Plastic Culture Vessels Research Articles

Abstract A35: Characterizing the role of the nuclear coactivator AIB1 in triple-negative breast cancer

Abstract Individuals diagnosed with triple-negative breast cancer (TNBC), a highly invasive and frequently metastatic breast cancer subtype, do not benefit from targeted therapies. TNBC is a heterogenous disease and can be further subdivided by intrinsic molecular subtyping into basal-like 1 or 2 (BL-1, BL-2), immunomodulatory (IM), mesenchymal (M), mesenchymal stem-like (MSL), and luminal androgen receptor (LAR). Amplified in Breast Cancer 1 (AIB1) is an oncogene that is frequently overexpressed in higher-grade and invasive breast cancers. AIB1 regulates the transcription of several genes through hormone or growth factor signaling. Many of these signaling pathways are involved in cellular growth, differentiation, migration, and metabolism—all of which become deregulated during oncogenesis. In this study, we silence AIB1 by shRNA and study the phenotypic effects in a panel of TNBC cell lines from different TNBC subtypes in vitro. We have established TNBC HCC1806 cell lines expressing low levels of AIB1 and have characterized their phenotype compared to scramble control cells. We observe significant reduction in AIB1 message and protein as detected by RT-qPCR and Western blot. shAIB1 TNBC cells have a significant reduced survival following the initial knockdown. However, serial passaging of surviving cells show differences in proliferation, especially in low serum conditions. These AIB1LOW cells have a significant increase in motility shown in chemotaxis experiments. However, we observe delayed adhesion of AIB1LOW cells when seeded onto plastic culture vessels. Furthermore, when grown onto Matrigel, AIB1LOW cells have a significant reduced tube formation capacity related to their delayed adhesion or reduced motility on substratum. Additionally, AIB1LOW cells have a differential expression of cell surface markers associated with cancer stem cells (i.e., CD24, CD44, CD133). Consistent with these findings, AIB1LOW cells generate tumorspheres more efficiently under serum free in suspension and when embedded in Matrigel. Moreover, AIB1LOW cells develop aggressive phenotypically different tumors in the cleared mammary fat pad of nude mice compared to controls. The AIB1LOW cells are significantly less sensitive to chemotherapy agents and may represent a resistant population that emerges from cancer stem cells following therapy. Interestingly, AIB1LOW cells have a different regulation of genes associated with hypoxia, glycolysis, and apoptosis and these changes may give guidance as to therapy following emergence of resistance in TNBC patients. Citation Format: Francisco R. Saenz, Anton Wellstein, Anna T. Riegel. Characterizing the role of the nuclear coactivator AIB1 in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A35.

Life cycle studies of the earthworm Lumbricus friendi (Cognetti, 1904)

The objective of this work was to obtain data on aspects of the lifecycle of Lumbricusfriendi. Field-collected specimens from NW Spain were grown to maturity and maintained under controlled laboratory conditions. Thereafter experiments were undertaken to determine mode of reproduction, reproductive output, cocoon viability and development, and hatchling growth rate. Effects of density and temperature on growth were also examined. Plastic culture vessels containing a substrate of Kettering loam were incubated in temperature-controlled chambers, with dried horse manure as food. Paired, mature animals were sampled monthly over a period of 9 months for cocoon production. Cocoons were incubated on moist filter papers in Petri dishes. Results showed that at 15 °C, individuals matured at a live mass of 3.5 g. Zero cocoons were produced by virgin animals maintained in isolation, but recently matured, paired L. friendi produced 2.16 cocoons individual−1 wk−1. Cocoons were lemon-shaped with a smooth pale yellow-coloured outer surface and had a mean mass of 28 mg. At 15 °C, cocoons required 90 days to hatch and hatchability from the first 5 months of cocoon production was 85% successful. The majority of cocoons produced a single hatchling (twins recorded < 1%). Growth of hatchlings to maturity was achieved within a period of 5 months, with tubercula pubertatis developed after 3 months. Increased temperatures in the range of 5–15 °C led to significantly increased growth rates, whereas increased earthworm density led to a significant decrease in rate of growth. These findings permit comparisons with more closely studied anecic species such as Lumbricus terrestris.

