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PubMed1965

MIXED-CULTURE INTERACTIONS. I. COMMENSALISM OF PROTEUS VULGARIS WITH SACCHAROMYCES CEREVISIAE IN CONTINUOUS CULTURE.

Shindala, Adnan (Virginia Polytechnic Institute, Blacksburg), Henry R. Bungay III, Noel R. Krieg, and Kathleen Culbert. Mixed-culture interactions. I. Commensalism of Proteus vulgaris with Saccharomyces cerevisiae in continuous culture. J. Bacteriol. 89:693-696. 1965.-A chemically defined medium was selected which supported the growth of Saccharomyces cerevisiae, but not Proteus vulgaris, in pure culture; however, P. vulgaris grew in mixed culture with the yeast. Steady-state populations of each organism in mixed culture at various dilution rates were enumerated with a Coulter electronic counter. The size differences in the organisms permitted easy resolution. An essential niacinlike factor elaborated by the yeast and required by the bacterium caused a dependence of the bacterium on the growth of the yeast. At high dilution rates causing wash-out, the bacterial population continued to reflect changes in the numbers of yeast. The numbers of S. cerevisiae were identical in pure culture or in mixed culture; thus, the interaction is a true commensalism. A single addition of niacin or related compound was made to a steady-state mixed culture, and the dependence of the bacterium on the yeast was upset. The numbers of the bacteria rose, causing a decrease in the yeast population, until continued pumping diluted the added niacin and restored the initial steady state.

Bacteria↗
PubMed1981

Organotypic culture of fetal rat lung: evaluation and comparison with organ culture.

Monodispersed suspensions of 19-day fetal rat lung when plated onto gelatin sponges reaggregated to form alveolarlike structures. These structures consisted almost entirely of alveolar type II cells, whereas the whole cultures contained about 65% type II cells. The phospholipid content and composition of the organotypic cultures after 8 days of incubation was very similar to that of explants of 19-day fetal rat lung grown in organ culture of 48 h, as was the pattern of incorporation of choline and acetate into phospholipids. Very little phosphatidylcholine was secreted by the organotypic cultures into the culture medium. Initial observations suggested that the organ cultures are more responsive to hormones. Organotypic cultures do not appear to offer any major advantage over the simpler and quicker organ cultures for studies of hormonal influences on fetal lung maturation. They may be useful for examining the metabolism and sympathetic pathways of the type II cell.

Acetates↗
PubMed2006

Congruence and functions of personal and cultural values: do my values reflect my culture's values?

Two studies are described examining the correlation between self- and culture-referenced values at a culture level (Study 1) and correlation between self- and culture-referenced values and self-reported behavior at an individual level (Study 2). It is found that values related to individual-group relationships (embeddedness) and expression and experience of affective feelings and emotions (affective autonomy) are significantly correlated at a culture level. In Study 2, culture-referenced values are shown to correlate with behaviors attached to social norms, whereas self-rated values are found to correlate with behaviors that are not norm-governed. Implications for measurement of cultural values and cultural and cross-cultural research designs are discussed.

Adult↗
PubMed1983

Differentiation-arrested rat fetal lung in primary monolayer cell culture. I. Development of a differentiation-arrested and growth-supporting culture system using carbon-stripped bovine fetal calf serum.

In vitro observations of fetal lung maturation are complicated by continued epithelial cell maturation in culture. Removal of trace quantities of cortisol from the culture system has been reported elsewhere by Torday to prevent epithelial cell differentiation, but also prevent cell growth. In this report we have developed monolayer cell cultures from 18-, 19-, 20-, and 22-day-gestation fetal rat lung in a culture system stripped of steroid and thyroid hormones. These cultures demonstrate lamellar body development comparable to that seen in vivo at the gestational age at which cultures were developed; the gestation-dependent increase in precursor incorporation into saturated lecithin observed in these cultures is similar to that reported in in vivo studies, yet cell growth in culture has been preserved.

Animals↗
PubMed1979

Long-term human peripheral blood monocyte cultures: establishment, metabolism and morphology of primary human monocyte-macrophage cell cultures.

