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Sample records for chemically defined serum-free

  1. Development of a chemically defined serum-free medium for differentiation of rat adipose precursor cells

    SciTech Connect

    Deslex, S.; Negrel, R.; Ailhaud, G.

    1987-01-01

    Stromal-vascular cells from the epididymal fat pad of 4-week-old rats, when cultured in a medium containing insulin or insulin-like growth factor, IFG-I, triiodothyronine and transferrin, were able to undergo adipose conversion. Over ninety percent of the cells accumulated lipid droplets and this proportion was reduced in serum-supplemented medium. The adipose conversion was assessed by the development of lipoprotein lipase (LPL) and glycerol-3-phosphate dehydrogenase (GPDH) activities, (/sup 14/)glucose incorporation into polar and neutral lipids, triacylglycerol accumulation and lipolysis in response to isoproterenol. Similar results were obtained with stromal-vascular cells from rat subcutaneous and retroperitoneal adipose tissues. Stromal-vascular cells required no adipogenic factors in addition to the components of the serum-free medium. Insulin was required within a physiological range of concentrations for the emergence of LPL and at higher concentrations for that of GPDH. When present at concentrations ranging from 2 to 50 nM, IGF-I was able to replace insulin for the expression of both LPL and and GPDH. The development of a serum free, chemically defined medium for the differentiation of diploid adiopose precursor cells opens up the possibility of characterizing inhibitors or activators of the adipose conversion process.

  2. Characterization of cultured rat oligodendrocytes proliferating in a serum-free, chemically defined medium

    SciTech Connect

    Saneto, R.P.; de Vellis, J.

    1985-05-01

    A serumless, chemically defined medium has been developed for the culture of oligodendrocytes isolated from primary neonatal rat cerebral cultures. Combined together, insulin, transferrin, and fibroblast growth factor synergistically induced an essentially homogeneous population (95-98%) of cells expressing glycerol-3-phosphate dehydrogenase activity to undergo cell division. Proliferating cells were characterized by several criteria: (i) ultrastructural analysis by transmission electron microscopy identified the cell type as an oligodendrocyte; (ii) biochemical assays showed expression of three oligodendrocyte biochemical markers, induction of both glycerol phosphate dehydrogenase and lactate dehydrogenase, and presence of 2',3'-cyclic nucleotide 3'-phosphodiesterase; and (iii) immunocytochemical staining showed cultures to be 95-98% positive for glycerol phosphate dehydrogenase, 90% for myelin basic protein, 60-70% for galactocerebroside, and 70% for A2B5.

  3. Characterization of cultured rat oligodendrocytes proliferating in a serum-free, chemically defined medium.

    PubMed Central

    Saneto, R P; de Vellis, J

    1985-01-01

    A serumless, chemically defined medium has been developed for the culture of oligodendrocytes isolated from primary neonatal rat cerebral cultures. Combined together, insulin, transferrin, and fibroblast growth factor synergistically induced an essentially homogenous population (95-98%) of cells expressing glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) activity to undergo cell division. Proliferating cels were characterized by several criteria: (i) ultrastructural analysis by transmission electron microscopy identified the cell type as an oligodendrocyte; (ii) biochemical assays showed expression of three oligodendrocyte biochemical markers, induction of both glycerol phosphate dehydrogenase and lactate dehydrogenase (EC 1.1.1.27), and presence of 2',3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37); and (iii) immunocytochemical staining showed cultures to be 95-98% positive for glycerol phosphate dehydrogenase, 90% for myelin basic protein, 60-70% for galactocerebroside, and 70% for A2B5. Few cells (less than 5%) stained positive for glial fibrillary acidic protein, and none were detected positive for fibronectin. Images PMID:2987930

  4. Norepinephrine stimulates progesterone production in highly estrogenic bovine granulosa cells cultured under serum-free, chemically defined conditions

    PubMed Central

    2012-01-01

    Background Since noradrenergic innervation was described in the ovarian follicle, the actions of the intraovarian catecholaminergic system have been the focus of a variety of studies. We aimed to determine the gonadotropin-independent effects of the catecholamine norepinephrine (NE) in the steroid hormone profile of a serum-free granulosa cell (GC) culture system in the context of follicular development and dominance. Methods Primary bovine GCs were cultivated in a serum-free, chemically defined culture system supplemented with 0.1% polyvinyl alcohol. The culture features were assessed by hormone measurements and ultrastructural characteristics of GCs. Results GCs produced increasing amounts of estradiol and pregnenolone for 144h and maintained ultrastructural features of healthy steroidogenic cells. Progesterone production was also detected, although it significantly increased only after 96h of culture. There was a highly significant positive correlation between estradiol and pregnenolone production in high E2-producing cultures. The effects of NE were further evaluated in a dose–response study. The highest tested concentration of NE (10 (−7) M) resulted in a significant increase in progesterone production, but not in estradiol or pregnenolone production. The specificity of NE effects on progesterone productio n was further investigated by incubating GCs with propranolol (10 (−8) M), a non-selective beta-adrenergic antagonist. Conclusions The present culture system represents a robust model to study the impact of intrafollicular factors, such as catecholamines, in ovarian steroidogenesis and follicular development. The results of noradrenergic effects in the steroidogenesis of GC have implications on physiological follicular fate and on certain pathological ovarian conditions such as cyst formation and anovulation. PMID:23171052

  5. Serum-free, chemically defined medium with TGF-beta(3) enhances functional properties of nucleus pulposus cell-laden carboxymethylcellulose hydrogel constructs.

    PubMed

    Reza, Anna T; Nicoll, Steven B

    2010-02-01

    Degeneration of the nucleus pulposus (NP) has been implicated as a major cause of low back pain. Tissue engineering strategies may provide a viable NP replacement therapy; however, culture conditions must be optimized to promote functional tissue development. In this study, a standard serum-containing medium formulation was compared to a chemically defined, serum-free medium to determine the effect on matrix elaboration and functional properties of NP cell-laden carboxymethylcellulose (CMC) hydrogels. Additionally, both media were further supplemented with transforming growth factor-beta 3 (TGF-beta(3)). Glycosaminoglycan (GAG) content increased in both TGF-beta(3)-treated groups and was highest for treated, serum-free constructs (9.46 +/- 1.51 microg GAG/mg wet weight), while there were no quantifiable GAGs in untreated serum-containing samples. Histology revealed uniform, interterritorial staining for chondroitin sulfate proteoglycan throughout the treated, serum-free constructs. Type II collagen content was greater in both serum-free groups and highest in treated, serum-free constructs. The equilibrium Young's modulus was highest in serum-free samples supplemented with TGF-beta(3) (18.54 +/- 1.92 kPa), and the equilibrium weight swelling ratio of these constructs approached that of the native NP tissue (22.19 +/- 0.46 vs. 19.94 +/- 3.09, respectively). Taken together, these results demonstrate enhanced functional matrix development by NP cells when cultured in CMC hydrogels maintained in serum-free, TGF-beta(3) supplemented medium, indicating the importance of medium formulation in NP construct development. PMID:19777586

  6. Ex Vivo Expansion of Human Mesenchymal Stem Cells in Defined Serum-Free Media

    PubMed Central

    Jung, Sunghoon; Panchalingam, Krishna M.; Rosenberg, Lawrence; Behie, Leo A.

    2012-01-01

    Human mesenchymal stem cells (hMSCs) are presently being evaluated for their therapeutic potential in clinical studies to treat various diseases, disorders, and injuries. To date, early-phase studies have indicated that the use of both autologous and allogeneic hMSCs appear to be safe; however, efficacy has not been demonstrated in recent late-stage clinical trials. Optimized cell bioprocessing protocols may enhance the efficacy as well as safety of hMSC therapeutics. Classical media used for generating hMSCs are typically supplemented with ill-defined supplements such as fetal bovine serum (FBS) or human-sourced alternatives. Ideally, culture media are desired to have well-defined serum-free formulations that support the efficient production of hMSCs while maintaining their therapeutic and differentiation capacity. Towards this objective, we review here current cell culture media for hMSCs and discuss medium development strategies. PMID:22645619

  7. Functional Myotube Formation from Adult Rat Satellite Cells in a Defined Serum-free System

    PubMed Central

    McAleer, Christopher W.; Rumsey, John W.; Stancescu, Maria; Hickman, James J.

    2016-01-01

    This manuscript describes the development of a culture system whereby mature contracting myotubes were formed from adult rat derived satellite cells. Satellite cells, extracted from the Tibialis Anterior (TA) of adult rats, were grown in defined serum-free growth and differentiation media, on a non-biological substrate, N-1[3-trimethoxysilyl propyl] diethylenetriamine. Myotubes were evaluated morphologically and immunocytochemically, using MyHC specific antibodies, as well as functionally using patch clamp electrophysiology to measure ion channel activity. Results indicated the establishment of the rapid expression of adult myosin isoforms that contrasts to their slow development in embryonic cultures. This culture system has applications in the understanding and treatment of age related muscle myopathy, muscular dystrophy, and for skeletal muscle engineering by providing a more relevant phenotype for both in vitro and in vivo applications. PMID:25683642

  8. A serum-free and defined medium for the culture of mammalian postimplantation embryos.

    PubMed

    Drakou, Katerina; Georgiades, Pantelis

    2015-12-25

    Whole embryo culture (WEC) of postimplantation rodent embryos is widely used for the study of mammalian embryogenesis and developmental toxicity testing. Its major advantage is that it allows direct access to embryos for experimental manipulations and the monitoring of their consequences that would otherwise not be possible or technically difficult to perform in utero. However, a major drawback of mammalian WEC is that the culture media currently in use display batch variations and are undefined, as they contain serum or serum replacements of unknown composition. Moreover, these media possess cell-signalling activities important for embryogenesis. Therefore, reproducibility of mammalian postimplantation WEC results may be affected by batch variation and their interpretation is complicated because the experimenter is unsure whether the embryo response to experimental perturbations is solely due to their action, or modified as a result of influences from undefined substances/signaling activities present in culture media. To alleviate these problems we investigated whether N2B27, a serum-free and defined medium, can support the in vitro development of postimplantation mammalian embryos. We show that N2B27 allows pre-gastrulation mouse embryos isolated at embryonic day 5.5 to develop to advanced gastrulation, reaching the mid- and late primitive streak stages. This is the first demonstration that postimplantation mammalian embryos can develop in vitro in a defined medium in the absence of serum and provides a novel WEC system for studying developmental mechanisms and testing for developmental toxicity during the early postimplantation period. PMID:26603939

  9. Fasciola hepatica in vitro: increased susceptibility to fasciolicides in a defined serum-free medium.

    PubMed

    Jenkins, D C; Topley, P; Rapson, E B

    1987-08-01

    The cidal properties of some phenolic, halogenated diphenyl, salicylanilide, benzimidazole and diaminophenoxyalkane anthelmintics, against 6-week-old worms of Fasciola hepatica were assessed in vitro. In a conventional fluke culture medium containing RPMI 1640, supplemented with serum with or without rabbit erythrocytes or pink-ghosts, only the halogenated diphenyl and salicylanilide compounds showed activity at concentrations equal to or less than 100 microM. However, when basal, serum and cell-free RPMI 1640 was used, all compounds other than diamphenethide were highly active, their minimum lethal concentrations being some 25-125 times lower under these conditions. The inclusion of rabbit liver microsomes in the basal culture medium resulted in diamphenethide exhibiting cidal activity equivalent to that seen when its free-amine active metabolite was assayed. The possibility that the activity of many of these compounds was masked in vitro because of their serum binding properties is discussed. Recommendations are made that in vitro screens for new fasciolicides should be carried out in serum-free medium and that additional replicates containing mammalian liver microsomes and liver cytosolic extracts be included as means for the metabolic activation of certain otherwise undetectable prodrugs. PMID:3670897

  10. A defined synthetic substrate for serum-free culture of human stem cell derived cardiomyocytes with improved functional maturity identified using combinatorial materials microarrays

    PubMed Central

    Patel, Asha K.; Celiz, Adam D.; Rajamohan, Divya; Anderson, Daniel G.; Langer, Robert; Davies, Martyn C.

    2016-01-01

    Cardiomyocytes from human stem cells have applications in regenerative medicine and can provide models for heart disease and toxicity screening. Soluble components of the culture system such as growth factors within serum and insoluble components such as the substrate on which cells adhere to are important variables controlling the biological activity of cells. Using a combinatorial materials approach we develop a synthetic, chemically defined cellular niche for the support of functional cardiomyocytes derived from human embryonic stem cells (hESC-CMs) in a serum-free fully defined culture system. Almost 700 polymers were synthesized and evaluated for their utility as growth substrates. From this group, 20 polymers were identified that supported cardiomyocyte adhesion and spreading. The most promising 3 polymers were scaled up for extended culture of hESC-CMs for 15 days and were characterized using patch clamp electrophysiology and myofibril analysis to find that functional and structural phenotype was maintained on these synthetic substrates without the need for coating with extracellular matrix protein. In addition, we found that hESC-CMs cultured on a co-polymer of isobornyl methacrylate and tert-butylamino-ethyl methacrylate exhibited significantly longer sarcomeres relative to gelatin control. The potential utility of increased structural integrity was demonstrated in an in vitro toxicity assay that found an increase in detection sensitivity of myofibril disruption by the anti-cancer drug doxorubicin at a concentration of 0.05 µM in cardiomyocytes cultured on the co-polymer compared to 0.5 µM on gelatin. The chemical moieties identified in this large-scale screen provide chemically defined conditions for the culture and manipulation of hESC-CMs, as well as a framework for the rational design of superior biomaterials. PMID:26005764

  11. Serum free hemoglobin test

    MedlinePlus

    ... page: //medlineplus.gov/ency/article/003677.htm Serum free hemoglobin test To use the sharing features on this page, please enable JavaScript. Serum free hemoglobin is a blood test that measures the ...

