In vitro cultivation of Hymenolepis diminuta: effect of antibiotics on the growth of three-day-old worms removed from the rat

The effects of several antibacterial or antifungal antibiotics on the growth of 3-day-old Hymenolepis diminuta cultivated in vitro were investigated. Worms were recovered from the rat and cultured in roller tubes for 4 days without a medium change. The medium contained horse serum, yeast extract, and liver extract; the gas phase was 5% CO₂ in N₂. It was found that penicillin and streptomycin did not inhibit the growth of worms at concentrations lower than 5000 units and μg/ml of medium, respectively. Cycloheximide was toxic to H. diminuta, retarding or inhibiting growth at levels higher than 1.5 μg/ml. The antifungal antibiotics nystatin and amphotericin B did not affect worm growth at concentrations lower than 1000 units and 123 μ/ml of medium, respectively.The use of penicillin and streptomycin, and nystatin or amphotericin B can be recommended for the control of bacterial and fungal contaminants in the cultivation of H. diminuta removed from the rat.     

Caspofungin reduces the incidence of fungal contamination in cell culture

Fungal contamination is a major problem in cell culture, and the antifungal compounds currently in use can affect cultured cells. Echinocandins are antifungal drugs that inhibit fungal cell wall synthesis by targeting an enzyme that has no counterpart in mammalian cells. We evaluated whether the echinocandin caspofungin affected the growth or morphology of six murine cell lines (a macrophage-like cell line (J774.16) and five hybridoma lines), or primary human endothelial cells. The antifungal did not influence cellular characteristics at concentrations less than 512 μg/ml, while effectively reducing the incidence of fungal contamination. Also, caspofungin did not affect the production of antibody by hybridoma cells, or alter the cytokine production of J774.16 cells, although modest increases in IL-4 and IFN-γ occurred upon LPS stimulation. Hence, echinocandins appear to be relatively non-toxic, and protect against fungal contamination in cell culture.     

Induction of precocious melanogenesis of pigment cells in cultures of neuroretinal cells of chick embryo by amphotericin B

Summary When dissociated neuroretinal cells of the 9-day-old chick embryo were cultured, the cells formed monolayer sheets of somewhat flattened epithelial cells within 15 days after inoculation. During 15 to 30 days, numerous foci of non-pigmented epithelial cells were formed. During 30 to 50 days, melanin appeared in the cells of these foci. When amphotericin B (1 g/ml) was added to the culture medium on day 25 of culture, brown pigments appeared precociously, i.e. within the first two days, in the cells. The brown pigments were identified as melanins by histochemical and electron-microscopic methods. Induction of melanogenesis required continuous treatment with amphotericin B. With the precocious appearance of melanins, tyrosinase activity increased rapidly. This rapid increase in tyrosinase activity was inhibited by the addition of phenylthiourea or diethyl-dithiocarbamate. It was not enhanced by iodoacetamide, but was blocked by a low concentration of cycloheximide or actinomycin D. These findings indicate that amphotericin B induces de novo synthesis of tyrosinase rather than activation of pre-existing tyrosinase.     

Determination of amphotericin B, liposomal amphotericin B, and amphotericin B colloidal dispersion in plasma by high-performance liquid chromatography

Amphotericin B is a potent polyene antifungal drug for intravenous treatment of severe infections. It is used as amphotericin B-deoxycholate and in order to reduce amphotericin B toxicity as lipid-formulated complex (liposomal or colloidal dispersion). A sensitive and specific analytical method is presented for the separation of lipid-complexed and plasma protein-bound amphotericin B in human heparinized plasma. This separation, which is required for pharmacokinetic studies, is achieved by solid-phase extraction (SPE) via Bond Elut C₁₈. The protein-bound amphotericin B has a higher affinity to the SPE material and is therefore retained, whereas the lipid-complexed amphotericin B is eluted in the first step. The recovery of the SPE was >75% for high concentrations and >95% for low concentrations. Quantification was performed by reversed-phase HPLC using a LiChrosorb-RP-8 column, UV detection (λ=405 nm) and a mixture of acetonitrile–methanol–0.010 M NaH₂PO₄ buffer (41:10:49, v/v) as mobile phase. The retention time for amphotericin B under the given conditions was 6.7 min. The calibration curves were found to be linear (r≥0.999) in two different ranges (5.0–0.50 μg/ml and 0.50–0.005 μg/ml). Intra- and inter-day precision and accuracy fulfilled the international requirements. No interference from other drugs (typical broad medication for intensive-care patients) or common plasma components was detected in >400 samples analyzed.     