Human peripheral blood monocytes were maintained in in vitro culture for periods up to 4 months using a non-human serum source. Monocytes were cultured in Dulbecco's modified Eagle's medium buffered with 20 mM HEPES and containing 10% horse serum and 10% foetal calf serum. The metabolic and morphological changes which occur in vitro were investigated using microtitre, Linbro and T 25 cultures. During culture, monocytes increased in size, had increased membrane activity as visualized by SEM, and differentiated into a morphologically heterogeneous population of fusiform and epithelioid shapes. These cell types retained the ability to phagocytose E glut and EA and to rosette with EA and EAC. Larger giant polynucleated cells were also observed during culture; many of these lacked the ability to bind or phagocytose inert or antibody-coated erythrocytes. Increases in lysozyme release and acid phosphatase activity also occurred during culture. Cultured monocytes exhibited characteristic profiles of leucine and uridine uptake with maximal activity observed by 5 days of culture. There was no detectable uptake of thymidine. Detailed analysis of regulatory processes involved in monocyte growth and differentiation could be performed with this in vitro system.

Cells, Cultured↗
PubMed1982

Primary epithelial cell cultures derived from canine prostate: isolation, culture, and characterization.

Epithelial-cell-enriched primary cultures were established from canine prostate. Minced tissue was dissociated with 750 units/ml of collagenase in F12K tissue culture medium containing 1% fetal bovine serum. This treatment resulted in the gradual removal of stromal elements from the base of the epithelial cells. After 30 minutes of digestion, aggregates of epithelial cells free of stroma were dislodged from the minced pieces of prostate. These aggregates were washed and plated at high density in F12K plus 10% fetal bovine serum. After 12-16 hours in vitro the unattached cellular aggregates were removed from the culture dishes, washed, and reinoculated into new culture vessels containing fresh medium. After 48 hours in vitro, the aggregates had attached to the culture vessels and spread out to yield discrete patches of epithelial cells. By 120 hours in vitro the patches of cells had grown and coalesced to form a confluent monolayer of epithelial cells. Ultrastructural examination of these cultures indicated that adjacent cells were joined by desmosomes and tight junctions and had tonofilaments and microvilli, giving the cells an epithelial appearance. The cells contained rough endoplasmic reticulum, Golgi apparatus, and secretory granules similar to those of the epithelial cells in the intact organ. In addition, intracellular "blebs" containing acid phosphatase were observed in the monolayers and were found to increase in size and number with time in vitro. Differentiated function of the cultures was demonstrated by the presence of ornithine decarboxylase and acid phosphatase and the ability of the cultures to metabolize testosterone to primarily 5 alpha-reduced metabolites.

Acid Phosphatase↗
PubMed1988

Serum-free culture of human hemopoietic progenitors in attenuated culture media.

To elucidate the precise mechanisms of molecular and cellular regulation of hemopoiesis, it is necessary to develop a chemically defined culture assay for purified hemopoietic progenitors. To approach this long-term goal, we attempted to develop a serum-free culture system for enriched human progenitors that permits expression of all hemopoietic lineages and stages of development. Preliminary studies indicated that alpha-medium was superior to Iscove's modified Dulbecco's medium (IMDM) and that culture under low (5%) oxygen condition was better than an ambient level of oxygen. We developed an attenuated (modified quarter-strength) alpha-medium and compared the colony-supporting ability of the three media by plating 1,000 bone marrow null cells per dish in the presence of a combination of recombinant human colony-stimulating factors (CSFs). The numbers of colonies supported in alpha-medium and attenuated alpha-medium were approximately 70% of those in serum-containing cultures. IMDM failed to support colony formation. While, in general, the colony sizes were smaller in the serum-free cultures than in the serum-containing cultures, a variety of types of single lineage and multilineage colonies were seen in serum-free culture. A linear relationship between cell number and colony formation was seen in 100-2,000 cells per dish. Serum-free cultures of enriched human progenitors should be an important tool for analysis of the mechanisms of recombinant CSFs.

Bone Marrow Cells↗
PubMed2005

Cell cycle analysis of Taxus suspension cultures at the single cell level as an indicator of culture heterogeneity.