  12. Purified human pancreatic duct cell culture conditions defined by serum-free high-content growth factor screening.

    PubMed

    Hoesli, Corinne A; Johnson, James D; Piret, James M

    2012-01-01

    The proliferation of pancreatic duct-like CK19+ cells has implications for multiple disease states including pancreatic cancer and diabetes mellitus. The in vitro study of this important cell type has been hampered by their limited expansion compared to fibroblast-like vimentin+ cells that overgrow primary cultures. We aimed to develop a screening platform for duct cell mitogens after depletion of the vimentin+ population. The CD90 cell surface marker was used to remove the vimentin+ cells from islet-depleted human pancreas cell cultures by magnetic-activated cell sorting. Cell sorting decreased CD90+ cell contamination of the cultures from 34±20% to 1.3±0.6%, yielding purified CK19+ cultures with epithelial morphology. A full-factorial experimental design was then applied to test the mitogenic effects of bFGF, EGF, HGF, KGF and VEGF. After 6 days in test conditions, the cells were labelled with BrdU, stained and analyzed by high-throughput imaging. This screening assay confirmed the expected mitogenic effects of bFGF, EGF, HGF and KGF on CK19+ cells and additionally revealed interactions between these factors and VEGF. A serum-free medium containing bFGF, EGF, HGF and KGF led to CK19+ cell expansion comparable to the addition of 10% serum. The methods developed in this work should advance pancreatic cancer and diabetes research by providing effective cell culture and high-throughput screening platforms to study purified primary pancreatic CK19+ cells. PMID:22442738

  13. Serum-free growth of human mammary epithelial cells: rapid clonal growth in defined medium and extended serial passage with pituitary extract

    SciTech Connect

    Hammond, S.L.; Ham, R.G.; Stampfer, M.R.

    1984-09-01

    A serum-free medium with bovine pituitary extract as the only undefined supplement has been developed for long-term culture of human mammary epithelial cells. This medium supports serial subculture of normal cells for 10-20 passages (1:10 splits) without conditioning or special substrates, and it supports rapid clonal growth with plating efficiencies up to 35%. It consists of an optimized basal nutrient medium, (MCDB 170, supplemented with insulin, hydrocortisone, epidermal growth factor, ethanolamine, phosphoethanolamine, and bovine pituitary extract. Replacement of pituitary extract with prostaglandin E/sub 1/ and ovine prolactin yields a defined medium that supports rapid clonal growth and serial subculture for three of four passages. Cultures initiated in these media from normal reduction mammoplasty tissue remain diploid and maintain normal epithelia morphology, distribution of cell-associated fibronectin, expression of keratin fibrils, and a low level of expression of milk fat globule antigen. Large cell populations can now be generated and stored frozen, permitting multiple experiments over a period of time with cells from a single donor. These media greatly extend the range of experiments that can be performed both conveniently and reproducibly with cultured normal and tumor-derived human mammary epithelial cells. 31 references, 3 figures, 4 tables.

  14. Growth of purified astrocytes in a chemically defined medium

    SciTech Connect

    Morrison, R.S.; De Vellis, J.

    1981-11-01

    Astrocytes purified from primary cultures of neonatal rat cerebrum can not be grown in a synthetic medium supplemented with putrescine, prostaglandin F/sub 2//sub ..cap alpha../, insulin, fibroblast growth factor, and hydrocortisone. These five supplements have a marked synergistic effect on growth when used in combination but have little effect when used individually. Astrocytes grown in the defined medium exhibit dramatic changes in morphological characteristics in comparison to cells grown in serum-free or serum-supplemented medium. In addition, these cells express the astrocyte-specific marker glial fibrillary acidic protein and are estimated by several criteria to be greater than 95% astrocytes.

  15. Chemically defined medium and Caenorhabditis elegans

    NASA Technical Reports Server (NTRS)

    Szewczyk, Nathaniel J.; Kozak, Elena; Conley, Catharine A.

    2003-01-01

    BACKGROUND: C. elegans has been established as a powerful genetic system. Use of a chemically defined medium (C. elegans Maintenance Medium (CeMM)) now allows standardization and systematic manipulation of the nutrients that animals receive. Liquid cultivation allows automated culturing and experimentation and should be of use in large-scale growth and screening of animals. RESULTS: We find that CeMM is versatile and culturing is simple. CeMM can be used in a solid or liquid state, it can be stored unused for at least a year, unattended actively growing cultures may be maintained longer than with standard techniques, and standard C. elegans protocols work well with animals grown in defined medium. We also find that there are caveats to using defined medium. Animals in defined medium grow more slowly than on standard medium, appear to display adaptation to the defined medium, and display altered growth rates as they change the composition of the defined medium. CONCLUSIONS: As was suggested with the introduction of C. elegans as a potential genetic system, use of defined medium with C. elegans should prove a powerful tool.

  16. Development of Serum-Free Media for Lepidopteran Insect Cell Lines.

    PubMed

    Chan, Leslie C L; Reid, Steven

    2016-01-01

    Baculovirus-based Insect Cell Technology (ICT) is widely used for the expression of recombinant heterologous proteins and baculovirus bioinsecticides, and has recently gained momentum as a commercial manufacturing platform for human and veterinary vaccines. The three key components of ICT are the Lepidopteran insect cell line, the baculovirus vector, and the growth medium. Insect cell growth media have evolved significantly in the past five decades, from basal media supplemented with hemolymph or animal serum, to highly optimized serum-free media and feeds (SFM and SFF) capable of supporting very high cell densities and recombinant protein yields. The substitution of animal sera with protein hydrolysates in SFM results in greatly reduced medium costs and much improved process scalability. However, both sera and hydrolysates share the disadvantage of lot-to-lot variability, which is detrimental to process reproducibility. Hence, the industrialization of ICT would benefit greatly from chemically defined media (CDM) for insect cells, which are not yet commercially available. On the other hand, applications such as baculovirus bioinsecticides would need truly low cost serum-free media and feeds (LC-SFM and LC-SFF) for economic viability, which require the substitution of a majority of expensive added amino acids with even higher levels of hydrolysates, hence increasing the risk of a variable process. CDM developments are anticipated to benefit both conventional and low cost ICT applications, by identifying key growth factors in hydrolysates for more targeted media and feed design. PMID:26820858

  17. Biliary lipids support serum-free growth of Giardia lamblia.

    PubMed Central

    Gillin, F D; Gault, M J; Hofmann, A F; Gurantz, D; Sauch, J F

    1986-01-01

    Giardia lamblia has been grown in vitro only in media containing serum or serum fractions. How this pathogen can grow in the human small intestinal lumen without serum is not known. We found that samples of human hepatic or gall bladder bile maintained G. lamblia survival for 24 to 48 h in medium without serum but did not support growth. By contrast, an artificial biliary lipid dispersion containing six bile salts, phosphatidylcholine (PC), and cholesterol, in the ratios characteristic of human bile, supported parasite growth in medium without serum or serum fractions. To define the requirements, we showed that 1-palmitoyl-2-linoleoyl-PC or 1-palmitoyl-2-oleoyl-PC (which predominate in human bile) satisfied the requirement for PC. Moreover, either glycocholate or glycodeoxycholate could be substituted for the bile salt mixture. The finding that biliary lipids can support serum-free growth of G. lamblia may help explain why this parasite colonizes the upper small intestine. PMID:3744557

  18. Fibrinogen synthesis in serum-free hepatocyte cultures: Stimulation by glucocorticoids

    PubMed Central

    Grieninger, Gerd; Hertzberg, Kathe M.; Pindyck, Johanna

    1978-01-01

    Fibrinogen synthesis was investigated in cultures of chicken embryo hepatocytes initiated and maintained in chemically defined, serum-free medium. 11-Hydroxy glucocorticoids caused a 3-fold stimulation of fibrinogen synthesis. Half-maximal stimulation was achieved with 1 nM corticosterone or hydrocortisone, as compared with only 0.1 nM dexamethasone. Increased fibrinogen production in the presence of these glucocorticoids was characterized by a 4-hr delay in onset, a sensitivity to actinomycin D, and a requirement for the continuous presence of the steroid. Crossed immunoelectrophoresis permitted analysis of the simultaneous effects of glucocorticoids on the synthesis of more than 20 plasma proteins secreted in culture. The absence of an effect on the synthesis of most of these proteins was in sharp contrast to the 3-fold increase in fibrinogen production. Sera from a variety of animals also stimulated an increase in fibrinogen synthesis that was similar in degree but less specific than that due to glucocorticoids and that partially masked the response of the cells to the steroid hormones. The presence of an anticoagulant in the medium was found to be necessary for detection of the fibrinogen secreted in culture. Although insulin was routinely included in the chemically defined medium, the cells synthesized fibrinogen and responded to glucocorticoids in the absence of hormonal supplementation of the medium. These findings are consistent with the thesis that variations in glucocorticoid levels contribute to the regulation of fibrinogen production in the intact animal. Images PMID:281699

  19. Fibrinogen synthesis in serum-free hepatocyte cultures: stimulation by glucocorticoids.

    PubMed

    Grieninger, G; Hertzberg, K M; Pindyck, J

    1978-11-01

    Fibrinogen synthesis was investigated in cultures of chicken embryo hepatocytes initiated and maintained in chemically defined, serum-free medium. 11-Hydroxy glucocorticoids caused a 3-fold stimulation of fibrinogen synthesis. Half-maximal stimulation was achieved with 1 nM corticosterone or hydrocortisone, as compared with only 0.1 nM dexamethasone. Increased fibrinogen production in the presence of these glucocorticoids was characterized by a 4-hr delay in onset, a sensitivity to actinomycin D, and a requirement for the continuous presence of the steroid. Crossed immunoelectrophoresis permitted analysis of the simultaneous effects of glucocorticoids on the synthesis of more than 20 plasma proteins secreted in culture. The absence of an effect on the synthesis of most of these proteins was in sharp contrast to the 3-fold increase in fibrinogen production. Sera from a variety of animals also stimulated an increase in fibrinogen synthesis that was similar in degree but less specific than that due to glucocorticoids and that partially masked the response of the cells to the steroid hormones. The presence of an anticoagulant in the medium was found to be necessary for detection of the fibrinogen secreted in culture. Although insulin was routinely included in the chemically defined medium, the cells synthesized fibrinogen and responded to glucocorticoids in the absence of hormonal supplementation of the medium. These findings are consistent with the thesis that variations in glucocorticoid levels contribute to the regulation of fibrinogen production in the intact animal. PMID:281699

  20. DEFINING THE CHEMICAL SPACE OF PUBLIC GENOMIC DATA (S)

    EPA Science Inventory

    The current project aims to chemically index the genomics content of public genomic databases to make these data accessible in relation to other publicly available, chemically-indexed toxicological information. By defining the chemical space of public genomic data, it is possibl...

  1. Serum-free primary human fibroblast and keratinocyte coculture.

    PubMed

    Mujaj, Sally; Manton, Kerry; Upton, Zee; Richards, Sean

    2010-04-01

    Research has shown that the inclusion of a fibroblast cell support layer is required for the isolation and expansion of primary keratinocytes. Recent advances have provided keratinocyte culture with fibroblast-free alternatives. However, these technologies are often undefined and rely on the incorporation of purified proteins/components. To address this problem we developed a medium that used recombinant proteins to support the serum-free isolation and expansion of human dermal fibroblasts and keratinocytes. The human dermal fibroblasts were able to be isolated serum free by adding recombinant human albumin to a collagenase solution. These fibroblasts were then expanded using a serum-free medium containing recombinant proteins: epidermal growth factor, basic fibroblast growth factor, chimeric vitronectin:insulin-like growth factor-I protein, and recombinant human albumin. These fibroblasts maintained a typical morphology and expressed fibroblast markers during their serum-free isolation, expansion, and freezing. Moreover, these fibroblasts were able to support the serum-free isolation and expansion of primary keratinocytes using these recombinant proteins. Real-time polymerase chain reaction and immunofluorescence analysis confirmed that there were no differences in expression levels of p63 or keratins 1, 6, and 10 when keratinocytes were grown in either serum-supplemented or serum-free medium. Using a three-dimensional human skin equivalent model we demonstrated that these keratinocytes also maintained their ability to reform an epidermal layer. In summary, the techniques described provide a valuable alternative for culturing fibroblasts and keratinocytes using recombinant proteins. PMID:19929322

  2. Helicobacter pylori Growth and Urease Detection in the Chemically Defined Medium Ham's F-12 Nutrient Mixture

    PubMed Central

    Testerman, Traci L.; McGee, David J.; Mobley, Harry L. T.

