We designed and fabricated a novel high throughput brain-on-chip with three dimensional structure with the aim to simulate the in vivo three-dimensional growth environment for brain tissues. The chip consists of a porous filter and 3D brain cell particles, and is loaded into a conventional 96-well plate for use. The filter and the particle molds were fabricated by using computer modeling, 3D printing of positive mold and agarose-PDMS double reversal mold. The 3D cell particles were made by pouring and solidifying a suspension of mouse embryonic brain cells with sodium alginate into a cell particle mold, and then cutting the resulting hydrogel into pieces. The loaded brain-on-chip was used to determine the neurotoxicity of pesticides. The cell particles were exposed to 0, 10, 30, 50, 100 and 200 µmol/L of chlorpyrifos or imidacloprid, separated conveniently from the medium by removing the porous filter after cultivation. Subsequently, cell proliferation, acetylcholinesterase activity and lactate dehydrogenase release were determined for toxicity evaluation. The embryonic brain cells were able to grow and proliferate normally in the hydrogel particles loaded into the filter in a 96-well plate. Pesticide neurotoxicity test showed that both chlorpyrifos and imidacloprid presented dose-dependent inhibition on cell growth and proliferation. Moreover, the pesticides showed inhibition on acetylcholinesterase activity and increase release of lactate dehydrogenase. However, the effect of imidacloprid was significantly weaker than that of chlorpyrifos. In conclusion, a novel brain-on-chip was developed in this study, which can be used to efficiently assess the drug neurotoxicity, pharmacodynamics, and disease mechanism by combining with a microtiterplate reader.
Animals ; Brain ; Chlorpyrifos/toxicity* ; Culture Media ; Mice ; Oligonucleotide Array Sequence Analysis ; Pesticides/toxicity*

Royal jelly (RJ) from honeybee has been widely used as a health promotion supplement. The major royal jelly proteins (MRJPs) have been identified as the functional component of RJ. However, the question of whether MRJPs have anti-senescence activity for human cells remains. Human embryonic lung fibroblast (HFL-I) cells were cultured in media containing no MRJPs (A), MRJPs at 0.1 mg/ml (B), 0.2 mg/ml (C), or 0.3 mg/ml (D), or bovine serum albumin (BSA) at 0.2 mg/ml (E). The mean population doubling levels of cells in media B, C, D, and E were increased by 12.4%, 31.2%, 24.0%, and 10.4%, respectively, compared with that in medium A. The cells in medium C also exhibited the highest relative proliferation activity, the lowest senescence, and the longest telomeres. Moreover, MRJPs up-regulated the expression of superoxide dismutase-1 (SOD1) and down-regulated the expression of mammalian target of rapamycin (MTOR), catenin beta like-1 (CTNNB1), and tumor protein p53 (TP53). Raman spectra analysis showed that there were two unique bands related to DNA synthesis materials, amide carbonyl group vibrations and aromatic hydrogens. These results suggest that MRJPs possess anti-senescence activity for the HFL-I cell line, and provide new knowledge illustrating the molecular mechanism of MRJPs as anti-senescence factors.
Animals ; Bees ; Cattle ; Cell Line ; Cell Proliferation ; Cellular Senescence/drug effects* ; Culture Media ; Dose-Response Relationship, Drug ; Fatty Acids/chemistry* ; Fibroblasts/drug effects* ; Humans ; Insect Proteins/chemistry* ; Lung/drug effects* ; Serum Albumin/metabolism* ; Spectrum Analysis, Raman ; Superoxide Dismutase-1/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; Tumor Suppressor Protein p53/metabolism* ; beta Catenin/metabolism*