Single cell growth and division was measured via flow cytometry in order to characterize the metabolic variability of Taxus cuspidata suspension cultures, which produce the valuable secondary metabolite Taxol. Good agreement was observed between the cell cycle distribution and biomass accumulation over the batch culture period. Specific growth rates of 0.13 days(-1) by fresh weight and 0.15 days(-1) by dry weight were measured. Elicitation with methyl jasmonate (MJ) significantly decreased both cell cycle progression and biomass accumulation, as the specific growth rate decreased to 0.027 days(-1) by fresh and dry weight. Despite the decrease in biomass accumulation for MJ elicited cultures, sucrose utilization was not significantly different from control cultures. MJ elicitation also increased the accumulation of paclitaxel and other taxanes. The accumulation of upstream taxanes (baccatin III and 10-deactylbaccatin III) increased during exponential growth, reached a maximum around day 12, and then declined throughout the stationary phase. The paclitaxel concentration increased during both exponential growth and stationary phase, reaching a maximum around days 20-25. Throughout the culture period, greater than 70% of the cells were in G(0)/G(1) phase of the cell cycle. Studies using bromodeoxyuridine (BrdU) incorporation showed that approximately 65% of the Taxus cells are noncycling, even during exponential growth. Although the role of these cells is currently unknown, the presence of a large, noncycling subpopulation can have a significant impact on the utilization of plant cell culture technology for the large-scale production of paclitaxel. These results demonstrate that there is a high degree of metabolic heterogeneity in Taxus cuspidata suspension cultures. Understanding this heterogeneity is important for the optimization of plant cell cultures, particularly the reduction of production variability.

Bromodeoxyuridine↗
PubMed2006

Examination of granule layer cell count, cell density, and single-pulse BrdU incorporation in rat organotypic hippocampal slice cultures with respect to culture medium, septotemporal position, and time in vitro.

Adult neurogenesis in the dentate gyrus is assuming an increasingly important role in supporting hippocampal-dependent learning and the modulation of mood and anxiety. Moreover, injury to the developing postnatal dentate gyrus has profound effects on neurogenesis and hippocampal learning throughout life. Organotypic hippocampal slice cultures represent an attractive model for studying neurogenesis both in the early postnatal and adult hippocampus, as they retain much of their anatomical and functional circuitry in vitro. Ongoing neurogenesis has been recently demonstrated in organotypic hippocampal slice cultures. However, cell proliferation, one of the critical components of neurogenesis, has yet to be characterized in this culture system. We examined single-pulse S-phase bromo-deoxyuridine (BrdU) labeling in the dentate granule layer with respect to the septotemporal position of origin of the slice culture, the medium the cultures were grown in, and the time the cultures were maintained in vitro up to 14 days, when they are believed to have matured to a near adult state. Using single 10-microm sections through a culture as our reference volume, we report significant effects of septotemporal position on the number of granule layer cells and the number of cells in S-phase, as estimated by short-survival (2 hours) BrdU studies. We report a declining rate of BrdU incorporation, evidence of significant structural changes within the granule cell layer, and differences in cell death between culture media over the first 14 days in vitro. We report caution with the use of BrdU cell density and changes in cell number to indirectly estimate proliferation.

Animals↗
PubMedDate not supplied

Culture of principal cells from the ram epididymis. A comparison of the morphology of principal cells in culture and in situ.

A procedure was developed to isolate and culture principal cells from the initial segment, central caput, distal caput, and proximal corpus epididymidis. The morphology of cultured cells and of cells in situ was compared. Over four to ten days, principal cells cultured in a floating collagen matrix with serum-free medium formed clusters that developed into either large sheets of columnar cells or tubular structures of cuboidal cells. Structural polarity was evident and junctional complexes reformed. The distribution and relative abundance of organelles in principal cells in situ differed depending on the region examined, and most of these regional characteristics were retained by principal cells in culture. Microvilli and membrane-bound vesicles were less conspicuous in cultured cells. Cultured principal cells were shorter than principal cells in situ, but were of similar volume. The high purity (90%) and viability (70%) of principal cells after seven to ten days in culture, their retention of morphologic characteristics unique to the region of origin, and the formation of function units are evidence that such cultures should be valuable for studying regional differences in the function of principal cells.