湛江等鞭金藻对抗生素的反应及无菌化培养

研究了5种抗生素在不同浓度下对湛江等鞭金藻(Isochrysis zhangjiangensis)生长的影响,采用混合添加抗生素法获得无菌藻株.结果表明,(1)不同抗生素对湛江等鞭金藻生长的影响存在差异,湛江等鞭金藻对氯霉素最敏感,浓度为50 μg·mL-1时抑制率为27.02%,浓度＞50 μg·mL-1时抑制率达80%,湛江等鞭金藻对青霉素、链霉素、卡那霉素和庆大霉素不敏感,后3种抗生素在低浓度时对藻细胞生长有促进作用;(2)除50 μg·mL-1青霉素处理外,青霉素、链霉素、卡那霉素和庆大霉素其它浓度处理均有明显的抑菌作用;(3)采用500 μg·mL-1链霉素,1000 μg·mL-1卡那霉素和50 μg·mL-1庆大霉素的4个组合混合添加抗生素,均获得了无菌藻株,且对生长有促进作用.     

Effect of Amphotericin B on Dopachrome Tautomerase Activity and Other Melanogenic Parameters in Cultured B16/F10 Melanoma Cells

The antifungal reagent Fungizone (amphotericin B and deoxycholate) caused an activation in dopachrome tautomerase and dopa oxidase activities of B16/F10 melanoma cells at the routine concentration (2.5 μg/ml) used for preventing molds and yeast growth in cultures of animal cells. However, higher amphotericin B concentrations caused a significant cell death and the inhibition of enzymatic activities. At the optimal concentration of Fungizone, the enzymatic activities and melanin content were augmented as incubation time increased. The detergent sodium deoxycholate alone exerted no effect on these melanogenic parameters, eliminating the possibility that this detergent was partially responsible for melanogenic modifications produced by Fungizone. After withdrawal of Fungizone from the reaction medium, the recovery of melanogenic parameters to normal values was slower for DCT than for tyrosinase. The behavior of dopa oxidase was very similar to that reported by Johnson and Bagnara (Pigment Cell Res. 3, 173–175) for tyrosine hydroxylase.     

Phenol red in the culture medium strongly affects the susceptibility of human MCF-7 cells to roscovitine

Estrogens play an important role in the growth and terminal differentiation of the mammary gland. Prolonged exposure to estrogens seems to predispose women to breast cancer. It recently became evident that not only the intrinsic hormonal status but also external factors such as the occurrence of pharmaceuticals and chemicals with hormone activity in the environment may put women at greater risk of developing breast cancer. We focused on the interference of endocrine disruptors in breast cancer therapy. We observed that phenol red added to the culture medium strongly promoted the cell proliferation and cell cycle progression of human cells expressing the estrogen receptor, and affected their susceptibility to chemotherapy.     

Neospora caninum: In vitro culture of tachyzoites in MCF-7 human breast carcinoma cells

The development of Neospora caninum tachyzoites, an apicomplexan protozoan parasite, was studied in vitro using the human breast carcinoma cell 7 (MCF-7) as the host cell line. The extracellular NC-1 tachyzoites in MCF-7 cells were observed and counted daily for 6 consecutive days post-infection to establish the growth curve. The intracellular parasites were observed by acridine orange staining using Laser scanning confocal microscope. The results indicated that NC-1 tachyzoites invaded MCF-7 cells and multiplied intracellularly. The number of extracellular NC-1 tachyzoites started to increase rapidly around day 3 and reached the maximum number around day 4. Results from the present study suggested that MCF-7 cells were susceptible to NC-1 tachyzoites and could be used as an alternative cell line for in vitro studies.     

Immobilized viable microbial cells: from the process to the proteome em leader or the cart before the horse

Biotechnological processes based on immobilized viable cells have developed rapidly over the last 30 years. For a long time, basic studies of the physiological behaviour of immobilized cells (IC) have remained in the shadow of the applications. Natural IC structures, i.e. biofilms, are being increasingly investigated at the cellular level owing to their definite importance for human health and in various areas of industrial and environmental relevance. This review illustrates this paradoxical development of research on ICs, starting from the initial rationale for IC emergence and main application fields of the technology—with particular emphasis on those that exploit the extraordinary resistance of ICs to antimicrobial compounds—to recent advances in the proteomic approach of IC physiology.     