Objective: To investigate whether in vitro culture medium (IVCM) for sparse spermatozoa can improve human sperm motility for the purpose of helping clinicians, laboratorians and patients choose a better strategy of assisted reproduction. METHODS: Semen samples were obtained from 178 males for routine semen examination from March to August 2016, including 151 cases of asthenozoospermia and 27 cases of normal sperm motility. A total of 200 μl was collected from each sample and divided into two equal portions and equal volumes of IVCM (experimental group) and F10 (1×) (control group) were added to the two portions, respectively, followed by 30-minute incubation at 37℃ in an incubator with 5% CO2. Sperm concentration, motility and viability and the percentages of progressively motile, non-progressively motile and immotile sperm were recorded before and after incubation. RESULTS: After activated with IVCM, neither the samples with asthenozoospermia nor those with normal sperm motility showed any statistically significant difference in sperm viability from the baseline or the control group (P>0.05). The rates of progressively and non-progressively motile sperm from the asthenozoospermia males were increased by 14.02% and 4.86% respectively, while that of immotile sperm decreased by 19.01% in the experimental group (P >0.01), and similar results were observed in the semen samples from the men with normal sperm motility. The percentage of reduced immotile viable sperm was positively correlated with that of immotile viable sperm in both the asthenozoospermia patients (r = 0.260, P <0.01) and the men with normal sperm motility (r = 0.679, P <0.01). CONCLUSIONS IVCM can increase sperm motility without affecting sperm viability in men with either asthenozoospermia or normal sperm motility. The larger the proportion of immotile viable sperm, the higher the percentages of progressively and non-progressively motile sperm in the semen after IVCM activation, and this correlation is more significant in men with normal sperm motility than in asthenozoospermia patients.
Asthenozoospermia ; physiopathology ; therapy ; Culture Media ; Culture Techniques ; Humans ; Male ; Semen ; Semen Analysis ; methods ; Sperm Count ; Sperm Motility ; Spermatozoa ; physiology

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.
Animals ; Brain/pathology ; *Cell Differentiation/drug effects ; Cells, Cultured ; Culture Media/chemistry/pharmacology ; Dental Pulp/*cytology ; Dopaminergic Neurons/*cytology/*metabolism/pathology ; Enzyme-Linked Immunosorbent Assay ; Glial Fibrillary Acidic Protein/genetics/metabolism ; Humans ; Mice ; Mice, Inbred ICR ; Myelin Basic Protein/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Stage-Specific Embryonic Antigens/genetics/metabolism ; Stem Cells/*cytology/*metabolism/pathology ; Tubulin/genetics/metabolism ; Tyrosine 3-Monooxygenase/analysis/genetics/metabolism

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.
Animals ; Brain/pathology ; *Cell Differentiation/drug effects ; Cells, Cultured ; Culture Media/chemistry/pharmacology ; Dental Pulp/*cytology ; Dopaminergic Neurons/*cytology/*metabolism/pathology ; Enzyme-Linked Immunosorbent Assay ; Glial Fibrillary Acidic Protein/genetics/metabolism ; Humans ; Mice ; Mice, Inbred ICR ; Myelin Basic Protein/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Stage-Specific Embryonic Antigens/genetics/metabolism ; Stem Cells/*cytology/*metabolism/pathology ; Tubulin/genetics/metabolism ; Tyrosine 3-Monooxygenase/analysis/genetics/metabolism

Through scale-up cultivation of Eriobotrya japonica suspension cells using WAVE bioreactor, the cell growth and ursolic acid (UA) accumulation were studied. The comparison test was carried out in the flask and the reactor with cell dry weight (DW) and UA content as evaluation indexes. The culture medium, DW and UA content were compared in 1 L and 5 L working volumes of bioreactor. The orthogonal test with main actors of inoculation amount, speed and angle of rotation was developed to find the optimal combination, in 1 L working volume of bioreactor. DW of the cell growth and the UA content in bioreactor were higher than those of the shaker by 105.5% and 27.65% respectively. In bioreactor, the dynamic changes of elements in the fluid culture, the dry weight of the cell growth and the UA content in 1 L and 5 L working volumes were similar. Inoculation of 80 g, rotational speed of 26 r · min(-1), and angle of 6 ° was the optimal combination, and the cell biomass of 19.01 g · L(-1) and the UA content of 27.750 mg · g(-1) were achieved after 100 h cultivation in 1 L working volume of bioreactor. WAVE Bioreactor is more suitable than flasks for the E. japonica cell suspension culture, and culture parameters can be achieved from 1 L to 5 L amplification.
Biomass ; Bioreactors ; Cell Culture Techniques ; instrumentation ; methods ; Culture Media ; chemistry ; metabolism ; Eriobotrya ; chemistry ; growth & development ; metabolism ; Triterpenes ; analysis ; metabolism

OBJECTIVETo investigate the effects of Dragon' s blood extract on proliferation and secret extracellular matrix function of fibroblasts in vitro.

METHODSDragon' s blood was extracted by chloroform, acetoacetic ester, alcohol. Human fibroblast were cultured in vitro in media containing gradient dilutions of Dragon' s blood extracts (0.002, 0.02, 0.2, 2, 20 mg/ml) , which was followed by cell proliferation assessed with MTT assay on 0, 12, 24, 36, 48, 60, 72 h. Under the optimal concentration, the cell growth curves were drawn and the flow cytometry (FCM) was used to determine the changes of cell cycle. On 0, 12, 24, 36, 48, 60, 72 h, the concentration of hyaluronic acid in the supernatant of fibroblast culture was measured by radioimmunoassay.