Animals↗
PubMed1989

Use of gel-well culture chambers as a liquid culture system to measure responses of hematopoietic colony-forming cells to growth factors.

This report presents the results of an investigation in which Gel-Well culture chambers were evaluated for their utility as a liquid culture assay system to measure the responses of hematopoietic colony-forming cells (CFC) to recombinant and cell-derived growth factors. Gel-Wells, designed for anchorage-independent cell growth and diffusion of media components, permitted the weekly replacement of media and growth factors without removing cells from the culture chambers. In these studies, changes in cellularity and CFC content in Gel-Well cultures of human umbilical cord blood cells induced by recombinant interleukin 3 (rIL-3) were quantified. After one week in culture without rIL-3, the number of erythroid burst-forming units (BFU-e) had decreased to 25 +/- 38% of pre-values. In contrast, addition of rIL-3 induced an increase in the number of BFU-e to 390 +/- 135% of pre-values. By three weeks with rIL-3, the number of granulocyte-macrophage colony-forming units (CFU-gm) had increased to 292 +/- 58% of pre-values. Also, the presence of a bone marrow stromal cell layer under the Gel-Well helped to maintain the survival of CFC in liquid culture. These studies demonstrated that Gel-Well culture chambers provide a useful liquid culture system for studying the responses of CFC to growth factors.

Culture Techniques↗
PubMed1982

The development of chick spinal cord in tissue culture. III. Neuronal precursor cells in culture.

On culturing fragments of neural tube of Hamilton and Hamburger (H & H) Stage 10 chick embryos, large multipolar neurons developed. The aim of this investigation was to determine whether these neurons in culture developed from dividing neuronal precursor cells, from postmitotic precursor cells, or both. Of the neurons formed during the 20 d of culturing in the presence of [3H]thymidine, 26% were unlabeled, indicating that they originated from cells that were already postmitotic at the time of explantation. By labeling cells of the neural tube in vivo and determining the total number of cells in the neural tube, we estimated that the neural tube of chick embryos of H & H Stage 10 contained approximately 1000 (3.3%) postmitotic cells. By estimating the total number of neurons that formed in 20-d cultures and the percentage of labeled and unlabeled neurons, we concluded that the postmitotic neuronal precursor cells survived well in cultures and proceeded on their predetermined path of differentiation. By considering the number of neurons found in the spinal cord in vivo and the number of labeled neurons found in cultures, we concluded that only a relatively small fraction of the dividing neuronal precursor cells entered the postmitotic stages of differentiation and formed neurons in cultures. The majority of cells that did this, entered the postmitotic stage of differentiation during the first 5 d in culture.

Animals↗
PubMed1984

Cell injury of cultured rat myocardial cells after reoxygenation of hypoxic cultures in the presence and absence of calcium.

Primary cultures of rat myocytes were deprived of oxygen (approximately 5 mm Hg, pO2) for 2 h in the presence or absence of calcium and were subsequently incubated for different time periods under normoxic (approximately 120 mm Hg, pO2) conditions. Myocyte calcium content was determined at the end of the oxygen-free period and after repletion of oxygen. Lactate dehydrogenase (LDH) release from the cells into the medium was used as an index of cell injury. Cultures deprived of oxygen in the absence of calcium showed a significant decrease in myocyte calcium content at the end of the oxygen-free period. However, the calcium content of myocyte cultures deprived of oxygen in the presence of calcium did not change significantly. The enzyme release of both groups of oxygen-deprived cultures was similar to that of controls. A progressive increase in myocyte calcium content was observed 5, 10, 15, and 30 min after repletion of oxygen in both groups. Reoxygenation of cultures subjected to hypoxia in the presence of calcium caused minimal LDH leakage. However, major enzyme release was observed 30 min after oxygen repletion in cultures previously subjected to hypoxia in a calcium-free environment. The present study shows that reoxygenation injury is more severe in myocyte cultures previously deprived of both oxygen and calcium than in cultures deprived of oxygen in the presence of calcium.

Animals↗
PubMed1983

Development of brown fat cells in monolayer culture. I. Morphological and biochemical distinction from white fat cells in culture.

