Days In Tissue Culture Research Articles

A simple method for differentiating vascular smooth muscle cells and fibroblasts in tissue culture.

The morphologic differentiation of vascular smooth muscle cells and fibroblasts in tissue culture is difficult if not impossible. By direct immunofluorescence, it is possible to distinguish between vascular smooth muscle cells and fibroblasts after 6 to 10 days in tissue culture. Microfilaments appear from the 6th to the 10th day. After an incubation period of 30 minutes with antibody against smooth muscle actomyosin at room temperature, microfilaments are demonstrable in smooth muscle cells. In contrast, fibroblasts, if incubated for the same period, show strong nuclear fluorescence and a primary fluorescence of the cytoplasm, but filaments are not visible. If fibroblasts are incubated with antiactomyosin for one hour at 37 degrees C, however microfilaments are easily detectable. With this method it is possible to differentiate in a simple manner vascular smooth muscle cells from fibroblasts in a heterologous tissue culture.

Evidence for the clonal abortion theory of B-lymphocyte tolerance.

This paper deals with the behavior of adult mouse bone marrow cells placed in tissue culture with or without antigen, and subsequently assessed for immune competence after adoptive transfer into lethally X-irradiated, syngeneic hosts. Attention was focussed on B lymphocytes through using hapten human gamma globulin (HGG) preparations as putative tolerogens in tissue culture, the T-cell-independent antigens DNP-POL and NIP-POL as challenge injections in adoptive hosts, and numbers of hapten-specific PFC in host spleens for the quantitation of immune competence. It was found that the capacity of bone marrow cells to mount an adoptive immune response rose by a factor of about fivefold over 3 days in tissue culture. This rise was completely abolished by the presence in the culture of hapten-HGG conjugates with about one mole of hapten per carrier molecule. The prevention of the emergence of immune competence amongst maturing B cells was termed clonal abortion tolerogenesis. Dose-response studies showed the lowest effective antigen concentration to be between 2.5 times 10- minus 10 and 2.5 times 10- minus 9 M, and a standard concentration of 2.5 times 10- minus 8 M was chosen as producing near maximal effects. The tolerance was antigen-specific and time-dependent, being maximal only when antigen was present continuously as the cultured cells was maturing. It did not depend on the presence of T lymphocytes in marrow, and was not of an "infectious" type. In contrast to tolerogenesis of mature B lymphocytes by high antigen concentrations, it could not be abolished by lipopolysaccharide. We speculate that clonal abortion may be a tolerance mechanism of great physiological significance for self-recognition, and discuss the results in the framework of other recent tolerance models, including those involving receptor blockade and suppressor T cells.

Progesterone, androstenedione, testosterone, estrone, and estradiol synthesis in hamster ovarian follicle cells.

Isolated granulosa cells and theca from proestrous hamsters alone and in recombination, were cultured in order to study steroidogenic capacity of this tissue. Cells from medium size antral follicles (100-300 mum diam.) and large preovulatory follicles (500 plus mum diam.) were used. Steroids were measured by radioimmunoassay. Cultures of cells derived from both sizes of follicles made significant amounts of progesterone for up to 6 days in tissue culture. The preparations from the medium sized antral follicles synthesized little or no estrogen. Of the cells harvested from the preovulatory follicles, the granulosa and theca made moderate amounts of estradiol-17beta while the recombined system made similar to 5 times the estradiol-17beta made by theca or granulosa alone. The results indicate that in the in vitro system used: 1) The hamster follicle cells are similar to other species in that they spontaneously luteinize in culture and secrete large amounts of progesterone, 2) Androgen accumulation is greatest in media from cultured theca of preovulatory follicle, 3) A synergism between theca and granulosa of the large preovulatory follicle exists to effect maximal estrogen synthesis, and 4) Estrogen synthesis is short-lived in vitro in contrast to continued progesterone production.

Schilder's disease: abnormal cholesterol retention and accumulation in cultivated fibroblasts.

Extract: Schilder's disease (SD) with adrenal insufficiency is a fatal X-linked recessive disorder for which the basic defect is as yet unknown. To investigate the hypothesis that Schilder's disease might be associated with a generalized defect in cholesterol metabolism, the uptake and disappearance of labeled cholesterol and cholesterol esters were studied in cultivated fibroblasts from patients with Schilder's disease, patients with familial hypercholesterolemia, and normal control subjects. Over a 10–12-day period of exposure to the labeled compound, SD fibroblasts accumulated significantly more cholesterol than did normal fibroblasts (P < 0.001). In addition, after a 24-hr exposure to the labeled cholesterol, the SD fibroblasts retained significantly more of the label than did control cells over a 5-day period of study (P < 0.001). The retained label was still located in the cholesterol fraction on thin layer chromatography in both SD and normal cells. The total cholesterol content of SD cells when measured directly after 12 days in tissue culture was significantly greater than that of normal cells.Speculation: The results of these studies support the concept that Schilder's disease may indeed be related to a generalized defect in cholesterol metabolism or transport. An alternative explanation for these findings is that Schilder's disease may be associated with a defect in membrane structure or turnover that is not directly related to cholesterol metabolism but can be followed in vitro by cholesterol accumulation and retention.