RESULTS0.2-2 mg/ml Dragon' s blood extracts enhanced the proliferation of fibroblasts in a dose-dependent manner. 2 mg/ml was the optimal dilution of Dragon's blood extract, and it increased the ratio of S cells in cell cycle [(25.80 ± 3.10)%] than control group [(7.50 ± 0.70)%, P < 0.01]. From 12 h to 72 h, in 2 mg/ml Dragon's blood group, concentration of Hyaluronic acid secreted by fibroblasts gradually increased, but were less than control (P < 0.01).

CONCLUSIONSDragon's blood acetoacetic ester extract improved the proliferation of cultured human fibroblasts in vitro, might be beneficial to promote wound healing.

Cell Cycle ; Cell Proliferation ; drug effects ; Culture Media ; chemistry ; Dose-Response Relationship, Drug ; Extracellular Matrix ; Fibroblasts ; cytology ; drug effects ; secretion ; Flow Cytometry ; Humans ; Hyaluronic Acid ; analysis ; secretion ; Plant Extracts ; pharmacology ; Resins, Plant ; Time Factors

Ginsenosides are the abundant secondary metabolites in American ginseng (Panax quinquefolium), it could be released into soil through root exudation and decomposition during plant growth. This study determined ginsenoside contents in American ginseng cultivated soil by HPLC. Three ginsenosides, Rb1, Rb2 and Rd, were detected in the rhizosphere soil of 3-4 years old American ginseng cultivated in Huairou District, Beijing, and their contents were 0.80-3.19 mg x kg(-1). Correspondingly, the contents of the three ginsenosides in soil solution were 4-16 mg x L(-1) at field water-holding capacity of 20%. According to the field soil test data, we designed the concentration of ginsenosides for bioassays (0.2-125 mg x L(-1) in solution or 0.2-125 mg x kg(-1) in soil). The results showed that radicle lengths of American ginseng were reduced by 6%-23% in solution containing 0.2-125 mg x L(-1) ginsenoside extract, and a significant difference was observed at concentration of 125 mg x L(-1) (P < 0.05). The shoot lengths of American ginseng were not significantly inhibited by 0.2-125 mg x L(-1) ginsenosides extractions. After 20 days of growth in nutrient solution amended with 25 mg x L(-1) ginsenosides extraction, plant height of 3-year-old American ginseng seedling was decreased by 28% compared to the control, and the biomass of aerial parts was also reduced by 50% (P < 0.05). However, the growth of newly-grown fibrous root was not significantly inhibited. Comparatively, when American ginseng embryos were cultivated into sterile or non-sterile soil, neither radicle lengths nor shoot lengths were significantly affected by 0.2-125 mg x kg(-1) ginsenoside extracts. In conclusion, ginsenosides showed autotoxic effect on growth of American ginseng radicle and adult seedling, however, this effect was weakened in field soil.
Chromatography, High Pressure Liquid ; Culture Media ; chemistry ; metabolism ; Ginsenosides ; analysis ; metabolism ; toxicity ; Panax ; chemistry ; drug effects ; growth & development ; metabolism ; Plant Roots ; chemistry ; drug effects ; growth & development ; metabolism ; Soil ; chemistry

Eleven compounds were isolated from the culture of Streptomyces sp. CPCC 202950 by a combination of various chromatographic techniques including column chromatography over macroporous resin HP-20, MCI, and reversed-phase HPLC. Their structures were identified as 1H-pyrrole-2-carboxamide(1),5'-deoxy-5'-methylthioinosine(2), vanillamide(3), trans-3-methylthioacrylamide(4), 1,2,3,4-Tetraydro-1H-pyrido[3,4-b]indole-3-carboxylic acid(5), cyclo(L-pro-L-tyr) (6), N-[2-(4-hydroxyphenyl)]ethylacetamide(7), benzamide (8), cyclo ('L-leucyl-trans-4-hydroxy-L-proline)(9), cyclo-(Phe-Gly) (10), and tryptophan (11). Among them, compounds 1 and 2 were new natural products. In the preliminary assays, none of the compounds exhibited obvious inhibition of HIV-1 protease activity (IC50 > 10 micromol x L(-1)).
Culture Media ; chemistry ; metabolism ; HIV Protease ; analysis ; HIV Protease Inhibitors ; chemistry ; isolation & purification ; Molecular Structure ; Spectrometry, Mass, Electrospray Ionization ; Streptomyces ; chemistry ; metabolism

OBJECTIVETo investigate the ability of invasion and migration of breast cancer MDA-MB-231 cells under serum starvation and hypoxia, and the effect of antiangiogenic drugs, rh-endostatin and bevacizumab, on the ability of invasion and migration of breast cancer cells under serum starvation and/or hypoxia, in order to explore the potential risk of antiangiogenic therapy in clinics.