    2001-01-01

    Obstacles continue to hinder in vitro studies of the gastric human pathogen Helicobacter pylori, including difficulty culturing the organism in the absence of serum or blood, rapid loss of viability following exponential growth due to autolysis, and the necessity for using high starting inocula. We demonstrate that H. pylori grows in the chemically defined broth medium Ham's F-12 nutrient mixture (F-12) in the absence of fetal bovine serum (FBS); this represents a breakthrough for studies in which serum components or proteins interfere with interpretation of results. Cultures can be continually passaged in fresh, FBS-free F-12 medium at an initial inoculum of only ∼103 CFU/ml. All H. pylori strains (n = 21), including fresh clinical isolates, grew in serum-free F-12. H. pylori grew poorly in the related medium, F-10, unless additional zinc was supplied. Enhanced growth of H. pylori in F-12 broth was obtained by addition of bovine serum albumin (BSA) (1 mg/ml), β-cyclodextrin (200 μg/ml), or cholesterol (50 μg/ml). H. pylori also grew in several simplified versions of F-12 broth lacking glucose and most vitamins but containing hypoxanthine, pyruvate, and all 20 amino acids. On F-12 medium solidified with agar, H. pylori only grew when BSA (98% pure; 1 mg/ml), cholesterol (50 μg/ml), β-cyclodextrin (200 μg/ml), or FBS (2 to 4%) was added; addition of urea and phenol allowed colorimetric detection of urease activity. Thus, F-12 agar plus cholesterol or β-cyclodextrin represents the first transparent chemically defined agar and the first urease indicator agar for H. pylori. Several lines of evidence suggested that BSA itself is not responsible for H. pylori growth enhancement in F-12 containing BSA or FBS. Taken together, these innovations represent significant advances in the cultivation and recovery of H. pylori using chemically defined media. Use of F-12 or its derivatives may lead to improved understanding of H. pylori metabolism, virulence factors, and

  3. Derivation of a novel undifferentiated human foetal phenotype in serum-free cultures with BMP-2

    PubMed Central

    Mirmalek-Sani, Sayed-Hadi; Stokes, Paula J; Tare, Rahul S; Ralph, Esther J; Inglis, Stefanie; Hanley, Neil A; Houghton, Franchesca D; Oreffo, Richard OC

    2009-01-01

    Skeletal stem and progenitor populations provide a platform for cell-based tissue regeneration strategies. Optimized conditions for ex vivo expansion will be critical and use of serum-free culture may allow enhanced modelling of differentiation potential. Maintenance of human foetal femur-derived cells in a chemically defined medium (CDM) with activin A and fibroblast growth factor-2 generated a unique undifferentiated cell population in comparison to basal cultures, with significantly reduced amino acid depletion, appearance and turnover, reduced alkaline phosphatase (ALP) activity and loss of type I and II collagen expression demonstrated by fluorescence immunocytochemistry. Microarray analysis demonstrated up-regulation of CLU, OSR2, POSTN and RABGAP1 and down-regulation of differentiation-associated genes CRYAB, CSRP1, EPAS1, GREM1, MT1X and SRGN as validated by quantitative real-time polymerase chain reaction. Application of osteogenic conditions to CDM cultures demonstrated partial rescue of ALP activity. In contrast, the addition of bone morphogenetic protein-2 (BMP-2) resulted in reduced ALP levels, increased amino acid metabolism and, strikingly, a marked shift to a cobblestone-like cellular morphology, with expression of SOX-2 and SOX-9 but not STRO-1 as shown by immunocytochemistry, and significantly altered expression of metabolic genes (GFPT2, SC4MOL and SQLE), genes involved in morphogenesis (SOX15 and WIF1) and differentiation potential (C1orf19, CHSY-2,DUSP6, HMGCS1 and PPL). These studies demonstrate the use of an intermediary foetal cellular model for differentiation studies in chemically defined conditions and indicate the in vitro reconstruction of the mesenchymal condensation phenotype in the presence of BMP-2, with implications therein for rescue studies, screening assays and skeletal regeneration research. PMID:19438813

  4. Developmental features of rat cerebellar neural cells cultured in a chemically defined medium

    SciTech Connect

    Gallo, V.; Ciotti, M.T.; Aloisi, F.; Levi, G.

    1986-01-01

    We studied some aspects of the differentiation of rat cerebellar neural cells obtained from 8-day postnatal animals and cultured in a serum-free, chemically defined medium (CDM). The ability of the cells to take up radioactive transmitter amino acids was analyzed autoradiographically. The L-glutamate analogue /sup 3/H-D-aspartate was taken up by astroglial cells, but not by granule neurons, even in late cultures (20 days in vitro). This is in agreement with the lack of depolarization-induced release of /sup 3/H-D-aspartate previously observed in this type of culture. In contrast, /sup 3/H-(GABA) was scarcely accumulated by glial-fibrillary-acidic-protein (GFAP)-positive astrocytes, but taken up by glutamate-decarboxylase-positive inhibitory interneurons and was released in a Ca2+-dependent way upon depolarization: /sup 3/H-GABA evoked release progressively increased with time in culture. Interestingly, the expression of the vesicle-associated protein synapsin I was much reduced in granule cells cultured in CDM as compared to those maintained in the presence of serum. These data would indicate that in CDM the differentiation of granule neurons is not complete, while that of GABAergic neurons is not greatly affected. Whether the diminished differentiation of granule cells must be attributed only to serum deprivation or also to other differences in the composition of the culture medium remains to be established. /sup 3/H-GABA was avidly taken up also by a population of cells which were not recognized by antibodies raised against GFAP, glutamate decarboxylase, and microtubule-associated protein 2. These cells have been characterized as bipotential precursors of oligodendrocytes and of a subpopulation of astrocytes bearing a stellate shape and capable of high-affinity /sup 3/H-GABA uptake.

  5. Chemically Defined Medium and Caenorhabditis elegans: A Powerful Approach

    NASA Technical Reports Server (NTRS)

    Szewczyk, N. J.; Kozak, E.; Conley, C. A.

    2003-01-01

    C. elegans has been established as a powerful genetic system. Growth in a chemically defined medium (C. elegans Maintenance Medium (CeMM)) now allows standardization and systematic manipulation of the nutrients that animals receive. Liquid cultivation allows automated culturing and experimentation and should be of me in large-scale growth and screening of animals. Here we present our initial results from developing culture systems with CeMM. We find that CeMM is versatile and culturing is simple. CeMM can be used in a solid or liquid state, it can be stored unused for at least a year, unattended actively growing cultures may be maintained longer than with standard techniques, and standard C. elegans protocols work well with animals grown in defined medium. We also find that there are caveats of using defined medium. Animals in defined medium grow more slowly than on standard medium, appear to display adaptation to the defined medium, and display altered growth rates as they change defined medium composition. As was suggested with the introduction of C. elegans as a potential genetic system, use of defined medium with C. elegans should prove a powerful tool.

  6. Lovastatin Biosynthesis by Aspergillus terreus in a Chemically Defined Medium

    PubMed Central

    Hajjaj, Hassan; Niederberger, Peter; Duboc, Philippe

    2001-01-01

    Lovastatin is a secondary metabolite produced by Aspergillus terreus. A chemically defined medium was developed in order to investigate the influence of carbon and nitrogen sources on lovastatin biosynthesis. Among several organic and inorganic defined nitrogen sources metabolized by A. terreus, glutamate and histidine gave the highest lovastatin biosynthesis level. For cultures on glucose and glutamate, lovastatin synthesis initiated when glucose consumption levelled off. When A. terreus was grown on lactose, lovastatin production initiated in the presence of residual lactose. Experimental results showed that carbon source starvation is required in addition to relief of glucose repression, while glutamate did not repress biosynthesis. A threefold-higher specific productivity was found with the defined medium on glucose and glutamate, compared to growth on complex medium with glucose, peptonized milk, and yeast extract. PMID:11375168

  7. Continuous Culture of Ruminal Microorganisms in Chemically Defined Medium1

    PubMed Central

    Quinn, Loyd Y.; Burroughs, Wise; Christiansen, William C.

    1962-01-01

    Ruminal ciliates have been grown in continuous culture in chemically defined media and in the absence of viable bacteria. Oligotrichic ruminal ciliates seem to require insoluble carbohydrates for growth; the holotrichic ciliates require soluble carbohydrates, but at low concentrations. Both groups of ciliates utilize amino acids as their principal nitrogen source when these are supplied in micromolar concentrations; at millimolar concentrations, amino acids are toxic, possibly from excessive ammonia formation arising from ciliate deaminase activity. Holotrichic ruminal ciliates are destroyed by overdeposition of amylopectin when glucose is present above 0.1% concentration in the medium. Ecological requirements of ruminal ciliates are also described. Images FIG. 1 FIG. 2 PMID:13972780

  8. Defining the Product Chemical Space of Monoterpenoid Synthases

    PubMed Central

    Tian, Boxue; Poulter, C. Dale; Jacobson, Matthew P.

    2016-01-01

    Terpenoid synthases create diverse carbon skeletons by catalyzing complex carbocation rearrangements, making them particularly challenging for enzyme function prediction. To begin to address this challenge, we have developed a computational approach for the systematic enumeration of terpenoid carbocations. Application of this approach allows us to systematically define a nearly complete chemical space for the potential carbon skeletons of products from monoterpenoid synthases. Specifically, 18758 carbocations were generated, which we cluster into 74 cyclic skeletons. Five of the 74 skeletons are found in known natural products; some of the others are plausible for new functions, either in nature or engineered. This work systematizes the description of function for this class of enzymes, and provides a basis for predicting functions of uncharacterized enzymes. To our knowledge, this is the first computational study to explore the complete product chemical space of this important class of enzymes. PMID:27517297

  9. Defining the Product Chemical Space of Monoterpenoid Synthases.

    PubMed

    Tian, Boxue; Poulter, C Dale; Jacobson, Matthew P

    2016-08-01

    Terpenoid synthases create diverse carbon skeletons by catalyzing complex carbocation rearrangements, making them particularly challenging for enzyme function prediction. To begin to address this challenge, we have developed a computational approach for the systematic enumeration of terpenoid carbocations. Application of this approach allows us to systematically define a nearly complete chemical space for the potential carbon skeletons of products from monoterpenoid synthases. Specifically, 18758 carbocations were generated, which we cluster into 74 cyclic skeletons. Five of the 74 skeletons are found in known natural products; some of the others are plausible for new functions, either in nature or engineered. This work systematizes the description of function for this class of enzymes, and provides a basis for predicting functions of uncharacterized enzymes. To our knowledge, this is the first computational study to explore the complete product chemical space of this important class of enzymes. PMID:27517297

  10. Nutritional models for space travel from chemically defined diets

    NASA Technical Reports Server (NTRS)

    Dufour, P. A.

    1984-01-01

    Human nutritional requirements are summarized, including recommended daily intake and maximum safe chronic intake of nutrients. The biomedical literature on various types of chemically defined diets (CDD's), which are liquid, formulated diets for enteral and total parenteral nutrition, is reviewed. The chemical forms of the nutrients in CDD's are detailed, and the compositions and sources of representative commercial CDD's are tabulated. Reported effects of CDD's in medical patients, healthy volunteers, and laboratory animals are discussed. The effects include gastrointestinal side effects, metabolic imbalances, nutrient deficiencies and excesses, and psychological problems. Dietary factors contributing to the side effects are examined. Certain human nutrient requirements have been specified more precisely as a result of long-term use of CDD's, and related studies are included. CDD's are the most restricted yet nutritionally complete diets available.

  11. Differentiation of purified astrocytes in a chemically defined medium

    SciTech Connect

    Morrison, R.S.; de Vellis, J.

    1981-01-01

    Homogeneous cultures of astrocytes and oligodendrocytes provide an excellent model system for studying the regulation of glial structure and function. Recently, a chemically defined (CD) medium was developed for purified cultures of astrocytes, thus eliminating the requirement for serum and providing a controlled system for the study of astroglial properties. Due to the widespread use of astrocyte cultures and the potential benefits to be gained from using a defined medium, astrocyte cultures raised in CD medium were analyzed for purity as well as morphological and biochemical properties. Purity was assessed using immunocytochemical staining for glial fibrillary acidic protein (GFAP) and fibronectin. Astrocytes raised in CD medium are 95% pure using the expression of GFAP as a criterion. Fewer than 1% of the cells in CD medium stained positive for fibronectin eliminating the possibility that CD medium is selective for meningeal or endothelial cells. Astrocytes raised in CD medium exhibit a striking degree of morphological differentiation as seen in scanning electron micrographs. They also exhibit a high degree of biochemical differentiation illustrated by increases in the specific activity of S-100 protein and the induction of glutamine synthetase by glucocorticoids. A defined medium that supports the proliferation of rat astrocytes and enhances numerous morphological and biochemical properties should greatly facilitate the study of factors controlling glial proliferation and differentiation.

  12. A critical synopsis: Continuous growth of proximal tubular kidney epithelial cells in hormone-supplemented serum-free medium

    NASA Technical Reports Server (NTRS)

    Chuman, L. M.; FINE; COHEN; Saier, M. H.

    1985-01-01

    The kidney forms urine and reabsorbs electrolytes and water. Kidney cell lines and hormone supplemented serum free medium were used for growth. The hormones were insulin, transferrin, vasopressin, cholesterol, prostaglandins, hydrocortisone, and triidothyronine. Epithelial cell lines are polar and form hemicysts. The Madin-Darby canine kidney(MDCK) cell line used is distal tubulelike. LLC-PK sub 1 cells are derived from pig kidneys and have the properties of different kidney segments. The LLC-PK sub 1 cells with proximal tubule properties were maintained in hormone-supplemented serum free medium. Seven factors (the aforementioned homrones and selenium) were needed for growth. Hormone-defined medium supported LLC-PK sub 1 cell growth, allowed transport (as seen by hemicyst formation), and influenced cell morphology. Vasopressin (used for growth and morphology) could be partially replaced by isobutylmethylxanthine or dibutyryl cAMP. The defined medium was used to isolate rabbit proximal tubule kidney epithelial cells free of fibroblasts.

  13. A chemically defined medium for the growth of Cowdria ruminantium.

    PubMed

    Zweygarth, E; Josemans, A I

    2001-03-01

    Chemically defined media, termed SFMC-23 and SFMC-36, were devised for the in vitro culture of Cowdria ruminantium, the causative agent of heartwater in domestic ruminants. Both media were based on Dulbecco's modified Eagle's medium nutrient mixture Ham F-12 (DME/F-12) containing various supplements. Medium SFMC-23 and SFMC-36 supported the long-term growth of the Welgevonden stock of C. ruminantium for a total of 55 and 28 passages, respectively, with regular passage intervals of 3 days. Using SFMC-23, split ratios varied from 5-10, depending on which host cell line was used. Other stocks of C. ruminantium (Sankat, Blaauwkrantz, Senegal) were successfully propagated for a test period of ten passages. PMID:11403428

  14. Chemically defined media for auxotyping of Campylobacter jejuni.

    PubMed

    Dickgiesser, N; Czylwik, D

    1985-08-01

    A set of chemically defined media has been developed for the cultivation of Campylobacter jejuni strains of human origin. A minimal medium, a complete medium and 5 different nutrient-deficient media (NDM1-NDM5) are described. Some of the strains investigated required L-methionine(lacking in NDM1), L-cystine and L-cysteine (NDM2), K2HPO4 (NDM 3), KH2PO4 (NDM4) and NAD, thiamine and calcium pantothenate (NDM5). 57.7% of the strains investigated required L-methionine. The strains grew at pH 6.6-7.7. The media described are not suitable for C. intestinalis. PMID:4060921

  15. Growth of hybridoma cells in serum-free medium: ethanolamine is an essential component.

    PubMed Central

    Murakami, H; Masui, H; Sato, G H; Sueoka, N; Chow, T P; Kano-Sueoka, T

    1982-01-01

    A serum-free medium supplemented with a few growth factors was devised to grow lymphocyte hybridomas. The medium was developed with the hybridoma line MPC11-BL, a fusion product between a mouse plasmacytoma cell line (MPC11TG70na3) and mouse (BALB/c) spleen cells. In the process of developing the medium, ethanolamine was found to be an essential growth factor for the hybridoma. Phosphoethanolamine at 10-fold higher concentration could substitute for ethanolamine. Long-term cultivation of the cells was achieved in the defined medium supplemented with insulin, transferrin, ethanolamine, and selenium. The defined medium supported the growth of various other mouse hybridoma cell lines, mostly at a rate comparable to that observed in a serum-containing medium. After one-step ammonium sulfate precipitation of the spent medium, more than 95% of the protein recovered was immunoglobulin as shown by NaDodSO4/polyacrylamide gel electrophoresis. Images PMID:7041116

  16. Growth, differentiation capacity, and function of mesenchymal stem cells expanded in serum-free medium developed via combinatorial screening.