Effect of formaldehyde and resveratrol on the viability of Vero, HepG2 and MCF-7 cells

A non-transformed (Vero) and two tumor cell lines (HepG2 and MCF-7) were treated with 10nM to 100 microM formaldehyde. Lower doses (10nM to 10 microM) enhanced the viability of the cultured cells, measured by MTT assay. Higher doses (75-100 microM) decreased viability of the cells by 50% or more. The 100 microM concentration of HCHO has been chosen for combination treatment of the three cell lines with a series of concentrations (0.2-100 microM) of resveratrol, a phytoestrogen occurring in various fruits. Resveratrol decreased the cytotoxicity of formaldehyde depending on cell line and point of time, especially in case of MCF-7 cells at 24 and 72 h, Vero cells at 24h and HepG2 cells at 48 h after treatment. Possible modes of interactions are discussed, considering the role of resveratrol in formaldehyde metabolism and also the estrogen receptor positivity of MCF-7 cells.     

草鱼前体脂肪细胞的原代培养

构建脂肪细胞体外培养体系,研究脂肪细胞分化过程,是探讨动物脂质代谢规律的重要手段.目前,哺乳动物方面,自20世纪60年代起至今,国内外已成功构建了大鼠[1,2]、人[3,4]、牛[5,6]和猪[7,8]的前体脂肪细胞体外培养模型,鱼类方面的工作则开始不久.     

毛脚鵟细胞体外培养体系建立及三种组织来源细胞特性分析

用组织块培养法对毛脚鵟不同组织进行原代培养,获得了3种不同组织来源的细胞,并成功对细胞进行了冷冻保存和复苏。在传代培养过程中,对比分析了3种组织来源细胞的形态学、生长曲线、贴壁率、核型等生物学特性。形态学方面,3种来源细胞均为成纤维样细胞。对于3种组织来源细胞的贴壁能力分析显示,输卵管源细胞最强,肺源细胞和气管源细胞次之。3种不同组织来源细胞的倍增时间分别为(29.91±0.39)、(33.18±0.21)和(30.67±0.28)h,群体倍增次数分别为3.54±0.01、4.52±0.02和4.38±0.03。毛脚鵟细胞的染色体数目为2n=68,性染色体为典型的ZW型。本实验为今后毛脚鵟细胞利用、遗传信息的保存及生物学特性的深入研究提供实验材料和依据。     

The minotaur proteome: Avoiding cross‐species identifications deriving from bovine serum in cell culture models

Cell culture is a fundamental tool in proteomics where mammalian cells are cultured in vitro using a growth medium often supplemented with 5–15% FBS. Contamination by bovine proteins is difficult to avoid because of adherence to the plastic vessel and the cultured cells. We have generated peptides from bovine serum using four sample preparation methods and analyzed the peptides by high mass accuracy LC‐MS/MS. Distinguishing between bovine and human peptides is difficult because of a considerable overlap of identical tryptic peptide sequences. Pitfalls in interpretation, different database search strategies to minimize erroneous identifications and an augmented contaminant database are presented.     

Alkaline phosphatase staining of pig and sheep epiblast cells in culture

To define better the characteristics of pig and sheep epiblast cells in culture, the cells were tested for the presence of alkaline phosphatase (AP), a biochemical marker characteristic of mouse embryonic stem cells. Pig and sheep epiblast cells were positive for AP staining both at isolation from the blastocyst and after primary in vitro culture. The innermost portion of the attendant endoderm surrounding the epiblast was also positive for AP staining during primary culture. AP staining was lost upon differentiation or senescence of the epiblast cells. Also, all differentiated epiblast-derived cell cultures were negative for AP staining, with the exception of neuron-like cultures. Epiblast-like cells were cultured from day 10 (pig) and day 13 (sheep) embryonic discs, and these cells were also AP positive until they differentiated. Trophectoderm-endoderm-like cells from embryonic discs were AP negative or weakly positive. AP is a convenient marker for undifferentiated pig and sheep epiblast cells in culture when used in conjunction with cell morphology analysis. © 1993 Wiley-Liss, Inc.     

Novel bioreactors for the culture and expansion of aggregative neural stem cells

Neural stem cells have been cultured as three-dimensional aggregates in a number of different types of bioreactors. The design and configuration of the bioreactor are shown to be crucial factors for the successful propagation of the cells. A novel bioreactor with liquid re-circulation and a working volume of 200 ml has been designed, tested and shown to be able to produce a higher cell vitality compared to those produced in multi-well plates, shake flasks and stirred flasks. The novel reactor was able to produce a total density of cells of 3.5 x 10(6) cells/ml consisting of a larger number of smaller and proliferative aggregates, compared to only 1.8 x 10(6) cells/ml produced in a multi-well plate. Shake flasks and stirred flasks commonly used for facilitating mass transfer in the culture of micro-organisms are shown to be unsuitable for the propagation of neural stem cells.     