METHODSThe cells were randomized into 4 groups, i.e., group A: 10% fetal bovine serum (FBS) group; group B: hypoxia + 10% FBS group; group C: serum starvation group; group D: hypoxia + serum starvation group; each group was further divided into three subgroups as blank control, treated with rh-endostatin and bevacizumab, respectively. Cell counting kit-8 (CCK-8) was used to assess the inhibition rate of cell growth induced by endostatin and bevacizumab, in order to determine the proper working concentration and time of the two drugs. Transwell assay was conducted to detect the cell invasion and migration in vitro. The expressions of c-Met and MMP-9 were detected by Western blot. The cells treated with rh-endostatin or bevacizumab under serum starvation were tested by hybridization using Exiqon miBase 18.0 microarray. The miRNAs which exibited significant differences (P < 0.05) in miRNA hybridization were verified by real-time PCR assay.

RESULTSCCK-8 assay showed that the inhibition rates of MDA-MB-231 cells cultured with 800 mg/L rh-endostatin for 48 h and 24 h were (32.2 ± 2.5)% and (27.0 ± 1.3)%, respectively, showing a significant difference (P = 0.023). The inhibition rates of MDA-MB-231 cells cultured with 80 mg/L bevacizumab for 48 h and 24 h were (30.5 ± 1.4) % and (26.1 ± 2.4) %, respectively, showing also a significant difference (P = 0.015). The Transwell assay showed that in the starvation blank group, the number of invaded and penetrated cells were 28.8 ± 2.2 and 31.4 ± 1.5, respectively, significantly different from that in the rh-endostatin and bevacizumab groups (P < 0.05). The relative expressions of c-Met and MMP-9 were 0.213 ± 0.017 and 0.542 ± 0.048, respectively, with a significant difference from those of the groups treated with each drug (P < 0.05 for both). The numbers of penetrated cells in the Transwell assay treated with rh-endostatin in hypoxia were 17.5 ± 2.1 and 16.5 ± 2.8, respectively, and the numbers of penetrated cells in the Transwell assay treated with bevacizumab were 16.3 ± 3.5 and 17.5 ± 2.4, respectively, showing no significant difference among them (P > 0.05 for both). The ability of migration and invasion of MDA-MB-231 cells and the expression of c-Met and MMP-9 were not impacted by hypoxia (P > 0.05). Real-time PCR assay showed that only the levels of miR-2355 and miR375 were significantly and stably decreased in the cells which had increased ability of invasion and migration. The relative expression levels of miR375 and miR-2355 in the serum starvation blank group were 0.550 ± 0.036 and 0.852 ± 0.121, respectively, significantly lower than that in the groups treated with rh-endostatin or bevacizumab (P<0.05). In the serum starvation group, the expression levels of miR375 and miR-2355 of cells treated with rh-endostatin were 0.295 ± 0.012 and 0.253 ± 0.011, and the expression levels of cells treated with bevacizumab were 0.234 ± 0.020 and 0.309 ± 0.022, respectively, (P > 0.05 for all). Compared with the serum starvation blank group, the expression levels of miR2355 and miR375 were significantly decreased when cells were treated with rh-endostatin/bevacizumab under serum starvation, but no significant difference was found between the two drugs (P > 0.05). However, hypoxia did not affect the expressions of miR2355 and miR375 (P > 0.05).

CONCLUSIONSThe results of this study suggest that serum starvation can increase the ability of invasion and migration of breast cancer cells. Furthermore, both rh-endostatin and bevacizumab may enhance their invasion and penetration ability under serum starvation condition.

Angiogenesis Inhibitors ; adverse effects ; Bevacizumab ; adverse effects ; Breast Neoplasms ; pathology ; Cell Hypoxia ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Culture Media, Serum-Free ; Endostatins ; adverse effects ; Female ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; MicroRNAs ; analysis ; Neoplasm Invasiveness ; Proto-Oncogene Proteins c-met ; metabolism ; Random Allocation ; Real-Time Polymerase Chain Reaction ; Time Factors