Fibronectin associated with the glial component of embryonic brain cell cultures

In a basic approach to investigations of neuronal–glial interactions during both normal brain development and its pathogenesis, embryonic brain cell populations were fractionated into purified neuronal and glial components. Using separation procedures based on differential adhesion and cytotoxicity, the isolated neuronal and glial phenotypes could be identified by distinct morphological and biochemical characteristics, including the visualization of glial fibrillary acid protein (GFA) within glial cells in immunohistochemical assays with monospecific anti-GFA serum. When unfractionated cerebrum cells dissociated from 10-day chick or 14-day mouse embryos were plated as monolayers and cultured for 1-14 days, monospecific antiserum against fibronectin (LETS glycoprotein) was found to react with many, but not all, of the cells as revealed by indirect immunofluorescence microscopy. The isolated neuronal and glial components of these populations were used to determine whether the appearance of membrane-associated fibronectin was characteristic of one cell type or the other, or both, and if neuronal–glial cell interaction was required for its expression. It was found that the surfaces of glial cells, completely isolated from neurons, showed an intense fluorescent reaction to the anti-fibronectin serum. In contrast, the purified neuronal cultures showed no fluorescence with either the anti-GFA or anti-fibronectin sera. These results demonstrate fibronectin as a cell surface protein associated primarily with glial cells and independent of neuronal–glial cell interaction for its expression. Furthermore, the results indicate that the fibronectin observed on glial cell surfaces in these cultures is produced endogenously and is not due to the preferential binding of fibronectin present in the culture medium. The role of fibronectin as an adhesive molecule in neuronal–glial interactions is discussed.     

In vitro development of theDrosophila chorion in a chemically defined organ culture medium

Summary TheDrosophila chorion is produced normally in isolated follicles in Robb's chemically defined culture medium. The complex architecture of the shell developed in vitro from follicles as young as early stage 10 is completely normal morphologically. In addition, the time required for in vitro development closely approximates that observed for in vivo development. Comparisons of insect culture media developed by Robb, Grace, Schneider, and Echalier show large variations in their ability to supportDrosophila chorion development.     

Transfer of cultured rabbit embryos

Embryo transfer experiments were carried out to study the developmental capacity of cultured rabbit embryos when transferred to recipients of variable postovulatory maturity. Rabbit embryos were flushed from the oviduct at 26 hours postcoitum (pc) and cultured in a modified Ham's F-10 medium supplemented with bovine serum albumin (BSA) for a period of 70 hours. At 96 hours pc the cultured embryos, which ranged from the early morula to the expanding blastocyst stage, were transferred to pseudopregnant recipients mated to vasectomized males 36 to 96 hours prior to the transfer procedure. Greatest embryo survival occurred when transfers were made to either the oviducts or uterine horns of recipients at 48 hours pc. Intermediate results for both implantation rates and number of young born were obtained with recipients at 36, 60, 72, and 84 hours pc. Transferred embryos consistently failed to survive the uterine environment of recipients 96 hours pc at transfer although this group was synchronous with embryonic chronological age. Oviductal transfers were generally more successful than uterine transfers. Markedly higher rates of embryo survival resulted from embryos that were collected 60 and 72 hours pc and transferred directly to synchronous recipients without an interim period of culture. Dissimilarity of development for in vivo grown rabbit embryos and those cultured in synthetic medium is demonstrated.     

The development of mouse spinal cord in tissue culture

Summary Whole mouse embryos were grown in vitro from Theiler stage 12 (1 to 7 somites) to Theiler stages 15 and 16 (25 to 35 somites). This procedure gives experimental access to precisely staged embryos during the early period of neurogenesis. To follow the further development of neurons in vitro, fragments of spinal primordia were set up from these cultured embryos. In such cultures, the proliferation of precursor cells, the formation of postmitotic cells and, finally, the cytodifferentiation of neurons were observed. A preliminary account of this work was given at the Tissue Culture Association Meeting in 1977, and the Canadian Federation of Biological Societies Meeting in 1977 (1,2). This work was supported by Grant MT 4235 from the Medical Research Council of Canada.     

Plasmodium falciparum in Culture: Improved Continuous Flow Method*

SYNOPSIS. A new design of flow vessel provides a method for continuous culture of P. falciparum in a settled layer of human erythrocytes with a slow flow of culture medium over them. The parasitemia is kept fluctuating from ? 1%, just after addition of fresh erythrocytes. to ? 10%, 2 or 3 days later. Each vessel provides each week 3 harvests, each containing ? 0.6–1 × 10⁹ parasites.     