Isolation and Characterization of Essential Life Factor on Immune System in Human Body

Essential Life Factor (Elfis) isolated from a certain beech sand is present in micro-trace quantities in soils and sands which have been exposed area by sun shine. Therapeutic effects of Elfis has been attributed include immuno-enhancement, free radical scavenging, and heavy detoxification. Clinical trials and use in private practices for over five years have demonstrated Elfis’ efficacy in treating a wide range of serious affections, including cancer, arthritis and senile osteoporosis. The Elfis has been characterized as a glycoprotein and/or an amino-sugar, Mw. Ca.12,000 plus cosmic energy. The research of Elfis has to be needed thoroughly by both the orthodox scientific and alternative holistic communities, with encouraging therapeutic results acceptable to the framework and philosophy of both these healing traditions. The versatility of Elfis’ therapeutic effects, and its virtual non-toxicity, make it a highly attractive substance to anyone interested in attaining and maintaining good health. The mass production of Elfis has been succeeded by using a plastic culture vessels with specific microorganisms, i.e., Lacto bacillus, bacillus brevis, and Saccheromyces, as well as rice bran as nutrients on exposing sun light to facilitate cosmic/quantum energy (Chinese term chi- energy). It has been scientifically established and accepted that our emotional state, stress level, and the food we eat all play an interrelated role in the function of our body's defense mechanism, the immune system, which to a large extent, determines our state of health. Nutrition, stress management, and preventive medicine are indispensable tools of general medical practice.

Cytokine modulation of murine stem cell engraftment: the role of adherence to plastic surfaces.

Murine marrow stem cells acquire an engraftment defect when cultured for 48 hours in cytokines, whereas the number of progenitor cells expands. Stem or progenitor cells have been noted to adhere to various surfaces, including plastic. Despite vigorous harvesting by cell scraping, the possibility existed that cytokines might induce selective adhesion of the rare engraftable stem cells to plastic surfaces. We have evaluated whether loss of engraftability by cytokine-treated marrow cells could be due to adhesion to plastic culture vessels. BALB/c marrow cells were cultured in the presence of interleukin 3 (IL-3), IL-6, IL-11, and steel factor for 48 hours in plastic tissue culture flasks from which cells were harvested by standard scraping and washing or after 5 or 10 minutes of additional exposure to trypsin (0.25%), or they were cultured with the same cytokines in nonadherent polytetrafluoroethylene (Teflon) culture bottles. Harvested cultured or fresh-starting male cells were then engrafted into nonirradiated female hosts or were placed in competition with fresh female BALB/c marrow in lethally irradiated female hosts. Defective engraftment was seen in nonmyeloablated or irradiated female hosts 7 to 24 weeks after marrow infusion in all cultured cell groups. These data indicate that cytokine-treated engraftable stem cells do not show significant adherence to plastic surfaces.

Enhanced Detection of Chemiluminescence Intensity during an Oxidative Burst Reaction in Suspension Cultured Nicotiana tabacum Cells through the Use of Ficoll

Glass petri dishes provide tight control of oxygen concentration during continuous nitrogen gassing to cultured cells. This control has led to a quick and highly reproducible simulated ischemic model of cortical neuron death in vitro. Twenty-five minutes of simulated ischemia was required for 92% death in cortical neuron cultures on glass. The contamination of leached oxygen from plastic culture vessels, in comparative experiments, necessitates longer times of simulated ischemia (120 min) to achieve similar cell death. Steady-state release of oxygen from plastic petri dishes was observed for up to 60 min of nitrogen gassing, providing a metabolic environment contrary to that of anoxia. Further investigations with models of simulated ischemia in glass culture vessels could answer some criticisms of in vitro models and their relevance to in vivo situations (10).