    PubMed

    Crapnell, Kirsten; Blaesius, Rainer; Hastings, Abel; Lennon, Donald P; Caplan, Arnold I; Bruder, Scott P

    2013-06-10

    The presence of serum in cell culture medium presents an obstacle to safe and efficient production of hMSCs for therapeutic purposes. Availability of defined medium will be crucial to elucidating the mechanism of action of hMSCs in many indications as well as a prerequisite to consistently produce cells with predictable performance characteristics. Using a bioinformatics driven approach, which we call the BD Discovery Platform, we have developed a novel serum-free medium that supports highly efficient growth while maintaining the surface markers and functional characteristics defining hMSCs. In a comparison with serum-containing and other commercially available serum-free formulations, all conditions led to expansion of cells that meet the minimal criteria for hMSCs as set by the International Society for Cellular Therapy (ISCT). However, differences in growth characteristics and gene expression patterns suggest that expansion in serum-free growth conditions can provide greater yields in a shorter time. The mRNA expression profile observed in cells grown without serum suggests upregulation of several genes implicated in hMSC function as well as downregulation of the proinflammatory cytokine IL6. PMID:23597555

  17. Chemically defined media modifications to lower tryptophan oxidation of biopharmaceuticals.

    PubMed

    Hazeltine, Laurie B; Knueven, Kristine M; Zhang, Yan; Lian, Zhirui; Olson, Donald J; Ouyang, Anli

    2016-01-01

    Oxidation of biopharmaceuticals is a major product quality issue with potential impacts on activity and immunogenicity. At Eli Lilly and Company, high tryptophan oxidation was observed for two biopharmaceuticals in development produced in Chinese hamster ovary cells. A switch from historical hydrolysate-containing media to chemically defined media with a reformulated basal powder was thought to be responsible, so mitigation efforts focused on media modification. Shake flask studies identified that increasing tryptophan, copper, and manganese and decreasing cysteine concentrations were individual approaches to lower tryptophan oxidation. When amino acid and metal changes were combined, the modified formulation had a synergistic impact that led to substantially less tryptophan oxidation for both biopharmaceuticals. Similar results were achieved in shake flasks and benchtop bioreactors, demonstrating the potential to implement these modifications at manufacturing scale. The modified formulation did not negatively impact cell growth and viability, product titer, purity, charge variants, or glycan profile. A potential mechanism of action is presented for each amino acid or metal factor based on its role in oxidation chemistry. This work served not only to mitigate the tryptophan oxidation issue in two Lilly biopharmaceuticals in development, but also to increase our knowledge and appreciation for the impact of media components on product quality. PMID:26560440

  18. A Chemically Defined Medium for Rabbit Embryo Cryopreservation

    PubMed Central

    Bruyère, Pierre; Baudot, Anne; Joly, Thierry; Commin, Loris; Pillet, Elodie; Guérin, Pierre; Louis, Gérard; Josson-Schramme, Anne; Buff, Samuel

    2013-01-01

    .066). We conclude that CRYO3 can be used as a chemically defined substitute for animal-based products in rabbit embryo cryopreservation solutions. PMID:23977074

  19. Is accuracy of serum free light chain measurement achievable?

    PubMed

    Jacobs, Joannes F M; Tate, Jillian R; Merlini, Giampaolo

    2016-06-01

    The serum free light chain (FLC) assay has proven to be an important complementary test in the management of patients with monoclonal gammopathies. The serum FLC assay has value for patients with plasma cell disorders in the context of screening and diagnosis, prognostic stratification, and quantitative monitoring. Nonetheless, serum FLC measurements have analytical limitations which give rise to differences in FLC reporting depending on which FLC assay and analytical platform is used. As the FLC measurements are incorporated in the International Myeloma Working Group guidelines for the evaluation and management of plasma cell dyscrasias, this may directly affect clinical decisions. As new certified methods for serum FLC assays emerge, the need to harmonise patient FLC results becomes increasingly important. In this opinion paper we provide an overview of the current lack of accuracy and harmonisation in serum FLC measurements. The clinical consequence of non-harmonized FLC measurements is that an individual patient may or may not meet certain diagnostic, prognostic, or response criteria, depending on which FLC assay and platform is used. We further discuss whether standardisation of serum FLC measurements is feasible and provide an overview of the steps needed to be taken towards harmonisation of FLC measurements. PMID:26641970

  20. Production of canine adenovirus type 2 in serum-free suspension cultures of MDCK cells.

    PubMed

    Castro, R; Fernandes, P; Laske, T; Sousa, M F Q; Genzel, Y; Scharfenberg, K; Alves, P M; Coroadinha, A S

    2015-09-01

    The potential of adherent Madin Darby Canine Kidney (MDCK) cells for the production of influenza viruses and canine adenovirus type 2 (CAV-2) for vaccines or gene therapy approaches has been shown. Recently, a new MDCK cell line (MDCK.SUS2) that was able to grow in suspension in a fully defined system was established. In this work, we investigated whether the new MDCK.SUS2 suspension cell line is suitable for the amplification of CAV-2 under serum-free culture conditions. Cell growth performance and CAV-2 production were evaluated in three serum-free media: AEM, SMIF8, and EXCELL MDCK. CAV-2 production in shake flasks was maximal when AEM medium was used, resulting in an amplification ratio of infectious particles (IP) of 142 IP out/IP in and volumetric and cell-specific productivities of 2.1 × 10(8) IP/mL and 482 IP/cell, respectively. CAV-2 production was further improved when cells were cultivated in a 0.5-L stirred tank bioreactor. To monitor infection and virus production, cells were analyzed by flow cytometry. A correlation between the side scatter measurement and CAV-2 productivity was found, which represents a key feature to determine the best harvesting time during process development of gene therapy vectors that do not express reporter genes. This work demonstrates that MDCK.SUS2 is a suitable cell substrate for CAV-2 production, constituting a step forward in developing a production process transferable to industrial scales. This could allow for the production of high CAV-2 titers either for vaccination or for gene therapy purposes. PMID:25994255

  1. Establishment of human ovarian serous carcinomas cell lines in serum free media.

    PubMed

    Pan, Zhuangyu; Hooley, Jeffrey; Smith, Douglas H; Young, Peter; Roberts, Penelope E; Mather, Jennie P

    2012-03-01

    Ovarian cancers are the fifth leading cause of cancer death among US woman. The majority of ovarian cancers belong to a category of serous adenocarcinomas. This type of cancer is often diagnosed at a late stage of the disease. Surgical debulking, followed by chemotherapy is the current treatment. Half of all patients will die within 5 years of diagnosis of the disease. Poor survival may be due to disease progression as a consequence of development of drug resistance, cancer cell heterogeneity within the tumor, or the persistence of cancer stem cells. Cancer stem cells (CSC) are defined as a minority cell type in the tumor, which retains the capacity, through asymmetric division, for self-renewal as well as differentiation into multiple cell types. Through this process, CSC can regenerate the entire tumor phenotype and subsequent metastases. Initial in vitro work in the area of solid tumor CSC biology has focused on the isolation and propagation of cells with CSC-like properties from breast and colon tumors. Breast and colon cell lines with CSC-like properties have been isolated and maintained in vitro for extended periods of time. The in vitro maintenance of these CSC requires growth in hormone-supplemented serum-free media and the use of matrix or growth as tumor spheres (Roberts, Ricci-Vitiani et al., Cammareri et al.). Based on the pioneering work generating breast and colon CSC, our lab has begun to develop methods for the establishment cell lines with CSC-like properties from additional solid tumors. In this article, we describe methods, using defined medium, which allow for the successful establishment of continuous cell cultures from a minority cell type within serous ovarian cancers. The cell lines established using these methods grow in serum-free hormone-supplemented medium either as a monolayer on a matrix, or as tumor spheres in suspension. These cells express markers previously reported for tumor stem cells, including CD44 and CD133, and form tumors

  2. Human dental pulp stem cells cultured in serum-free supplemented medium

    PubMed Central

    Bonnamain, Virginie; Thinard, Reynald; Sergent-Tanguy, Solène; Huet, Pascal; Bienvenu, Géraldine; Naveilhan, Philippe; Farges, Jean-Christophe; Alliot-Licht, Brigitte

    2013-01-01

    Growing evidence show that human dental pulp stem cells (DPSCs) could provide a source of adult stem cells for the treatment of neurodegenerative pathologies. In this study, DPSCs were expanded and cultured with a protocol generally used for the culture of neural stem/progenitor cells. Methodology: DPSC cultures were established from third molars. The pulp tissue was enzymatically digested and cultured in serum-supplemented basal medium for 12 h. Adherent (ADH) and non-adherent (non-ADH) cell populations were separated according to their differential adhesion to plastic and then cultured in serum-free defined N2 medium with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Both ADH and non-ADH populations were analyzed by FACS and/or PCR. Results: FACS analysis of ADH-DPSCs revealed the expression of the mesenchymal cell marker CD90, the neuronal marker CD56, the transferrin receptor CD71, and the chemokine receptor CXCR3, whereas hematopoietic stem cells markers CD45, CD133, and CD34 were not expressed. ADH-DPSCs expressed transcripts coding for the Nestin gene, whereas expression levels of genes coding for the neuronal markers β-III tubulin and NF-M, and the oligodendrocyte marker PLP-1 were donor dependent. ADH-DPSCs did not express the transcripts for GFAP, an astrocyte marker. Cells of the non-ADH population that grew as spheroids expressed Nestin, β-III tubulin, NF-M and PLP-1 transcripts. DPSCs that migrated out of the spheroids exhibited an odontoblast-like morphology and expressed a higher level of DSPP and osteocalcin transcripts than ADH-DPSCs. Conclusion: Collectively, these data indicate that human DPSCs can be expanded and cultured in serum-free supplemented medium with EGF and bFGF. ADH-DPSCs and non-ADH populations contained neuronal and/or oligodendrocyte progenitors at different stages of commitment and, interestingly, cells from spheroid structures seem to be more engaged into the odontoblastic lineage than the ADH

  3. Proliferation and responsiveness to estrogen of human endometrial cancer cells under serum-free culture conditions.

    PubMed

    Holinka, C F; Anzai, Y; Hata, H; Kimmel, N; Kuramoto, H; Gurpide, E

    1989-06-15

    Studies of hormonal growth regulation in cultured human endometrial cancer cells are limited by the requirement of exogenous growth factors, usually supplied by addition of serum. The present report provides evidence that estradiol can stimulate proliferation of endometrial cancer cells of the Ishikawa line in the absence of serum or added growth factors. Mitogenic effects of estrogen were demonstrated in two different experimental systems, in cells attached to the substratum of mammalian tissue culture dishes, and in cells forming colonies in soft agar under anchorage-independent conditions. Addition of estradiol to a mixture of serum-free, phenol red-free Dulbecco's minimal essential medium and Ham's F-12 medium, supplemented with L-glutamine and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [basal medium: (BM)] significantly increased the proliferation of cells attached to culture dishes. Dose-response experiments revealed maximal estradiol stimulation at 10 nM; significant responses were also observed at 1 nM and at 100 nM concentrations. The mitogenic effect of 10 nM estradiol was comparable to that of 1% charcoal-treated fetal bovine serum and the two effects were additive. The presence of estradiol in serum-free BM resulted in a shortening of the doubling time of exponentially proliferating cells from 38 to 29 h. From the labeling index, measured after exposure to a pulse of [3H]thymidine, and from the mitotic index, both determined in exponentially proliferating cells, the lengths of the S and M phases were calculated to be 11 and 1 h, respectively. From these data it was estimated that estradiol shortened the G1 phase by approximately 40%, from 22 to 13 h. Estradiol doubled the colony formation efficiency of cells plated in BM containing 0.3% agar in the absence of serum as well as in the presence of 1% charcoal-treated fetal bovine serum. The stimulation of colony formation by estradiol was influenced by medium components, since no effects were

  4. Culture of fetal alveolar epithelial type II cells in serum-free medium.

    PubMed

    Fraslon, C; Rolland, G; Bourbon, J R; Rieutort, M; Valenza, C

    1991-11-01

    A serum-free culture medium (defined medium = DM) was elaborated by adding to Eagle's minimum essential medium (MEM), non-essential amino acids, transferrin, putrescine, tripeptide glycyl-histidyl-lysine, somatostatin, sodium selenite, ethanolamine, phosphoethanolamine, sodium pyruvate, and metal trace elements. This medium was tested for its ability to support sustained surfactant biosynthesis in fetal alveolar epithelial type II cells. For up to 8 days, ultrastructure was maintained with persistence of lamellar inclusion bodies. Thymidine incorporation into DNA was enhanced about 50% in DM as compared with MEM, whereas it was enhanced 300% in 10% fetal bovine serum. With DM, the incorporation of tritiated choline into phosphatidylcholine (PC) of isolated surfactant material was about twice that with MEM. Deletion experiments evidenced the prominent role of pyruvate, transferrin, and selenium in the stimulation of surfactant PC biosynthesis. The addition of biotin to DM enhanced surfactant PC biosynthesis slightly and nonsurfactant PC biosynthesis markedly. The presence of nucleosides seemed unfavorable to the synthesis of surfactant PC. Type II cells responded to the addition of epidermal growth factor and insulinlike growth factor-I both by increased thymidine incorporation into DNA and choline incorporation into PC. It is concluded that DM represents a useful tool for cultivating type II cells without loss of their specialized properties and for studying the regulation of cell proliferation and surfactant biosynthesis in a controlled environment. PMID:1748624

  5. Serum-free transfection of CHO-cells with tailor-made unilamellar vesicles

    PubMed Central

    Sevcsik, Eva; Vorauer-Uhl, Karola; Lohner, Karl; Katinger, Hermann; Kunert, Renate

    2007-01-01

    At present, a number of transfection techniques are available to introduce foreign DNA into cells, but still minimal intrusion or interference with normal cell physiology, low toxicity, reproducibility, cost efficiency and successful creation of stable transfectants are highly desirable properties for improved transfection techniques. For all previous transfection experiments done in our labs, using serum-free cultivated host cell lines, an efficiency value of ∼0.1% for selection of stable cell lines has not been exceeded, consequently we developed and improved a transfection system based on defined liposomes, so-called large unilamellar vesicles, consisting of different lipid compositions to facilitate clone selection and increase the probability for creation of recombinant high-production clones. DNA and DOTAP/DOPE or CHEMS/DOPE interact by electrostatic means forming so-called lipoplexes (Even-Chen and Barenholz 2000) and the lipofection efficiency of those lipoplexes has been determined via confocal microscopy. In addition, the expression of the EGFP was determined by FACS to investigate transient as well as stable transfection and the transfection efficiency of a selection of different commercially available transfection reagents and kits has been compared to our tailor-made liposomes. PMID:19003008

  6. In vitro maintenance of spermatogenesis in Xenopus laevis testis explants cultured in serum-free media

    SciTech Connect

    Risley, M.S.; Miller, A.; Bumcrot, D.A.