Atrazine and PCB 153 and their effects on the proteome of subcellular fractions of human MCF-7 cells

Several man-made organic pollutants including polychlorinated biphenyls (PCBs) and several pesticides may exhibit endocrine disrupting (ED) properties. These ED molecules can be comparatively persistent in the environment, and have shown to perturb hormonal activity and several physiological functions. The objective of this investigation was to study the impact of PCB 153 and atrazine on human MCF-7 cells, and to search for marker proteins of their exposure. Cells were exposed to environmentally high but relevant concentrations of atrazine (200ppb), PCB 153 (500ppb), 17-β estradiol (positive control, 10nM) and DMSO (0.1%, negative control) for t=36h (n=3 replicates/exposure group). Proteins from cell membrane and cytosol were isolated, and studied by 2D-DiGE. Differentially regulated proteins were trypsin-digested and identified by MALDI-ToF-ToF and NCBInr database. A total of 36 differentially regulated proteins (>|1.5| fold change, P<0.05) were identified in the membrane fraction and 22 in the cytosol, and were mainly involved in cell structure and in stress response, but also in xenobiotic metabolism. 67% (membrane) and 50% (cytosol) of differentially regulated proteins were more abundant following atrazine exposure whereas nearly 100% (membrane) and 45% (cytosol) were less abundant following PCB 153 exposure. Western blots of selected proteins (HSBP1, FKBP4, STMN1) confirmed 2D-DiGE results. This study emphasizes the numerous potential effects that ED compounds could have on exposed humans.     

Effect of androstenedione on growth of untransfected and aromatase-transfected MCF-7 cells in culture

Aromatase is present in human breast tumors and in breast cancer cell lines suggesting the possibility of in-situ estrogen production via the androstenedione to estrone and estradiol pathway. However, proof of the biologic relevance of aromatase in breast cancer tissue requires the demonstration that this enzyme mediates biologic effects on cell proliferation. Accordingly, we studied the effects of the aromatase substrate, androstenedione, on the rate of proliferation of wild-type and aromatase-transfected MCF-7 breast cancer cells. Androstenedione did not increase cell growth in wild-type MCF-7 cells which contained relatively low aromatase activity and produced 4-fold more estrone than estradiol. In contrast, aromatase-transfected cell contained higher amounts of aromatase, produced predominantly estradiol, and responded to androstenedione with enhanced growth. An aromatase inhibitor fadrozole hydrochloride, blocked the proliferative effects of androstenedione providing evidence for the role of aromatase in this process. As further evidence of the requirement for aromatase, cells transfected with the neomycin resistance expression plasmid but lacking the aromatase cDNA did not respond to androstenedione. These studies provide evidence that aromatase may have a biologic role for in-situ synthesis of estrogens of breast cancer tissue.     

猪胎儿肾脏成纤维细胞体外培养体系的建立

本研究旨在建立猪胎儿肾脏成纤维细胞体外培养体系,并探讨其作为猪体细胞克隆供体的可能性。使用组织块培养法从体长为10cm以上的猪胎儿分离得到猪胎儿肾脏成纤维细胞,绘制了生长曲线,鉴定了细胞类型并且进行了细胞周期同期化效果的研究。结果表明:该培养体系可以支持猪胎儿肾脏成纤维细胞的体外生长,单个细胞均为梭形细胞,抗波形蛋白免疫荧光染色显示为阳性,而抗角形蛋白免疫荧光染色为阴性,分离到的细胞为胎儿肾脏成纤维细胞。使用血清饥饿法和接触抑制法诱导细胞进入G0/G1期,并且分别比较两者同期化效率,结果显示:血清饥饿2d和4d的同期化效率差异不显著,但都比8d组的高(88.97%和87.69%比82.45%,P<0.05);接触抑制4d、6d组间同期化效率差异不显著,但都比0d组的高(85.56%和85.89%比81.82%,P<0.05)。本研究在国内首次分离得到猪胎儿肾脏成纤维细胞,已经在体外传代培养到32代,其同期化效果好,可以作为体细胞克隆供体。     

Scanning electron microscopy of bone cells in culture

Summary Embryonic and young rat bone cells have been grown in culture and examined in the scanning electron microscope (SEM). Compared with cells fixed in situ and taken directly from the animal, the cultured osteoblastic cells were smoother, flatter and more extensive and showed tighter intercellular contacts. Some matrix is formed in culture and undergoes at least partial mineralization as judged by the accumulation of Ca and P measured by energy dispersive x-ray analysis. Findings concerning the morphology of the collagen arrangement were indecisive. Some superficial cells, free of surrounding matrix, resembled osteocytes in normal in vivo bone. This may indicate that a proportion of the extracellular matrix produced by the cultured cells failed to polymerise into recognizable bone matrix, and that osteocytic morphology is not dependent upon the physical characteristics of the bone matrix.