A simplified protocol for micropropagation of guayule (Parthenium argentatum gray)

A simple, efficient protocol for in vitro micropropagation of guayule is reported. Shoot cultures were maintained on MS (Murashige and Skoog, 1962) medium supplemented with 1.0 mg l−1 (4.4 μM) 6-benzylaminopurine and 0.025 mg l−1 (0.3 μM) α-naphthaleneacetic acid. Excised shoots were treated for 14–18 h with 100 mg l−1 (492.1 μM) indole-3-butyric acid in 0.5 x MS salts to induce rooting. The shoots were subsequently inserted into cellulose plugs which were packed in sterile, ventilated plastic culture vessels and moistened with 0.5 x MS medium without growth regulators. Use of cellulose plugs, liquid medium and ventilated culture vessels facilitated acclimation. Rooted shoots were transplanted into potting medium and acclimated to greenhouse conditions by covering with a cloche for 2 d, followed by daily watering for the first week.

Ventilation of culture vessels. I. Increased ’growth in vitro and survival ex vitro of Delphinium

SummaryInclusion of small circular apertures covered with filters in the sides of plastic culture vessels led to a small but significant increase in the multiplication rate of Delphinium cultured in vitro and greater survival following transfer ex vitro. Filters had no effect on the multiplication rate and survival of Hosta. In vessels with apertures there was a large increase in the rate of water loss, but relative humidity was greater than 95% in both intact vessels and in vessels with filters. It is suggested that in vessels with apertures there was an increase in the flow of water vapour from the vessel atmosphere to the external atmosphere, due to a reduced diffusive resistance. The improved performance of Delphinium plants could have resulted from an increase in transpiration and movement of water (and therefore some nutrients) through the plants.

Soluble iron is lost from MS medium pre-exposed to light but growth of potato plantlets is not inhibited

Plant tissue culture medium which contained FeEDTA as sole iron source was incubated aseptically in light (16-h photoperiod, 100 μmol m-2 s-1 PAR) at 20°C without plant tissue. Soluble iron dropped from an initial concentration of 4 mg 1-1 to less than 0.1 mg 1-1 in 4 weeks. This occurred in both glass and plastic culture vessels. No loss occurred when medium was incubated at 20°C in darkness. A further experiment showed that soluble iron concentration fell to <0.2 mg 1-1 in only 4 days but the loss was slower at lower irradiances.

Stable heparin-producing cell lines derived from the Furth murine mastocytoma.

Stable cell lines that synthesize heparin have been established from the Furth murine mastocytoma. The parental line (MST) divides in suspension every 14-18 h in growth medium supplemented with fetal bovine serum or defined growth factors. Adherent subclones were selected by adhesion to plastic culture vessels. Both adherent and nonadherent cells contain about 0.4 micrograms of glycosaminoglycan hexuronic acid per 10(6) cells, composed of 80% heparin and 20% chondroitin sulfate E. Deaminative cleavage of MST heparin by HNO2 at pH 1.5 released disaccharides that were similar in composition to those obtained from commercial heparin, except that disaccharides containing 3,6-O-desulfated GlcN units were not found. Greater than 90% of the glycosaminoglycans were stored in cytoplasmic granules, and challenge of the cells with dinitrophenylated bovine serum albumin and anti-dinitrophenyl IgE released a portion of the stored material. Growth studies of subclones showed that MST cells tolerate a 10-fold variation in glycosaminoglycan content. Incubation of cells with sodium chlorate reduced glycosaminoglycan sulfation by > 95% without affecting cell growth. Thus, granule glycosaminoglycans appear to be nonessential for growth of MST cells.