    1987-05-01

    Spermatogenesis has been maintained for extended periods in Xenopus laevis testis explants cultured in serum-free media supplemented with bovine serum albumin, insulin, transferrin, follicle-stimulating hormone, dihydrotestosterone, testosterone, retinol, ascorbate, and tocopherol. The organization of the testis fragments was maintained for 28 days, and all stages of development were present throughout the culture period. /sup 3/H-Thymidine-labeled secondary (Type B) spermatogonia developed in 28 days into spermatids at the acrosomal vesicle stage whereas labeled zygotene spermatocytes became mature spermatids in 28 days. Spermatogonial proliferation also continued in vitro for 28 days. Germ cell differentiation was not dependent upon exogenous testosterone, ascorbate, or tocopherol since /sup 3/H-labeled spermatogonia became mature spermatids in testes cultured 35 days in media lacking these supplements. Autoradiography demonstrated that 55% of the luminal sperm present in explants cultured 10 days had differentiated in vitro. Sperm from testes cultured 10-35 days were similar to sperm from freshly dissected testes with regard to motility and fecundity, and eggs fertilized with sperm from explant cultures developed normally into swimming tadpoles. The results demonstrate the feasibility of maintaining vertebrate spermatogenesis in culture and suggest that in vitro analysis of Xenopus spermatogenesis using defined media may provide important insights into the evolution of regulatory mechanisms in spermatogenesis.

  7. Serum-Free Suspension Culture of MDCK Cells for Production of Influenza H1N1 Vaccines

    PubMed Central

    Huang, Ding; Peng, Wen-Juan; Ye, Qian; Liu, Xu-Ping; Zhao, Liang; Fan, Li; Xia-Hou, Kang; Jia, Han-Jing; Luo, Jian; Zhou, Lin-Ting; Li, Bei-Bei; Wang, Shi-Lei; Xu, Wen-Ting; Chen, Ze; Tan, Wen-Song

    2015-01-01

    Development of serum-free suspension cell culture processes is very important for influenza vaccine production. Previously, we developed a MDCK suspension cell line in a serum-free medium. In the present study, the growth kinetics of suspension MDCK cells and influenza virus production in the serum-free medium were investigated, in comparison with those of adherent MDCK cells in both serum-containing and serum-free medium. It was found that the serum-free medium supported the stable subculture and growth of both adherent and suspension cells. In batch culture, for both cell lines, the growth kinetics in the serum-free medium was comparable with those in the serum-containing medium and a commercialized serum-free medium. In the serum-free medium, peak viable cell density (VCD), haemagglutinin (HA) and median tissue culture infective dose (TCID50) titers of the two cell lines reached 4.51×106 cells/mL, 2.94Log10(HAU/50 μL) and 8.49Log10(virions/mL), and 5.97×106 cells/mL, 3.88Log10(HAU/50 μL), and 10.34Log10(virions/mL), respectively. While virus yield of adherent cells in the serum-free medium was similar to that in the serum-containing medium, suspension culture in the serum-free medium showed a higher virus yield than adherent cells in the serum-containing medium and suspension cells in the commercialized serum-free medium. However, the percentage of infectious viruses was lower for suspension culture in the serum-free medium. These results demonstrate the great potential of this suspension MDCK cell line in serum-free medium for influenza vaccine production and further improvements are warranted. PMID:26540170

  8. Culture of porcine hepatocytes or bile duct epithelial cells by inductive serum-free media

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A serum-free, feeder-cell-dependent, selective culture system for the long-term culture of porcine hepatocytes or cholangiocytes was developed. Liver cells were isolated from 1 wk old pigs or young adult pigs (25 and 63 kg live weight) and were placed in primary culture on feeder-cell layers of mit...

  9. EMBRYONIC PALATAL RESPONSES TO TERATOGENS IN SERUM-FREE ORGAN CULTURE

    EPA Science Inventory

    This study examines development of rat, mouse and human embryonic palates in submerged, serum-free organ culture. he concentration-response profiles for retinoic acid (RA), triamcinolone (TRI), hydrocortisone (HC), dexamethasone (DEX), and 2,3,7,11- tetrachlorodibenzo-p-dioxin (T...

  10. A Pilot Study To Define Chemical Coping in Cancer Patients Using the Delphi Method

    PubMed Central

    Hui, David; Bruera, Eduardo

    2015-01-01

    Abstract Background: “Chemical coping” is a commonly used term in the pain and palliative care literature, but is heterogeneously defined. We conducted a Delphi survey among palliative care and pain specialists internationally to identify a consensus definition for “chemical coping with opioids” and warning signs for chemical coping. Methods: This Delphi survey consisted of two rounds on the following: (1) concepts and definition related to chemical coping, (2) warning signs for chemical coping, and (3) demographics. Consensus in this study was defined as agreement by a minimum of 70% of the experts. Results: Participating in the first round were 14/19 (74%) physicians; 12/14 (86%) participated in the second round. The international experts reached the following consensus definition for chemical coping with opioids (92% agreement): “The use of opioids to cope with emotional distress, characterized by inappropriate and/or excessive opioid use.” They also identified depression (consensus 93%); psychiatric disease (86%); a history of substance abuse (86%); a positive score for the Cut-down, Annoyed, Guilty, and Eye-opener (CAGE) alcoholism screening test (79%); a history of alcoholism (79%); and a history of smoking (71%) as important warning signs for chemical coping. Conclusion: Our expert panel reached a consensus definition for chemical coping and related warning signs, which may help clinicians and researchers to identify patients at risk of opioid misuse. PMID:25922879

  11. Interactions between Skeletal Muscle Myoblasts and their Extracellular Matrix Revealed by a Serum Free Culture System

    PubMed Central

    Chaturvedi, Vishal; Dye, Danielle E.; Kinnear, Beverley F.; van Kuppevelt, Toin H.; Grounds, Miranda D.; Coombe, Deirdre R.

    2015-01-01

    Decellularisation of skeletal muscle provides a system to study the interactions of myoblasts with muscle extracellular matrix (ECM). This study describes the efficient decellularisation of quadriceps muscle with the retention of matrix components and the use of this matrix for myoblast proliferation and differentiation under serum free culture conditions. Three decellularisation approaches were examined; the most effective was phospholipase A2 treatment, which removed cellular material while maximizing the retention of ECM components. Decellularised muscle matrices were then solubilized and used as substrates for C2C12 mouse myoblast serum free cultures. The muscle matrix supported myoblast proliferation and differentiation equally as well as collagen and fibronectin. Immunofluorescence analyses revealed that myoblasts seeded on muscle matrix and fibronectin differentiated to form long, well-aligned myotubes, while myoblasts seeded on collagen were less organized. qPCR analyses showed a time dependent increase in genes involved in skeletal muscle differentiation and suggested that muscle-derived matrix may stimulate an increased rate of differentiation compared to collagen and fibronectin. Decellularized whole muscle three-dimensional scaffolds also supported cell adhesion and spreading, with myoblasts aligning along specific tracts of matrix proteins within the scaffolds. Thus, under serum free conditions, intact acellular muscle matrices provided cues to direct myoblast adhesion and migration. In addition, myoblasts were shown to rapidly secrete and organise their own matrix glycoproteins to create a localized ECM microenvironment. This serum free culture system has revealed that the correct muscle ECM facilitates more rapid cell organisation and differentiation than single matrix glycoprotein substrates. PMID:26030912

  12. The Effect of a Three-Month Physical Fitness Program on Serum Free Cholesterol.

    ERIC Educational Resources Information Center

    Montgomery, D. L.; Ismail, A. H.

    This study investigates the effect of a three-month physical fitness program on serum-free cholesterol concentration in four age and fitness adult groups. Twenty-four men were divided into the following groups: (a) young, low-fit; (b) old, low-fit; (c) young, high-fit; and (d) old, high-fit. All subjects had normal resting glucose and triglyceride…

  13. Conditions for growing Mycoplasma canadense and Mycoplasma verecundum in a serum-free medium.

    PubMed

    Muñoz, G; Sotomayor, P

    1990-07-01

    Mycoplasma canadense and Mycoplasma verecundum were cultured in a serum-free medium containing bovine serum albumin, cholesterol, oleic acid, and palmitic acid in order to avoid the addition of horse serum. Growth was detected by measurement of A640 and by colony formation. The level of growth attained in this medium was less than that obtained in the horse serum-supplemented media, but colonies retained their distinctive morphology. PMID:2202260

  14. Conditions for growing Mycoplasma canadense and Mycoplasma verecundum in a serum-free medium.

    PubMed Central

    Muñoz, G; Sotomayor, P

    1990-01-01

    Mycoplasma canadense and Mycoplasma verecundum were cultured in a serum-free medium containing bovine serum albumin, cholesterol, oleic acid, and palmitic acid in order to avoid the addition of horse serum. Growth was detected by measurement of A640 and by colony formation. The level of growth attained in this medium was less than that obtained in the horse serum-supplemented media, but colonies retained their distinctive morphology. Images PMID:2202260

  15. Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture.

    PubMed

    Schulz, Thomas C; Noggle, Scott A; Palmarini, Gail M; Weiler, Deb A; Lyons, Ian G; Pensa, Kate A; Meedeniya, Adrian C B; Davidson, Bruce P; Lambert, Nevin A; Condie, Brian G

    2004-01-01

    The use of human embryonic stem cells (hESCs) as a source of dopaminergic neurons for Parkinson's disease cell therapy will require the development of simple and reliable cell differentiation protocols. The use of cell cocultures, added extracellular signaling factors, or transgenic approaches to drive hESC differentiation could lead to additional regulatory as well as cell production delays for these therapies. Because the neuronal cell lineage seems to require limited or no signaling for its formation, we tested the ability of hESCs to differentiate to form dopamine-producing neurons in a simple serum-free suspension culture system. BG01 and BG03 hESCs were differentiated as suspension aggregates, and neural progenitors and neurons were detectable after 2-4 weeks. Plated neurons responded appropriately to electrophysiological cues. This differentiation was inhibited by early exposure to bone morphogenic protein (BMP)-4, but a pulse of BMP-4 from days 5 to 9 caused induction of peripheral neuronal differentiation. Real-time polymerase chain reaction and whole-mount immunocytochemistry demonstrated the expression of multiple markers of the midbrain dopaminergic phenotype in serum-free differentiations. Neurons expressing tyrosine hydroxylase (TH) were killed by 6-hydroxydopamine (6-OHDA), a neurotoxic catecholamine. Upon plating, these cells released dopamine and other catecholamines in response to K+ depolarization. Surviving TH+ neurons, derived from the cells differentiated in serum-free suspension cultures, were detected 8 weeks after transplantation into 6-OHDA-lesioned rat brains. This work suggests that hESCs can differentiate in simple serum-free suspension cultures to produce the large number of cells required for transplantation studies. PMID:15579641

  16. Systematic development and optimization of chemically defined medium supporting high cell density growth of Bacillus coagulans.