The ability of cells from the stromal-vascular fraction of rat brown adipose tissue to develop into adipocytes in primary cell cultures was investigated. Comparison was made with precursor cells isolated by the same procedure from the white adipose tissue of the same animals and cultured in parallel under identical conditions. The culture procedure used allowed the cells isolated from both tissues to rapidly proliferate and differentiate. During the first week in culture the brown fat cells grew to confluence and accumulated fat in a multilocular way. During the second week, further fat was accumulated, but the cells remained multilocular. Analysis of the parallel white fat cell cultures revealed clear differences between the two adipocyte types, although the rates of cell growth were identical. Measurement of the size of the cellular lipid inclusions as a function of the time in culture indicated a much higher number of fat droplets larger than 30 micron in the white adipocytes. Moreover, after isolation of pelleting fractions of both cultured cell types, comparative functional analysis of their mitochondria by oxygen consumption measurement, as well as direct cytochrome-c-oxidase determinations, showed a significantly higher amount of mitochondria in the brown fat cell fractions than in the white fat cell fractions. It was concluded that mature brown fat contains precursor cells which can proliferate and develop into adipocytes in monolayer cell culture and which have inherent characteristics distinct from those of white fat precursor cells.

Adipose Tissue↗
PubMed1995

In vitro murine embryotoxicity of cyclophosphamide in embryos co-cultured with maternal hepatocytes: development and application of a murine embryo-hepatocyte co-culture model.

The technique of whole embryo culture provides a sensitive model to evaluate both the effects, and their underlying mechanisms, of drugs and environmental chemicals on embryonic development, independent of maternal influences. However, before teratogenic expression, many teratogens must be enzymatically bioactivated to toxic reactive intermediates. To detect such proteratogens, the embryo culture model may need to be coupled with an exogenous bioactivating system if maternal and/or placental metabolism is involved. We developed a similar embryo-hepatocyte co-culture system using embryos and maternal hepatocytes from mice, which often are more sensitive than rats to chemical teratogens, and which may have a balance of phase II drug metabolising enzymes more similar to humans. This murine system was then used to evaluate the relative maternal and embryonic contributions to cyclophosphamide embryopathy. Day 9.5 (morning of plug = day 1) murine embryos were co-cultured for 24 h in vitro with primary cultures of murine maternal hepatocytes (> 85% viability). Murine embryos were exposed to cyclophosphamide concentrations (0, 7.5, 15, 25 micrograms/ml), similar to those used in rat embryo culture studies. Murine embryos co-cultured with murine maternal hepatocytes developed normally, as did embryos exposed to cyclophosphamide in the absence of hepatocytes. Maternal hepatocytes were necessary for the expression of cyclophosphamide embryotoxicity, which was concentration-dependent, as demonstrated by increasing severity of reductions in crown rump length, yolk sac diameter and somite number. These results show that the co-culture of murine maternal hepatocytes and embryos is feasible, and suggest that maternal bioactivation is required for murine cyclophosphamide embryopathy.

Animals↗
PubMedDate not supplied

Leiomyoma primary cultures have elevated transcriptional response to estrogen compared with autologous myometrial cultures.

OBJECTIVE: We tested the hypothesis that uterine leiomyomas are hypersensitive to estrogen as compared with autologous human myometrium. METHODS: The estrogen-induced transcriptional responses of uterine leiomyoma and myometrial primary cultures were determined by transient expression assays. The relative levels of estrogen receptor (ER) in myometrial and leiomyoma tissues were determined by immunoblot. RESULTS: Myometrial and leiomyoma primary cultures were transcriptionally responsive to the estrogen ethinyl estradiol (eE2). The partial agonist tamoxifen did not elicit a positive transcriptional response and antagonized estrogen-induced transcription in the cultured cells. The responses of hormone-treated leiomyoma cells averaged 4.5-fold higher than those in controls with no hormone (P = .0001). The myometrial cells from women in the follicular phase exhibited little if any transcriptional response to eE2, whereas myometrial cells from women in the luteal phase had a transcriptional response to eE2 averaging threefold higher than that in no-hormone controls (P = .0083). Differences in response between autologous myometrial and leiomyoma cultures were statistically significant by the two-tailed Wilcoxon paired nonparametric signed-rank test (n = 11; P = .0137). These differences were more pronounced in cultures from women in the follicular or early luteal phase. In addition, the levels of ER increased in follicular and early luteal phase myometrial tissues, which correlated well with the number of days from the last menstrual period (n = 8; r = 0.9046; P = .002). Estrogen-receptor levels in myometrial tissues decreased during the late luteal phase. Levels in leiomyoma tissues did not follow the same pattern as in the myometrium and were elevated in tissues taken from women in the follicular phase. CONCLUSIONS: Autologous leiomyoma cultures have a significantly higher response to estrogen than do matched myometrial cultures, especially if the cultures are derived from the follicular phase. The levels of ER in leiomyoma tissue from women in the follicular phase are significantly elevated.