Proliferative response and macromolecular synthesis by ocular cells cultured on extracellular matrix materials

To investigate the effects of extracellular matrix components on cellular function, we cultured several types of ocular cells on substrates composed of extracellular matrix materials that were layered on culture dishes either as dried films or as gels. We measured cellular proliferation on these substrates and on a series of gels composed of varying proportions of rat tail tendon type I collagen and Matrigel, a commercially available extract of a basement membrane-producing murine tumor. In addition, we studied the biosynthesis of collagens and of proteoglycans by these cultured cells using [3H]-L-proline and [35S]-sulfate. The proliferative abilities of the various types of ocular cells on the dried film substrates, on uncoated plastic culture vessels, and on pure type I collagen gel, were similar. However, proliferation of ocular cells cultured on gels composed of greater than or equal to 90% Matrigel was markedly reduced. There was little or no inhibition of growth of two types of non-ocular cells: rat C6 astrocytoma cells, and human dermal fibroblasts. Histologic studies showed that the ocular cells tested often formed long strands and capillary-like tubes, and tended to "burrow" beneath the surface of substrates containing high percentages of Matrigel. Fibroblasts infrequently formed tubes, and exhibited the burrowing property also on gels containing primarily type I collagen, while C6 cells showed neither of these behaviors on any of the matrices tested. The elution pattern of newly synthesized [3H]-labeled and [35S]-labeled macromolecules produced by all of the cultured cell types, and detected by Sepharose CL-4B chromatography in the medium and in the cell layer plus matrix fractions did not vary following culture on the different substrates. Approximately twofold more of the newly synthesized collagens and proteoglycans were deposited in the cell layer plus matrix, and proportionately less appeared in the medium, when cells were cultured on type I collagen gels and on Matrigel than on the dried film substrates. These experiments demonstrate the influence of the extracellular matrix on several aspects of cell behavior, and provide further evidence that modification of the composition of the extracellular matrix may be an important determinant of normal or pathological cell function.

The effect of tissue culture agar on chromosome breakage, sister-chromatid exchanges and clonogenicity in human cells

To investigate the cytogenetic effects of electromagnetic fields, a system containing an agar gel was developed to support the growth of various human cell types (peripheral lymphocytes, lymphoblasts, and fibroblasts). When compared to alioquots of identical cells, grown in plastic culture vessels, statistically significant increases in the frequencies of chromosome breakage, sister-chromatid exchange and decreased cloning efficiency were observed in those cells cultured in the agar. These results suggest a possible clastogenic and/or cytotoxic component in the agar gel.

In vitro models of differentiated Sertoli cell structure and function.

Primary cultures of Sertoli cells maintained in conventional cultures on plastic culture vessels do not retain many of the structural and functional properties of their in vivo counterparts. Sertoli cell phenotype is better maintained by incorporating certain environmental parameters, intrinsic to the testis, into the Sertoli cell culture system. These environmental parameters include a) high cell density, b) a unique extracellular matrix, c) a semipermeable support between the basal plasma membrane of the cells and blood-derived nutrients in the interstitium, d) chemically distinct microenvironments at the apical and basal surfaces of the cells, and e) cell-to-cell interactions among Sertoli cells and other testicular cell types. Using three variations of Sertoli cell culture we have demonstrated the importance of each of these environmental parameters in obtaining a better Sertoli cell culture model.

The effect of lipids on the subculture of differentiated cells from primary cultures of grasshopper embryonic tissues

Cells from mechanically dissociated postblastokinetic embryos of the rangeland grasshopper,Melanoplus sanguinipes, were seeded in Falcon Primaria culture dishes and monitored for attachment, growth, and differentiation. Various sonicated nutrient phospholipid combinations, including both natural complexes and specific synthetic lipid types, were tested on these embryonic tissues as supplements in combination with three serum-containing basal media. The combined use of Primaria culture vessels and lipid supplementation supported the growth of various epithelioid as well as fibroblastoid and other cell types. Some cells became tightly associated into partially differentiated tissuelike growths that included networks of ducted tracheolarlike cells, ducted granular epithelioid cells, and networks and sheets of slowly contracting muscle cells. The tissue-associated cell types and two individually growing cell types (a plasmatocytoid or hemocytic cell and a peculiar blak granule-containing cell) were serially subcultured from three to nine times in Primaria vessels. Tissues subcultured in standard plastic culture vessels were selected by this growth surface for fibroblastoid cell types or expressed a fibroblastoid morphology, or both, within 1 to 3 passages. The growth of cells bearing neuritelike and glialike processes was stimulated in the primary cultures by certain medium and lipid combinations, but these cell types were not subcultivable.