    PubMed

    Chen, Yu; Dong, Fengqing; Wang, Yonghong

    2016-09-01

    With determined components and experimental reducibility, the chemically defined medium (CDM) and the minimal chemically defined medium (MCDM) are used in many metabolism and regulation studies. This research aimed to develop the chemically defined medium supporting high cell density growth of Bacillus coagulans, which is a promising producer of lactic acid and other bio-chemicals. In this study, a systematic methodology combining the experimental technique with flux balance analysis (FBA) was proposed to design and simplify a CDM. The single omission technique and single addition technique were employed to determine the essential and stimulatory compounds, before the optimization of their concentrations by the statistical method. In addition, to improve the growth rationally, in silico omission and addition were performed by FBA based on the construction of a medium-size metabolic model of B. coagulans 36D1. Thus, CDMs were developed to obtain considerable biomass production of at least five B. coagulans strains, in which two model strains B. coagulans 36D1 and ATCC 7050 were involved. PMID:27262567

  17. Cofilin Phosphorylation Decreased by Serum-free Starvation with Low Glucose in the L6 Myoblasts.

    PubMed

    Kim, Mee-Young; Kim, Ju-Hyun; Lee, Jeong-Uk; Lee, Lim-Kyu; Yang, Seung-Min; Park, Byoung-Sun; Jeon, Hye-Joo; Lee, Won-Deok; Noh, Ji-Woong; Kwak, Taek-Yong; Jang, Sung-Ho; Lee, Tae-Hyun; Kim, Ju-Young; Kim, Tae-Whan; Kim, Bokyung; Kim, Junghwan

    2014-10-01

    [Purpose] Many studies have been using cell culture models of muscle cells with exogenous cytokines or glucocorticoids to mimic atrophy in in vivo and in vitro tests. However, the changes in the phosphorylation of atrophy-related cofilin are still poorly understood in starved skeletal muscle cells. In this study, we first examined whether or not phosphorylation of cofilin is altered in L6 myoblasts after 3, 6, 12, 24, 48, and 72 hours of serum-free starvation with low glucose. [Methods] We used Western blotting to exam protein expression and phosphorylation in atrophied L6 myoblasts. [Results] L6 cell sizes and numbers were diminished as a result of serum-free starvation in a time-dependent manner. Serum-free starvation for 3, 6, 12, 24, 48, and 72 hours significantly decreased the phosphorylation of cofilin, respectively. [Conclusion] These results suggest that starvation-induced atrophy may be in part related to changes in the phosphorylation of cofilin in L6 myoblasts. PMID:25364107

  18. Mouse retina explants after long-term culture in serum free medium.

    PubMed

    Caffé, A R; Ahuja, P; Holmqvist, B; Azadi, S; Forsell, J; Holmqvist, I; Söderpalm, A K; van Veen, T

    2001-11-01

    The neonatal mouse retina remains viable as an explant in serum-supplemented growth media for more than 4 weeks. Interpretation of drug effects on this tissue is compromised by the enigmatic composition of the serum. We sought to remove this ambiguity by culturing neonatal as well as late postnatal mouse retina in serum-free nutrient medium. In this study three important observations were made, (1) there is histotypic development of neonatal as well as preservation of late postnatal mouse retinal structure during long-term culture in serum-free medium, although the late postnatal tissue tends to show some loss of cells in the outer nuclear layer. (2) Protein expression in explant photoreceptor cells was similar to that in the litter-matched ones, except for green cone opsin and interphotoreceptor retinoid-binding protein, although mRNA of the latter is present at similar amounts as in age-matched in vivo controls. (3) Cells of the inner retina stained by antibodies to calcium-binding proteins display some novel sprouting of processes. The results show that the mouse retina can be cultured as an explant for more than 4 weeks in a serum-free medium. This represents an important step forward because, (1) the possibility of interference of drug effects by unknown serum factors has been eliminated; and (2) the spent culture medium can be analyzed to investigate biomolecules released by the retina in vitro. PMID:11719023

  19. [Metabolic characteristics and kinetic model of recombinant CHO cells in serum-free suspension batch culture].

    PubMed

    Liu, Xingmao; Liu, Hong; Ye, Lingling; Li, Shichong; Wu, Benchuan; Wang, Haitao; Xie, Jing; Chen, Zhaolie

    2010-01-01

    By using the cell density, cell viability, Pro-UK activity, specific consumption rate of glucose (q(glc)), specific production rate of lactate (q(lac)), yield of lactate to glucose (Y(lac/glc)) and as the evaluation indexes, the growth and metabolism characteristics of pro-urokinase (Pro-UK) expressing CHO cells in serum-free suspension batch culture were examined and compared to those in serum-containing suspension batch culture. We observed hardly differences in growth and metabolism characteristics between the CHO cell populations grown in serum-free suspension batch culture and serum-containing suspension batch culture. The optimal mathematical model parameters for the CHO cells grown in suspension batch culture were obtained by non-linear programming of data representing the growth, substrate consumption and product formation of the CHO cells during logarithmic growth phase using MATLAB software, and the kinetic model of the cell growth and metabolism in serum-free culture were established. PMID:20353097

  20. Iron dose-dependent differentiation and enucleation of human erythroblasts in serum-free medium.

    PubMed

    Byrnes, Colleen; Lee, Y Terry; Meier, Emily R; Rabel, Antoinette; Sacks, David B; Miller, Jeffery L

    2016-02-01

    Improvements in ex vivo generation of enucleated red blood cells are being sought for erythroid biology research, toward the ultimate goal of erythrocyte engineering for clinical use. Based upon the high levels of iron-saturated transferrin in plasma serum, it was hypothesized that terminal differentiation in serum-free media may be highly dependent on the concentration of iron. Here adult human CD34(+) cells were cultured in a serum-free medium containing dosed levels of iron-saturated transferrin (holo-Tf, 0.1-1.0 mg/ml). Iron in the culture medium was reduced, but not depleted, with erythroblast differentiation into haemoglobinized cells. At the lowest holo-Tf dose (0.1 mg/ml), terminal differentiation was significantly reduced and the majority of the cells underwent apoptotic death. Cell survival, differentiation and enucleation were enhanced as the holo-Tf dose increased. These data suggest that adequate holo-Tf dosing is critical for terminal differentiation and enucleation of human erythroblasts generated ex vivo in serum-free culture conditions. Published 2013. This article is a US Government work and is in the public domain in the USA. PMID:23606586

  1. Chemically Defined and Small Molecule-Based Generation of Human Cardiomyocytes

    PubMed Central

    Burridge, Paul W.; Matsa, Elena; Shukla, Praveen; Lin, Ziliang C.; Churko, Jared M.; Ebert, Antje D.; Lan, Feng; Diecke, Sebastian; Huber, Bruno; Mordwinkin, Nicholas M.; Plews, Jordan R.; Abilez, Oscar J.; Cui, Bianxiao; Gold, Joseph D.; Wu, Joseph C.

    2014-01-01

    Existing methodologies for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require the use of complex, undefined medium constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed a highly optimized cardiac differentiation strategy, employing a chemically defined medium consisting of just three components: the basal medium RPMI 1640, L-ascorbic acid 2-phosphate, and rice-derived recombinant human albumin. Along with small molecule-based differentiation induction, this protocol produced contractile sheets of up to 95% TNNT2+ cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell, and was effective in 11 hiPSC lines tested. This is the first fully chemically defined platform for cardiac specification of hiPSCs, and allows the elucidation of cardiomyocyte macromolecular and metabolic requirements whilst providing a minimally complex system for the study of maturation and subtype specification. PMID:24930130

  2. Optimization of chemically defined feed media for monoclonal antibody production in Chinese hamster ovary cells.

    PubMed

    Kishishita, Shohei; Katayama, Satoshi; Kodaira, Kunihiko; Takagi, Yoshinori; Matsuda, Hiroki; Okamoto, Hiroshi; Takuma, Shinya; Hirashima, Chikashi; Aoyagi, Hideki

    2015-07-01

    Chinese hamster ovary (CHO) cells are the most commonly used mammalian host for large-scale commercial production of therapeutic monoclonal antibodies (mAbs). Chemically defined media are currently used for CHO cell-based mAb production. An adequate supply of nutrients, especially specific amino acids, is required for cell growth and mAb production, and chemically defined fed-batch processes that support rapid cell growth, high cell density, and high levels of mAb production is still challenging. Many studies have highlighted the benefits of various media designs, supplements, and feed addition strategies in cell cultures. In the present study, we used a strategy involving optimization of a chemically defined feed medium to improve mAb production. Amino acids that were consumed in substantial amounts during a control culture were added to the feed medium as supplements. Supplementation was controlled to minimize accumulation of waste products such as lactate and ammonia. In addition, we evaluated supplementation with tyrosine, which has poor solubility, in the form of a dipeptide or tripeptide to improve its solubility. Supplementation with serine, cysteine, and tyrosine enhanced mAb production, cell viability, and metabolic profiles. A cysteine-tyrosine-serine tripeptide showed high solubility and produced beneficial effects similar to those observed with the free amino acids and with a dipeptide in improving mAb titers and metabolic profiles. PMID:25678240

  3. A thermoresponsive and chemically defined hydrogel for long-term culture of human embryonic stem cells

    PubMed Central

    Zhang, Rong; Mjoseng, Heidi K.; Hoeve, Marieke A.; Bauer, Nina G.; Pells, Steve; Besseling, Rut; Velugotla, Srinivas; Tourniaire, Guilhem; Kishen, Ria E. B.; Tsenkina, Yanina; Armit, Chris; Duffy, Cairnan R. E.; Helfen, Martina; Edenhofer, Frank; de Sousa, Paul A.; Bradley, Mark

    2013-01-01

    Cultures of human embryonic stem cell typically rely on protein matrices or feeder cells to support attachment and growth, while mechanical, enzymatic or chemical cell dissociation methods are used for cellular passaging. However, these methods are ill defined, thus introducing variability into the system, and may damage cells. They also exert selective pressures favouring cell aneuploidy and loss of differentiation potential. Here we report the identification of a family of chemically defined thermoresponsive synthetic hydrogels based on 2-(diethylamino)ethyl acrylate, which support long-term human embryonic stem cell growth and pluripotency over a period of 2–6 months. The hydrogels permitted gentle, reagent-free cell passaging by virtue of transient modulation of the ambient temperature from 37 to 15 °C for 30 min. These chemically defined alternatives to currently used, undefined biological substrates represent a flexible and scalable approach for improving the definition, efficacy and safety of human embryonic stem cell culture systems for research, industrial and clinical applications. PMID:23299885

  4. Photolithographic Patterning of C2C12 Myotubes using Vitronectin as Growth Substrate in Serum-Free Medium

    PubMed Central

    Molnar, Peter; Wang, Weishi; Natarajan, Anupama; Rumsey, John W.; Hickman, James J.

    2008-01-01

    The C2C12 cell line is frequently used as a model of skeletal muscle differentiation. In our serum-free defined culture system differentiation of C2C12 cells into myotubes required surface-bound signals such as substrate adsorbed vitronectin or laminin. Based on this substrate-requirement of myotube formation, we developed a photolithography-based method to pattern C2C12 myotubes, where myotubes formed exclusively on vitronectin surface patterns. We have determined that the optimal line width to form single myotubes is approximately 30 μm. In order to illustrate a possible application of this method, we patterned myotubes on the top of commercial substrate-embedded microelectrodes. In contrast to previous experiments where cell patterning was achieved by selective attachment of the cells to patterned surfaces in a medium that contained all the factors necessary for differentiation, this study illustrates that surface patterning of a signaling molecule, which is essential for skeletal muscle differentiation in a defined system, can result in the formation of aligned myotubes on the patterns. This technique is being developed for applications in cell biology, tissue engineering and robotics. PMID:17269697

  5. [Serum free thyroid hormones and response of TSH to TRH in nonthyroidal illnesses].

    PubMed

    Kokei, S; Inoue, T; Iino, S

    1986-11-20

    The change in the levels of free thyroid hormones and the pathophysiology of the hypothalamo-pituitary-thyroid axis of patients with nonthyroidal illness (NTI) have not been clearly elucidated so far. Therefore, it was thought of interest to investigate this problem by determining free thyroid hormones and TSH in serum and the response of TSH to TRH in these patients. The subjects employed in this study were 71 cases with hemodialysis, 40 cases with diabetes mellitus, 24 cases with liver cirrhosis, 12 cases with various cancers, 10 cases with anorexia nervosa and 110 normal subjects as controls. The serum total protein, albumin, free T4, free T3, TSH and other parameters of thyroid function were determined, and the TRH test was performed on about 10 patients of each group. Serum TSH was not only determined by a conventional assay system, but with a highly sensitive method, and the data were compared with one another. It was found that the serum free T3 levels were significantly low in all the groups investigated, but the serum free T4 levels were significantly low only in the groups with hemodialysis, decompensated liver cirrhosis, cancers and anorexia nervosa. No significant lowering of serum free T4 was observed in the patients with diabetes mellitus, acute hepatitis and compensated liver cirrhosis. However, serum TSH levels tended to be higher in all the groups studied, though they were not significant. The response of TSH to TRH was low or delayed in about 20-50% of patients with hemodialysis, diabetes mellitus, liver cirrhosis, cancers and anorexia nervosa. It was observed that the serum rT3 concentration was significantly high in the patients with diabetes mellitus and anorexia nervosa but significantly low in the patients on hemodialysis. In the rest of the groups, there were found many cases who showed high levels of serum rT3 although they were not statistically significant. These results indicate that low concentrations of serum free T3 observed in the

  6. Performance goals for immunoglobulins and serum free light chain measurements in plasma cell dyscrasias can be based on biological variation.

    PubMed

    Hansen, Charlotte Toftmann

    2016-06-01

    Measurements of immunoglobulins and serum free light chains (sFLC) are frequently used in patients with monoclonal plasma cell dyscrasia (PCD). For optimum patient care, well-defined performance standards or goals for the measured concentrations of immunoglobulins and sFLC are required. Generally, data based on biological variation is a good and reliable method for setting desirable performance standards; this also applies for the measurements of paraprotein and sFLC. The benefits of this approach are several. Among others, it is independent of the clinician, and it provides us with information about reference change value and index of individuality. Several studies on biological variation of both immunoglobulins and sFLC have been published, and mostly the studies are well performed. The studies normally show small within-subject biological variation resulting in strict analytical goals, which in most cases are difficult to meet. Nevertheless, we still need further information on biological variation of immunoglobulins and sFLC in patients with PCD and in the elderly, which are the main target populations for the two measurands. Furthermore, to improve data on biological variation of immunoglobulins and sFLC, studies accounting for number of individuals, samples, and replicates, as well as time length of the studies are needed. PMID:26824979

  7. Growth control of prostatic carcinoma cells in serum-free media: interrelationship of hormone response, cell density, and nutrient media.

    PubMed Central

    Kaighn, M E; Kirk, D; Szalay, M; Lechner, J F

    1981-01-01

    Two established prostatic carcinoma cell lines have been grown in long-term culture in a defined medium (PFMR-4) free of serum, hormones, or growth factors. Growth of both lines in serum-free medium was population dependent. This cell-density requirement could be replaced by mitomycin C-inactivated feeder cells, homologous conditioned medium, or fetal bovine serum, but not by hormones or growth factors. The cells responded to these factors only at high density. The nature of this hormonal response was dependent on the kind of basal nutrient medium used. Growth in PFMR-4 with added insulin was more rapid than that in DME/F12 medium with any combination of hormones or growth factors and was substantially greater than growth in DME/F12 medium with insulin alone. The results demonstrate that whereas these two prostatic carcinoma lines (PC-3 and DU 145) do not require hormones for survival or growth, they do respond to certain hormones under appropriate conditions. These conditions include both the type of basal nutrient medium used and the population density. PMID:7029542

  8. Overexpression and purification of HSV-2 glycoprotein D in suspension CHO cells with serum-free medium and immunogenicity analysis.