Light and electron microscope study of neurons and synapses in neonatal mouse olfactory bulb cultured in vitro

Explants of neonatal mouse olfactory bulb were maintained for 7–60 days in tissue culture, and the morphology of neurons and synapses were studied by Bodian's silver method and electron microscopy. In Bodian silver preparations two principal types of neurons, mitral cells and granule cells, were identified. Electron microscopic observations revealed that the neurons contained the orgelles usually found in adult neurons. Besides conventional axosomatic and axodendritic synapses, four types of atypical synaptic junctions were demonstrated: (a) Somatoaxonic or somatodendritic synapses; (b) reciprocal synapses which were identified as the dendrodendritic synapses; (c) serial synapses; (d) synapses in which synaptic vesicles were localized on both sides of synaptic junctions. These findings, particularly the occurrence of the atypical synaptic junctions, indicate that isolated fragments of mouse olfactory bulb can be maintained for extended periods of time and achieve a remarkable degree of morphological development under tissue culture conditions.

Human lymphocyte antigen reactivity modified by neuraminidase.

Human lymphocytes treated with neuraminidase (from Vibrio cholera) are more susceptible to lysis with antiserums directed against HL-A antigens in the cytotoxicity test than are the corresponding cells incubated in buffer. Enzymetreated cells are also lysed by antibodies other than those directed against HL-A, but control cells are not. The extra sensitivity to antibody disappears after 2 to 6 days in tissue culture.

RETENTION OF MULTIPLE DEVELOPMENTAL POTENTIALITIES BY CELLS OF A MOUSE TESTICULAR TERATOCARCINOMA DURING PROLONGED CULTURE in vitro AND THEIR EXTINCTION UPON HYBRIDIZATION WITH CELLS OF PERMANENT LINES

Ever since the early days of tissue culture, one of the chief debates has centered on the question of the maintenance and expression of tissueand organ-specific functions by animal cells grown in vitro (cf. ref. 1-4). This debate on versus dedifferentiation, often confused by lack of both adequate experimental data and adequate definitions, can now be considered as settled by recent demonstrations of the possibility, in at least a few cases, of obtaining cultures capable of (a) expressing, over prolonged periods of time, differentiated functions characteristic of the tissue of origin and (b) re-expressing them after growth under conditions interfering with their expression.5-s These experiments thus demonstrate that in vitro cultured cells can oscillate between a state of overt differentiation (i.e. active performance of a given function) and a state of covert maintenance of the potentiality to express the same function under appropriate conditions (called modulation by Paul Weiss). The mechanism underlying the two states is sought today in the type of phenomena of regulation of gene expression which has recently been uncovered in bacteria and with which they have many features in common.3 Research along these lines is in progress in this and several other laboratories (cf. ref. 10). The debate on differentiation-dedifferentiation in vitro has meanwhile ob-

Effect of inhibitors on the metabolism of cells in tissue culture and on foot-and-mouth disease virus synthesis

Metabolic studies have been made with suspensions of pig-kidney cells grown in tissue culture. Oxygen uptake, carbon dioxide output and glycolysis have been measured in the presence of arsenite, fluoride, cyanide, 2,4-dinitrophenol, and upon the addition of glucose, succinate and glutamate. The cells possessed the conventional glycolytic, the Krebs cycle and terminal oxidative pathways of metabolism, together with some aspects of respiratory control as demonstrated by the Pasteur and Crabtree effects. Cells between 2 and 9 days in tissue culture showed a progressive decline in oxygen uptake and an increase in glycolytic activity. It was found that the energy for a single multiplication cycle of foot-and-mouth disease virus could be supplied by the oxidation of glucose or of endogenous fat. This could be completely replaced by glycolysis in the presence of inhibitors of terminal oxidations. Arsenite and fluoride inhibition of virus multiplication was not primarily due to their action on the Krebs cycle and glycolysis respectively. Virus synthesis was inhibited by p-fluorophenylalanine but not by ethionine, 2,6-diaminopurine, azaguanine, thiouracil and azathymine.

Studies on Thrombopoiesis I. Thrombocytopoiesis in Vitro: Experiments with Animal and Normal Human Material

Abstract Thrombocyte production from megakaryocytes of healthy humans, dogs, guinea pigs and mice was observed continuously for one to six days in tissue culture. Approximately 70 per cent of the explanted megakaryocytes broke down to give rise to numerous platelets, while the remaining 30 per cent of the cells remained unchanged. The newly formed thrombocytes were separated from the rest of the bone marrow tissue, counted and their serotonin absorbing capacity determined. There was invariably a gradual increase in both the number of thrombocytes and in their serotonin absorbing capacity during the one to six days of observation. The results obtained were similar in human megakaryocytes and in those of experimental animals.

Effects of Osmotic Pressure on Normal and Malignant Fibroblasts

In view of the importance of osmotic pressure as a factor in the internal environment of the organism, the investigation here described was made for the purpose of noting the reactions of surviving and growing cells in vitro to changes in osmotic conditions. Little previous work has touched on this problem. Hogue in 1919 (1) grew tissue cultures of chick embryo tissues and established the limits of hypertonicity and hypotonicity in which they will survive and grow. She found that they grow (or at least migrate) more rapidly in slightly hypotonic solutions and grow well in a medium consisting of NaCl diluted to 0.5 per cent, and made a number of interesting morphological observations on cells grown under these conditions. In the early days of tissue culture, Lewis (2) had observed that tissue cells would migrate under a wide range of inorganic salt concentrations with various degrees of tonicity. Fischer (3) discusses the permeability of macrophages and fibroblasts briefly, with particular reference to the phenomenon of phagocytosis. Shear and Fogg, in two recent papers (4, 5), have made observations on the peripheral cells of excised tissues placed in various solutions, and noted in the case of malignant tissues and some normal mouse tissues that cells show globular swelling around their free margins in hypertonic and hypotonic solutions alike, beginning within the first few minutes of immersion and continuing for hours. This phenomenon can be retarded by increasing the protein content of the solution and is minimal in a solution of 10 or 15 per cent horse serum protein. Swellings which had already appeared in saline solutions were seen to disappear on increasing the protein content of the solutions.