Platinum-shadowed, whole-mounts for transmission electron microscopy of cells cultured on plastic.

A simple and convenient method is described whereby cells cultured on polystyrene plastic substrata may be critical point-dried, rotary-shadowed with platinum/carbon and finally released by means of propylene oxide for examination whole in the transmission electron microscope. The technique is particularly useful for the localization of colloidal gold probes, both intra- and extra-cellularly. The problem of premature melting of polystyrene at about 307 K after prolonged exposure to carbon dioxide is solved by limiting the residence time in the critical point-drier to 1 h. Details for cleaning and mounting the released films of cells on specimen grids are given. The technique expands the application of the 'whole-mount' approach of Hopkins and co-workers (Hopkins et al., 1981; Hopkins, 1985) to cells cultured directly on polystyrene substrata, that is, in conventional plastic culture vessels.

Apolipoprotein E is a biologically active constituent of the normal immunoregulatory lipoprotein, LDL-In.

A normal plasma lipoprotein, termed LDL-In, has been shown to be a potent inhibitor of mitogen-driven human lymphocyte proliferation in vitro and of primary antibody responses in the mouse. To determine whether the immunoregulatory activity of LDL-In resided with the protein rather than the lipid constituents of LDL-In, one of the apoproteins of LDL-In, apoprotein E, was isolated from plasma and was analyzed for its inhibitory activity. Apoprotein E, isolated after delipidization of lipoproteins with either methyl ethyl ketone or ethanol and ethyl ether, was immunosuppressive. Furthermore, the characteristics of inhibition of cellular [3H]thymidine uptake by isolated apoprotein E were identical to those characteristics obtained with suppression by LDL-In. Inhibition by apoprotein E and LDL-In required preincubation of the cells with either apoprotein or lipoprotein for 24 hr before exposure of the cells to mitogen for maximal expression of suppressive activity, and this inhibition could not be reversed by removal of non-cell-associated inhibitor before stimulation. Neither apoprotein E or LDL-In was inhibitory when they were added to the cells after mitogen stimulation. The only difference noted between suppression by apoprotein E and LDL-In was that of dose. Compared with quantitative estimates of the apoprotein E content of LDL-In, significantly more isolated apoprotein E was required than lipoprotein-associated E for comparable levels of suppression. The potency of apoprotein E could be increased by adding it to cells in the presence of dimyristoylphosphatidylcholine/cholesterol vesicles. The data suggesting that phospholipid increased the specific activity of apoprotein E by altering its molecular dispersion was obtained from analyses of the interaction of apo E with cells, as well as the plastic culture vessels. The results suggested that the molecular dispersion and perhaps organization of isolated apoprotein E in an aqueous system is critical to its interaction with lymphocytes and subsequently its biological activity.

Interference of serum and serum components with attachment of HeLa 71 cells to the surface of plastic culture vessels

The attachment of HeLa 71 cells to plastic surfaces has been investigated. Without serum in the growth medium the attachment of cells was rapid and not reduced at temperatures down to 25 °C. The cells were not detached by treatment with trypsin-EDTA up to 48 h after incubation. With calf serum in the medium the cells attached equally well to the substrate, but the process was temperature dependent. These cells were detached by trypsin-EDTA treatment. The same results were found when the culture flasks were precoated with serum. The serum coat could be removed by rinsing with potassium bicarbonate at pH 9.7. Preliminary fractionation of the calf serum suggested that the effect was specific.