    PubMed

    Qi, Yong; Xu, Yueyue; Pan, Ying; Li, Suqin; Li, Bingjun; Pan, Mingjie; Zhang, Shumin; Li, Yuexi

    2016-05-01

    Glycoprotein D (gD2) is the most important candidate antigen for herpes simplex virus type 2 (HSV-2) vaccine development. Establishment of a stable eukaryotic cell line to overexpress gD2 and an efficient purification process to purify is essential for the development of subunit vaccine against HSV-2. The DNA sequence of the extracellular epitope-rich fragment of gD2 was optimized, chemically synthesized, and cloned into plasmid pMD902. The recombinant plasmid pMD902-gD was stably transfected into CHO-DG44 cells, and cell lines with high levels of expression of gD2 were established. The recombinant gD2 was purified efficiently using an anion exchange column and a Sephadex G-25 desalting column. The yield of the purified gD2 was 57 mg/L of serum-free culture medium, and its purity was determined to be about 95% by HPLC analysis. Finally, the immunogenicity of the purified gD2 was measured and it induced strong and specific humoral immunity and higher level of cellular immune response than gD2 expressed in prokaryotic cells. We established a stable, secretory, and high-yield gD2-expression cell line and an easy and efficient gD2-purification process, which lays the foundation for preparation of large amount of gD2 that is essential for HSV-2 subunit vaccine development. PMID:25906680

  9. The serial cultivation of suspended BHK-21/13 cells in serum-free Waymouth medium.

    PubMed

    Guskey, L E; Jenkin, H M

    1976-01-01

    A simple medium system was developed to obtain growth of BHK-21 cells in shaker cultures in the absence of serum. These cells have now undergone over 80 serial passages in serum-free Waymouth medium and have been recovered from the frozen state after storage for over 1 month in medium containing 10% dimethyl sulfoxide (DMSO) and 1% bovine serum albumin (BSA). Various amounts of exogenous lipid in the form of sodium oleate were added to cultures of cells growing in serum-free Waymouth medium. Concentrations of 10-50 mug of sodium oleate/ml had no detrimental effects on the cells as measured by trypan blue uptake. Furthermore, the cells were serially passed ten times in the presence of 10 mug sodium oleate/ml. Depletion of calf serum from the growth medium and addition of known quantities of lipids to the system provides a means of revealing subtle changes in lipid synthesis and lipid turnover during cellular growth. PMID:1250851

  10. Development of a serum-free liquid medium for Bartonella species.

    PubMed

    Müller, Andreas; Reiter, Michael; Mantlik, Katrin; Schötta, Anna-Margarita; Stockinger, Hannes; Stanek, Gerold

    2016-09-01

    The genus Bartonella comprises numerous species with at least 13 species pathogenic for humans. They are fastidious, aerobic, Gram negative, and facultative intracellular bacteria which cause a variety of human and non-human diseases. This study focused on the development of a serum-free liquid medium for culture of Bartonella species. Some liquid media are available commercially but all of them use undefined supplements such as fetal calf serum or defibrinated sheep blood. Our intention was to create a reproducible liquid medium for Bartonella species that can simply be prepared. We tested several supplements that could potentially support the growth of Bartonella species. Slight growth improvement was achieved with glucose and sucrose. However, hemin in particular improved the growth rate. At a temperature of 37 °C, a CO2 concentration of 5 %, a humidified atmosphere, and the use of the supplements glucose, sucrose, and hemin, we developed a medium that does not need serum as an undefined supplement any more. In conclusion, the newly developed medium supports growth of Bartonella species equal to the commercially available media but with the advantage that it has a serum-free formulation. It can be prepared fast and easy and is a useful tool in studying these bacteria. PMID:26842394

  11. Survival and neurite growth of chick embryo spinal cord cells in serum-free culture.

    PubMed

    Tanaka, H; Obata, K

    1982-07-01

    Cell survival and neurite growth were investigated in serum-free spinal cord cell cultures on polyornithine coating (PORN). Cells were obtained from 6- or 7-day-old chick embryos. Isolated spinal cord cells required promoting factors for their survival and neurite growth. The survival-promoting factors were initially present in spinal cord cells. High density cultures, co-cultures with spinal cord explants, and spinal cord extract promoted survival of isolated spinal cord cells in MEM with no additives. Other tissue extracts (brain, liver, heart and skeletal muscle), serum, and serum-free conditioned medium (SF-CM) of muscle or glioma C6 cells also promoted survival. The active substances in the brain extract and SF-CM were shown to be protein and were separated into 3 fractions (approximately molecular weight 150,000, 70,000, 40,000) by gel filtration chromatography. Survival and neurite growth were suggested to be promoted by different factors because: (1) survival was promoted by both tissue extract and SF-CM, but neurite growth was promoted only by SF-CM; (2) the neurite growth-stimulating activity of SF-CM was lost following dialysis and heat (100 degrees C, 2 min) treatment; however, the survival-promoting activity was not. It was also suggested that spinal cord cells produce neurite growth promoting factors, but did not initially contain these factors. PMID:7104764

  12. It is what it eats: Chemically defined media and the history of surrounds.

    PubMed

    Landecker, Hannah

    2016-06-01

    The cultivation of living organs, cells, animals, and embryos in the laboratory has been central to the production of biological knowledge. Over the twentieth century, the drive to variance control in the experimental setting led to systematic efforts to generate synthetic, chemically defined substitutes for complex natural foods, housing, and other substrates of life. This article takes up the history of chemically defined media with three aims in mind. First, to characterize patterns of decontextualization, tinkering, and negotiation between life and experimenter that occur across disparate histories of cultivation. Second, to highlight the paradoxical historicity of cultivated organisms generated to be freed from context, as they incorporate and embody the purified amino acids, vitamins, plastics, and other artificial supports developed in the name of experimental control. Third, to highlight the figure-ground reversal that occurs as these cells and organisms are reconsidered as accidentally good models of life in industrialized conditions of pollution and nutrient excess, due to the man-made nature of their surrounds. Methodologically, the history of surrounds is described as an epigenetic approach that focuses on the material relations between different objects and organisms previously considered quite separately, from explanted organs to bacteria to plant cells to rats to human embryos. PMID:26992285

  13. Chemically defined polyethylene glycol siRNA conjugates with enhanced gene silencing effect

    PubMed Central

    Gaziova, Zuzana; Baumann, Volker; Winkler, Anna-Maria; Winkler, Johannes

    2014-01-01

    The therapeutic application of siRNA suffers from poor bioavailability caused by rapid degradation and elimination. The covalent attachment of PEG is a universal concept to increase molecular size and enhance the pharmacokinetic properties of biomacromolecules. We devised a facile approach for attachment of PEG molecules with a defined molecular weight, and successful purification of the resulting conjugates. We directly conjugated structurally defined PEG chains with twelve ethylene glycol units to the 3′-terminal hydroxyl group of both sense and antisense strands via an aminoalkyl linker. The conjugates were easily purified by HPLC and successful PEGylation and molecule integrity were confirmed by ESI-MS. The evaluation of in vitro gene knockdown of two different targets in MCF-7 breast cancer cells showed stable pharmacologic activity when combined with a standard transfection reagent. Sense strand PEGylation even increased the silencing potency of a CRCX4-siRNA which had modest activity in its wild-type form. The results indicate that PEG chains at the 3′-terminus of both strands of siRNA are well tolerated by the RNAi effector. The attachment of short, chemically defined PEG chains is a feasible approach to improve the pharmacokinetic properties of siRNA, and can be combined with other targeted and untargeted delivery vehicles. PMID:24613624

  14. Critical Chemical-Mechanical Couplings that Define Permeability Modifications in Pressure-Sensitive Rock Fractures

    SciTech Connect

    Derek Elsworth; Abraham Grader; Susan Brantley

    2007-04-25

    This work examined and quantified processes controlling changes in the transport characteristics of natural fractures, subjected to coupled thermal-mechanical-chemical (TMC) effects. Specifically, it examined the effects of mineral dissolution and precipitation mediated by mechanical effects, using laboratory through-flow experiments concurrently imaged by X-ray CT. These were conducted on natural and artificial fractures in cores using water as the permeant. Fluid and mineral mass balances are recorded and are correlated with in-sample saturation, porosity and fracture aperture maps, acquired in real-time by X-ray CT-imaging at a maximum spatial resolution of 15-50 microns per pixel. Post-test, the samples were resin-impregnated, thin-sectioned, and examined by microscopy to define the characteristics of dissolution and precipitation. The test-concurrent X-ray imaging, mass balances, and measurements of permeability, together with the post-test microscopy, were used to define dissolution/precipitation processes, and to constrain process-based models. These models define and quantify key processes of pressure solution, free-face dissolution, and shear-dilation, and the influence of temperature, stress level, and chemistry on the rate of dissolution, its distribution in space and time, and its influence on the mechanical and transport properties of the fracture.

  15. Newly-derived neuroblastoma cell lines propagated in serum-free media recapitulate the genotype and phenotype of primary neuroblastoma tumours.

    PubMed

    Bate-Eya, Laurel T; Ebus, Marli E; Koster, Jan; den Hartog, Ilona J M; Zwijnenburg, Danny A; Schild, Linda; van der Ploeg, Ida; Dolman, M Emmy M; Caron, Huib N; Versteeg, Rogier; Molenaar, Jan J

    2014-02-01

    Recently protocols have been devised for the culturing of cell lines from fresh tumours under serum-free conditions in defined neural stem cell medium. These cells, frequently called tumour initiating cells (TICs) closely retained characteristics of the tumours of origin. We report the isolation of eight newly-derived neuroblastoma TICs from six primary neuroblastoma tumours and two bone marrow metastases. The primary tumours from which these TICs were generated have previously been fully typed by whole genome sequencing (WGS). Array comparative genomic hybridisation (aCGH) analysis showed that TIC lines retained essential characteristics of the primary tumours and exhibited typical neuroblastoma chromosomal aberrations such as MYCN amplification, gain of chromosome 17q and deletion of 1p36. Protein analysis showed expression for neuroblastoma markers MYCN, NCAM, CHGA, DBH and TH while haematopoietic markers CD19 and CD11b were absent. We analysed the growth characteristics and confirmed tumour-forming potential using sphere-forming assays, subcutaneous and orthotopic injection of these cells into immune-compromised mice. Affymetrix mRNA expression profiling of TIC line xenografts showed an expression pattern more closely mimicking primary tumours compared to xenografts from classical cell lines. This establishes that these neuroblastoma TICs cultured under serum-free conditions are relevant and useful neuroblastoma tumour models. PMID:24321263

  16. Chemically Defined and Xeno-Free Cryopreservation of Human Adipose-Derived Stem Cells

    PubMed Central

    López, Melany; Bollag, Roni J.; Yu, Jack C.; Isales, Carlos M.; Eroglu, Ali

    2016-01-01

    The stromal compartment of adipose tissue harbors multipotent cells known as adipose-derived stem cells (ASCs). These cells can differentiate into various lineages including osteogenic, chrondrogenic, adipogenic, and neurogenic; this cellular fraction may be easily obtained in large quantities through a clinically safe liposuction procedure. Therefore, ASCs offer exceptional opportunities for tissue engineering and regenerative medicine. However, current practices involving ASCs typically use fetal bovine serum (FBS)-based cryopreservation solutions that are associated with risks of immunological reactions and of transmitting infectious diseases and prions. To realize clinical applications of ASCs, serum- and xeno-free defined cryopreservation methods are needed. To this end, an animal product-free chemically defined cryopreservation medium was formulated by adding two antioxidants (reduced glutathione and ascorbic acid 2-phosphate), two polymers (PVA and ficoll), two permeating cryoprotectants (ethylene glycol and dimethylsulfoxide), a disaccharide (trehalose), and a calcium chelator (EGTA) to HEPES-buffered DMEM/F12. To limit the number of experimental groups, the concentration of trehalose, both polymers, and EGTA was fixed while the presence of the permeating CPAs and antioxidants was varied. ASCs suspended either in different versions of the defined medium or in the conventional undefined cryopreservation medium (10% dimethylsulfoxide+10% DMEM/F12+80% serum) were cooled to -70°C at 1°C/min before being plunged into liquid nitrogen. Samples were thawed either in air or in a water bath at 37°C. The presence of antioxidants along with 3.5% concentration of each penetrating cryoprotectant improved the freezing outcome to the level of the undefined cryopreservation medium, but the plating efficiency was still lower than that of unfrozen controls. Subsequently, increasing the concentration of both permeating cryoprotectants to 5% further improved the plating

  17. Sorafenib suppresses the cell cycle and induces the apoptosis of hepatocellular carcinoma cell lines in serum-free media

    PubMed Central

    TOMIZAWA, MINORU; SHINOZAKI, FUMINOBU; SUGIYAMA, TAKAO; YAMAMOTO, SHIGENORI; SUEISHI, MAKOTO; YOSHIDA, TAKANOBU

    2010-01-01

    To suppress the invasion of hepatocellular carcinoma (HCC) cells into surrounding connective tissues during metastasis, we investigated the usefulness of sorafenib. In order to search for model cell lines, cell numbers were counted to reveal cell lines with the potential to proliferate in serum-free media. Cell proliferation and cell motility were analyzed with the MTS and wound assay, respectively. 5-Bromo-2′-deoxyuridine (BrdU) labeling and mitotic and apoptotic indices were analyzed to assess the cell cycle and apoptosis. The expression levels of cyclin D1 and the cleavage of caspase-3 were analyzed by Western blotting. HLF cells exhibited growth in the serum-free medium, while the other cell lines examined did not. Sorafenib suppressed the cell proliferation and motility of the HLF cells in the serum-free media. Both indices of BrdU and mitotic potential decreased and the apoptotic index was increased in the serum-free media with sorafenib, suggesting that the cell cycle was suppressed and apoptosis was induced. The expression levels of cyclin D1 decreased and the cleavage of caspase-3 was noted in the serum-free media with sorafenib. Sorafenib may be suitable for molecular therapy to suppress the metastasis of HCC. PMID:22993610

  18. Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum-free microcarrier process.