Adult↗
PubMed2000

Acyl lipid composition variation related to culture age and nitrogen concentration in continuous culture of the microalga Phaeodactylum tricornutum.

The influence of culture age and nitrogen concentration on the distribution of fatty acids among the different acyl lipid classes has been studied in continuous cultures of the microalga Phaeodactylum tricornutum. The culture age was tested in the range of 1.15-7 days, controlled by adjusting the dilution rate of fresh medium supplied. The effect of nitrogen concentration was tested from saturating conditions to starvation by modifying nitrate concentration in the fresh medium. Culture age had almost no influence on the fatty acid content; 16:0, 16:3 and 20:5 increased moderately wherein the level of 16:1 decreased when the culture age decreased. Culture age had no effect on the total fatty acid content that remained around 11% of dry weight. Conversely, culture age had a greater impact on lipid classes, producing changes in amounts of triacylglycerols (TAG) which ranged between 43% and 69%, and galactolipids (GLs) that oscillated between 20% and 40%. In general, the content of polar lipids of the biomass decreased with culture age. The other factor assayed, nitrogen content, affected the fatty acid profile. Saturated and monounsaturated fatty acids accumulated when the nitrogen concentration was decreased. The experiments regarding the effect of nitrogen concentration on lipid species were carried out with cells of an average age of 3.5 days. A decrease of the nitrogen concentration caused the GL fraction to decrease from 21 to 12%. Conversely, both neutral lipids (NLs) and phospolipids (PLs) increased from about 73 to 79% and from 6 to 8%, respectively. In these experiments, TAG was the lipid class with the highest increase, from 69 to 75%.

Culture Media↗
PubMed2002

Two-dimensional cell sheet manipulation of heterotypically co-cultured lung cells utilizing temperature-responsive culture dishes results in long-term maintenance of differentiated epithelial cell functions.

Here we report two-dimensional cell sheet manipulation (2D CSM) of heterotypically co-cultured lung cell sheets and the maintenance of differentiated phenotypes of lung epithelial cells over prolonged periods of up to 70 days. This was facilitated by poly(N-isopropylacrylamide) (PIPAAm)-grafted tissue culture dishes. PIPAAm-grafted dishes are responsive to temperature changes and offer a unique surface on which cells adhere and multiply like on ordinary tissue culture dishes under the permissive temperature of 37 degrees C, but on lowering of temperature resulting in changes in hydration of the polymer the cells spontaneously detach from the surface without use of enzymes like trypsin which is the common procedure. It has been well documented that type II pneumocytes of the lung lose many of their special features rapidly in culture. The culture system detailed here comprises random co-culture of epithelial and mesenchymal cells of lung. The heterotypic cell culture system promotes cell-cell interactions maintaining a harmonized physiology. When this heterotypic monolayer on PIPAAm-grafted dishes was subjected to lower temperature of 20 degrees C and 2D CSM we were able to transfer the monolayer as a single contiguous sheet with cell-cell connections intact to other surfaces. This non-invasive transfer of cell sheet resulted in shrinkage of the monolayer, enabling the type II cells to regain their cuboidal morphology and specialized characters like Maclura pomifera lectin binding and surfactant protein A (SP-A) expression. The active dome formation also observed subsequent to transfer reaffirms the uniqueness of the culture conditions and 2D CSM in future for developing tissue like architecture in vitro.

Acrylic Resins↗