. 3. A comparison has been made of the amino acid requirements of various types of tissue cultures, of the chick, and of man and certain differences in these requirements have been discussed. (+info)
Mucopolysaccharides produced in tissue culture.
(27/1955)
1. A method of mass tissue culture has been devised by which, in a relatively short period of time, samples large enough for chemical isolation of mucopolysaccharides can be obtained. 2. Chemical isolation of acid mucopolysaccharides from mass cultures of human fetal skin, human fetal bone, bovine fetal skin, and rat subcutaneous tissue has been carried out. It has been found that the fibroblasts of each of these tissues produce in tissue culture more than one mucopolysaccharide, namely, hyaluronic acid, and a chondroitin sulfate. 3. The chondroitin sulfate produced by fibroblasts of the above tissues in tissue culture was not fully sulfated. The possible significance of this finding is discussed. (+info)
Clonal growth in vitro of human cells with fibroblastic morphology; comparison of growth and genetic characteristics of single epithelioid and fibroblast-like cells from a variety of human organs.
(28/1955)
A methodology has been described for reliable cultivation in vitro of dispersed fibroblastic cells obtained from normal human organs. The procedure has permitted establishment of stable cell lines from almost every sample taken, among which the following organs were represented: skin, spleen, amnion, lung, liver, bone marrow, brain, muscle, and heart. Equally good growth has been achieved with cells from embryonic or adult tissues. The methods previously developed whereby single cells plated in Petri dishes grow into isolated macroscopic colonies can successfully be applied to the plating of human fibroblastic stocks. Plating efficiencies in the neighborhood of 50 to 60 per cent are readily achieved with such strains. The resulting colonies can be picked and clonal stocks established. Fibroblastic morphology is maintained in the colonies arising from every single cell of such clonal stocks. All of the single cells from epithelioid clonal strains also maintain their integrity throughout repeated subculture. Since the difference between clonal stocks of these two types is always maintained whenever the respective single cells are plated in the same medium, regardless of the previous history of these stocks, it may be concluded that a true genetic difference exists in these cell lines. In addition to the morphological differences between epithelioid and fibroblastic cell strains, the latter have more demanding nutritional requirements for single cell growth. Thus, single cells of fibroblastic lines almost never produce colonies with high efficiency unless the growth medium which is sufficient for epithelioid cells is supplemented with embryo extract, or a cell feeder layer. Fibroblastic cells are also more resistant to tryptic digestion of the bond uniting the cells to glass surfaces. By use of differential media, growth of both fibroblastic and epithelioid cells, respectively, has been obtained, from dispersed single cells obtained by trypsinization of a specimen of human embryonic lung. (+info)
The amino acid requirements of rabbit fibroblasts, strain RM3-56.
(29/1955)
Strain RM3-56 of rabbit fibroblasts was found to require arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine for growth in a medium containing 2 per cent dialyzed serum as the only undefined component. The requirement for serine is less specific than that of the other 13 amino acids and it is partially replaced by glycine, or alanine, or by several combinations of so called accessory amino acids. The concentrations of essential amino acids which permit maximal proliferation range from 0.005 to 0.3 mM. Cystine, glutamine, lysine, tryptophan, tyrosine, valine are toxic at concentrations of 5 mM. The rate of proliferation of RM3-56 in a medium containing all 14 essential amino acids is increased significantly by the addition of alanine and to a lesser extent by the addition of aspartic and glutamic acids and glycine. A deficiency of cystine or glutamine results in cellular degeneration within 3 to 5 days, whereas the cells remain in good condition for 2 to 3 weeks in the absence of each of the remaining 12 essential amino acids. The results obtained with RM3-56 are compared with strains HeLa, L, and U12, whose amino acid requirements have been investigated under similar conditions. (+info)
Microkinetospheres and VP satellites of pinocytic cells observed in tissue cultures of Gey's strain HeLa with phase contrast cinematographic techniques.
(30/1955)
By tissue culture methods and with the use of phase contrast, interference color contrast, and time-lapse cinematographic equipment, the activity of a cytoplasmic organoid, termed the microkinetosphere, has been followed and correlated with pinocytosis. A transformation of several microkinetospheres by coherence and coalescence into the solitary VP satellite was observed in cells undergoing pinocytosis in serum nutrients. A correlation of both of these structures to cytoplasmic organoids described by others, notably with the electron microscope, and a hypothesis on the nature of the microkinetosphere and its transformation was presented. (+info)
Electron microscopy of HeLa cells after the ingestion of colloidal gold.
(31/1955)
Tissue cultures of HeLa cells were grown in media containing colloidal gold, and after various intervals, the cells were fixed, embedded, and sectioned for electron microscopy. Uncoated grids with small holes were used in many of the experiments. Intracellular particles of gold were identified in areas surrounded by single membranes, in moderately dense granules, in globoid bodies, and in the cytoplasmic matrix. Gold particles were not found in typical mitochondria, Golgi complex, ergastoplasm (granular forms of endoplasmic reticulum), or nuclei. The phenomenon of pinocytosis was considered to be the most likely means by which the gold particles were ingested, and the locations of gold particles appeared to have significance concerning theories that membranous organelles of the cytoplasm may be derived from the cell membrane. (+info)
Action of x-rays on mammalian cells. II. Survival curves of cells from normal human tissues.
(32/1955)
Survival curves of normal human cells from a variety of tissues exposed to varying doses of x-irradiation have been constructed, which permit definition of the intrinsic radiation sensitivity of the reproductive power of each cell type. The mean lethal dose of x-irradiation for all the cells employed, including those from normal and cancerous organs, those exhibiting diploid and polyploid chromosome number; those from embryonic and adult tissues, including recently isolated cells and cultures which had been maintained in vitro for many years, and cells exhibiting either epithelioid or fibroblastic morphology, was found to be contained between the limits of 50 to 150 r. Other similarities in the pattern of radiation effects, such as giant formation and abortive colonial growth, in these cells and that of the HeLa S3, previously studied, confirm the hypothesis that the pattern of reaction to x-irradiation previously elucidated, is representatative, at least in over-all outline, for a large variety of human cells. While the radiation survival curves of various human cells are similar in the gross, small but important characterizing differences have been found. All epithelioid cells so far studied are approximately 2-hit, and more radioresistant than the fibroblast-like cells whose survival data correspond to a mean lethal dose of around 60 r, and which so far can be fitted by either 1-hit or 2-hit curves. The earlier prediction that the major radiobiologic damage to mammalian cells is lodged in the genetic apparatus was confirmed by the demonstration of high frequency of mutants among the survivors of doses of 500 to 900 r. All the data on the x-radiosensitivity of these cells can be explained on the basis of a defect resulting from primary damage localized in one or more chromosomes. These considerations afford a convincing explanation of several aspects of the mammalian radiation syndrome. (+info)