Long-term culture of human aortas

Summary Segments of human thoracic aorta were maintained in long-term explant culture for 18 weeks in serum-supplemented medium. The aortas were grossly normal in appearance, and random samples fixed for light microscopy prior to culture revealed a normal morphology. The intima contained no more than five layers of smooth muscle cells. After 7 days in culture, the intima was noticeably thicker than the uncultured segments. The increased thickness was due to proliferating smooth muscle cells and production of extracellular material. After several months in culture, extracellular material consisting of collagen and flocculent material was present in areas resembling atherosclerotic fibrous plaques. A peripheral growth, which formed around the explant, was composed of fibroblastlike cells and added to the overall thickness of the intima. However, aortic segment maintained for up to 2 months in serum-free culture medium showed no cellular proliferation. This study demonstrates that changes resembling early stages of atherosclerosis occur in human aortas maintained in explant culture using routine culture procedures. Supported in part by the Pangborn Fund and the Graduate School of the University of Maryland. This is publication 443 from the Cellular Pathobiology Laboratory.     

Mutagenic effect on L5178Y mouse lymphoma cells by growth in ethylene oxide-sterilized polycarbonate flasks

Summary The mutation frequency of L5178Y mouse lymphoma cells to resistance to 5′-bromo-2′-deoxyuridine increased 6-to 14-fold after growth in ethylene oxide-sterilized polycarbonate culture flasks compared to growth in glass flasks. No comparable increase was observed when L5178Y cells were growth in identical polycarbonate culture flasks sterilized by autoclaving.     

Proliferation and morphology of chick embryo cells cultured in the presence of horse serum and hemoglobin

Summary We have shown previously that hemoglobin greatly stimulates chick embryo cell proliferation in Eagle's minimal essential medium supplemented with horse serum. In the present study we compared the effects of horse serum plus 10 μM hemoglobin to those of fetal bovine serum on subcultures of chick embryo cells serially propagated at high cell densities. The cells became elongated in the presence of fetal bovine serum and their rate of proliferation progressively decreased, whereas they became polygonal in the presence of horse serum plus hemoglobin and proliferated well in successive cell passages. The polygonal cell obtained in the presence of horse serum plus hemoglobin rapidly elongated if cultured at low cell densities in the presence of fetal bovine serum, but, in contrast, elongated cells did not yield polygonal cells if cultured at low densities in the presence of horse serum plus hemoglobin. It is possible that the polygonal and elongated cells are undifferentiated cells and differentiating myogenic cells, respectively.     

Long-term in vitro cell culture of sinclair swine melanoma

Summary The melanoma of Sinclair swine exhibits several characteristics similar to human melanoma but demonstrates an unusually high incidence of spontaneous regression. A total of 66 finite cell lines derived from 21 swine melanotic lesions, both cutaneous and visceral, were studied in vitro over their life spans of up to 14 months. The growth characteristics of the cultures varied with the age of the swine from which the tumors were obtained. Cell cultures of tumors obtained from swine aged less than 2 months grew steadily in culture with a population-doubling time of 120 to 180 hr until growth and division ceased after a maximum of 25 to 35 population doublings (6 to 8 passages). Cell culture of tumors obtained from swine aged 3 months or older showed a biphasic growth pattern with an early slow growth rate (population-doubling time 120 to 160 hr), which shifted after 3 to 6 passages to a faster rate (80 to 110 hr population-doubling time) until termination of growth and division after a maximum of 75 to 85 population doublings (18 to 20 passages). The cultures were morphologically heterogeneous including cuboidal, spindle and dendritic cell types. Electron microscopy showed classic melanosomes only in the primary and passage 1 cultures although vesicular inclusions were numerous in later-passage cells. However, continued melanin synthesis was indicated by the spectroscopic characteristics of material obtained from medium of passage 8 cultures and by DOPA staining of cultures as advanced as passage 18. This work was supported by a grant from the NIH, NCI (2 P01 CA 08023-11A1).     

Volatile emissions of plant tissue cultures

Summary The low-molecular-weight volatiles released by a variety of plant tissue cultures were examined by gas chromatography. Callus cultures invariably produced carbon dioxide, ethylene, acetaldehyde and ethanol. In cultures with developed shoots, ethanol was absent and acetaldehyde was detected only rarely.