    PubMed

    Heathman, Thomas R J; Glyn, Veronica A M; Picken, Andrew; Rafiq, Qasim A; Coopman, Karen; Nienow, Alvin W; Kara, Bo; Hewitt, Christopher J

    2015-08-01

    Human mesenchymal stem cell (hMSC) therapies are currently progressing through clinical development, driving the need for consistent, and cost effective manufacturing processes to meet the lot-sizes required for commercial production. The use of animal-derived serum is common in hMSC culture but has many drawbacks such as limited supply, lot-to-lot variability, increased regulatory burden, possibility of pathogen transmission, and reduced scope for process optimization. These constraints may impact the development of a consistent large-scale process and therefore must be addressed. The aim of this work was therefore to run a pilot study in the systematic development of serum-free hMSC manufacturing process. Human bone-marrow derived hMSCs were expanded on fibronectin-coated, non-porous plastic microcarriers in 100 mL stirred spinner flasks at a density of 3 × 10(5) cells.mL(-1) in serum-free medium. The hMSCs were successfully harvested by our recently-developed technique using animal-free enzymatic cell detachment accompanied by agitation followed by filtration to separate the hMSCs from microcarriers, with a post-harvest viability of 99.63 ± 0.03%. The hMSCs were found to be in accordance with the ISCT characterization criteria and maintained hMSC outgrowth and colony-forming potential. The hMSCs were held in suspension post-harvest to simulate a typical pooling time for a scaled expansion process and cryopreserved in a serum-free vehicle solution using a controlled-rate freezing process. Post-thaw viability was 75.8 ± 1.4% with a similar 3 h attachment efficiency also observed, indicating successful hMSC recovery, and attachment. This approach therefore demonstrates that once an hMSC line and appropriate medium have been selected for production, multiple unit operations can be integrated to generate an animal component-free hMSC production process from expansion through to cryopreservation. PMID:25727395

  19. Chemically defined conditions for human iPS cell derivation and culture

    PubMed Central

    Chen, Guokai; Gulbranson, Daniel R.; Hou, Zhonggang; Bolin, Jennifer M.; Ruotti, Victor; Probasco, Mitchell D.; Smuga-Otto, Kimberly; Howden, Sara E.; Diol, Nicole R.; Propson, Nicholas E.; Wagner, Ryan; Lee, Garrett O.; Antosiewicz-Bourget, Jessica; Teng, Joyce M. C.; Thomson, James A.

    2011-01-01

    We reexamine the individual components for human ES and iPS cell culture, and formulate a cell culture system in which all protein reagents for liquid media, attachment surfaces, and splitting are chemically defined. A major improvement is the lack of a serum albumin component, as variations in either animal or human sourced albumin batches have previously plagued human ES and iPS cell culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces, we demonstrate improved derivation efficiencies of vector-free human iPS cells with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ES and iPS cells and their derivatives, and should be applicable to other reprogramming methods. PMID:21478862

  20. Antimycobacterial activity of chemically defined natural substances from the Caribbean flora in Guadeloupe.

    PubMed

    Rastogi, N; Abaul, J; Goh, K S; Devallois, A; Philogène, E; Bourgeois, P

    1998-04-01

    Eight chemically defined, naturally occurring compounds were extracted from the tropical flora of the Caribbean island of Guadeloupe: pilocarpine, an alkaloid from Pilocarpus racemosus; heraclenol and isomeranzin, coumarins from Triphasia trifolia; lochnerin, an indole alkaloid from Rauwolfia biauriculata; ibogaine and voacangine, indole alkaloids from Tabernaemontana citrifolia; texalin, an oxazole from Amyris elemifera; and canellal, a sesquiterpene dialdehyde from Canella winterana. An essential oil fraction from Canella winterana was also tested. The antimycobacterial activity of these substances was tested against Mycobacterium tuberculosis, M. avium and M. kansasii using the Middlebrook 7H11 agar medium, the Bactec 460-TB radiometric methodology, and determination of bacterial viable counts. Three compounds, namely ibogaine, voacangine and texalin, showed antimycobacterial activity. Investigations on the structure-modification and structure-activity relationships of these compounds may help determine new targets for future drug development. PMID:9626931

  1. Chemically well-defined self-assembled monolayers for cell culture: toward mimicking the natural ECM

    PubMed Central

    Hudalla, Gregory A.

    2014-01-01

    The extracellular matrix (ECM) is a network of biological macromolecules that surrounds cells within tissues. In addition to serving as a physical support, the ECM actively influences cell behavior by providing sites for cell adhesion, establishing soluble factor gradients, and forming interfaces between different cell types within a tissue. Thus, elucidating the influence of ECM-derived biomolecules on cell behavior is an important aspect of cell biology. Self-assembled monolayers (SAMs) have emerged as promising tools to mimic the ECM as they provide chemically well-defined substrates that can be precisely tailored for specific cell culture applications, and their application in this regard is the focus of this review. In particular, this review will describe various approaches to prepare SAM-based culture substrates via non-specific adsorption, covalent immobilization, or non-covalent sequestering of ECM-derived biomolecules. Additionally, this review will highlight SAMs that present ECM-derived biomolecules to cells to probe the role of these molecules in cell-ECM interactions, including cell attachment, spreading and ‘outside-in’ signaling via focal adhesion complex formation. Finally, this review will introduce SAMs that can present or sequester soluble signaling molecules, such as growth factors, to study the influence of localized soluble factor activity on cell behavior. Together, these examples demonstrate that the chemical specificity and variability afforded by SAMs can provide robust, well-defined substrates for cell culture that can simplify experimental design and analysis by eliminating many of the confounding factors associated with traditional culture substrates. PMID:25214878

  2. Acetoin Catabolism and Acetylbutanediol Formation by Bacillus pumilus in a Chemically Defined Medium

    PubMed Central

    Xiao, Zijun; Ma, Cuiqing; Xu, Ping; Lu, Jian R.

    2009-01-01

    Background Most low molecular diols are highly water-soluble, hygroscopic, and reactive with many organic compounds. In the past decades, microbial research to produce diols, e.g. 1,3-propanediol and 2,3-butanediol, were considerably expanded due to their versatile usages especially in polymer synthesis and as possible alternatives to fossil based feedstocks from the bioconversion of renewable natural resources. This study aimed to provide a new way for bacterial production of an acetylated diol, i.e. acetylbutanediol (ABD, 3,4-dihydroxy-3-methylpentan-2-one), by acetoin metabolism. Methodology/Principal Findings When Bacillus pumilus ATCC 14884 was aerobically cultured in a chemically defined medium with acetoin as the sole carbon and energy source, ABD was produced and identified by gas chromatography – chemical ionization mass spectrometry and NMR spectroscopy. Conclusions/Significance Although the key enzyme leading to ABD from acetoin has not been identified yet at this stage, this study proposed a new metabolic pathawy to produce ABD in vivo from using renewable resources – in this case acetoin, which could be reproduced from glucose in this study – making it the first facility in the world to prepare this new bio-based diol product. PMID:19461961

  3. Gut microbes define liver cancer risk in mice exposed to chemical and viral transgenic hepatocarcinogens

    PubMed Central

    Fox, J G; Feng, Y; Theve, E J; Raczynski, A R; Fiala, J L A; Doernte, A L; Williams, M; McFaline, J L; Essigmann, J M; Schauer, D B; Tannenbaum, S R; Dedon, P C; Weinman, S A; Lemon, S M; Fry, R C; Rogers, A B

    2013-01-01

    Background and aims Hepatocellular carcinoma (HCC) frequently results from synergism between chemical and infectious liver carcinogens. Worldwide, the highest incidence of HCC is in regions endemic for the foodborne contaminant aflatoxin B1 (AFB1) and hepatitis B virus (HBV) infection. Recently, gut microbes have been implicated in multisystemic diseases including obesity and diabetes. Here, the hypothesis that specific intestinal bacteria promote liver cancer was tested in chemical and viral transgenic mouse models. Methods Helicobacter-free C3H/HeN mice were inoculated with AFB1 and/or Helicobacter hepaticus. The incidence, multiplicity and surface area of liver tumours were quantitated at 40 weeks. Molecular pathways involved in tumourigenesis were analysed by microarray, quantitative real-time PCR, liquid chromatography/mass spectrometry, ELISA, western blot and immunohistochemistry. In a separate experiment, C57BL/6 FL-N/35 mice harbouring a full-length hepatitis C virus (HCV) transgene were crossed with C3H/HeN mice and cancer rates compared between offspring with and without H hepaticus. Results Intestinal colonisation by H hepaticus was sufficient to promote aflatoxin- and HCV transgene-induced HCC. Neither bacterial translocation to the liver nor induction of hepatitis was necessary. From its preferred niche in the intestinal mucus layer, H hepaticus activated nuclear factor-κB (NF-κB)-regulated networks associated with innate and T helper 1 (Th1)-type adaptive immunity both in the lower bowel and liver. Biomarkers indicative of tumour progression included hepatocyte turnover, Wnt/β-catenin activation and oxidative injury with decreased phagocytic clearance of damaged cells. Conclusions Enteric microbiota define HCC risk in mice exposed to carcinogenic chemicals or hepatitis virus transgenes. These results have implications for human liver cancer risk assessment and prevention. PMID:19850960

  4. Zinc Levels in Seminal Fluid in Infertile Males and its Relation with Serum Free Testosterone

    PubMed Central

    Chaudhari, Ajay Rajeshwar

    2016-01-01

    Introduction The role of zinc is critical to reproduction potential. Seminal zinc is thought to be derived almost exclusively from prostatic secretions. Sperm motility is significantly influenced by zinc. Zinc deficiency has been linked with male sterility and subfertility. Aim To assess the influence of seminal plasma zinc on seminogram characteristics and whether endogenous testosterone affects the seminal levels of zinc. Materials and Methods The semen samples were obtained from 150 male partners of infertile couples who attended the Reproductive Biology Unit of the Department of Physiology, within the age 21-50 years and semen samples were analysed for the routine seminogram parameters. All the subjects were classified into two main groups, A- the subjects with normal ejaculates (n=62) and B- the subjects with abnormal ejaculates, who were further sub divided into the following groups: i) Asthenoteratozoospermics (n=43); ii) Oligoasthenoteratozoospermics (n=24); and iii) Azoospermics (n=21). The seminal plasma zinc was measured spectrophotometrically. The sample for serum free testosterone was sent to Thyrocare laboratory. Results The seminal plasma zinc was found to be significantly lower in the abnormal ejaculates than in the normal ejaculates. A statistically significant positive correlation was observed between the seminal plasma zinc and serum free testosterone (p<0.05, r=0.449). Statistically significant correlation was also found between seminal plasma zinc and all the seminogram parameters such as the sperm concentration, sperm motility and sperm morphology (p<0.05, r= 0.86, 0.87 and 0.86 respectively). Conclusion Low seminal plasma zinc might be a significant causative factor in impairing sperm functions and its dependence on endogenous free testosterone, is observed from a positive correlation between the two. PMID:27437207

  5. Physiological Study of Lactobacillus delbrueckii subsp. bulgaricus Strains in a Novel Chemically Defined Medium

    PubMed Central

    Chervaux, Christian; Ehrlich, S. Dusko; Maguin, Emmanuelle

    2000-01-01

    We developed a chemically defined medium called milieu proche du lait (MPL), in which 22 Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) strains exhibited growth rates ranging from 0.55 to 1 h−1. MPL can also be used for cultivation of other lactobacilli and Streptococcus thermophilus. The growth characteristics of L. bulgaricus in MPL containing different carbon sources were determined, including an initial characterization of the phosphotransferase system transporters involved. For the 22 tested strains, growth on lactose was faster than on glucose, mannose, and fructose. Lactose concentrations below 0.4% were limiting for growth. We isolated 2-deoxyglucose-resistant mutants from strains CNRZ397 and ATCC 11842. CNRZ397-derived mutants were all deficient for glucose, fructose, and mannose utilization, indicating that these three sugars are probably transported via a unique mannose-specific-enzyme-II-like transporter. In contrast, mutants of ATCC 11842 exhibited diverse phenotypes, suggesting that multiple transporters may exist in that strain. We also developed a protein labeling method and verified that exopolysaccharide production and phage infection can occur in MPL. The MPL medium should thus be useful in conducting physiological studies of L. bulgaricus and other lactic acid bacteria under well controlled nutritional conditions. PMID:11097906

  6. Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming.

    PubMed

    Moreau, Thomas; Evans, Amanda L; Vasquez, Louella; Tijssen, Marloes R; Yan, Ying; Trotter, Matthew W; Howard, Daniel; Colzani, Maria; Arumugam, Meera; Wu, Wing Han; Dalby, Amanda; Lampela, Riina; Bouet, Guenaelle; Hobbs, Catherine M; Pask, Dean C; Payne, Holly; Ponomaryov, Tatyana; Brill, Alexander; Soranzo, Nicole; Ouwehand, Willem H; Pedersen, Roger A; Ghevaert, Cedric

    2016-01-01

    The production of megakaryocytes (MKs)-the precursors of blood platelets-from human pluripotent stem cells (hPSCs) offers exciting clinical opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chemically defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 10(5) mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufacturing practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro production of platelets for transfusion and basic research in MK and platelet biology. PMID:27052461

  7. Attachment, Growth, and Detachment of Human Mesenchymal Stem Cells in a Chemically Defined Medium

    PubMed Central

    Salzig, Denise; Leber, Jasmin; Merkewitz, Katharina; Lange, Michaela C.; Köster, Natascha; Czermak, Peter

    2016-01-01

    The manufacture of human mesenchymal stem cells (hMSCs) for clinical applications requires an appropriate growth surface and an optimized, preferably chemically defined medium (CDM) for expansion. We investigated a new protein/peptide-free CDM that supports the adhesion, growth, and detachment of an immortalized hMSC line (hMSC-TERT) as well as primary cells derived from bone marrow (bm-hMSCs) and adipose tissue (ad-hMSCs). We observed the rapid attachment and spreading of hMSC-TERT cells and ad-hMSCs in CDM concomitant with the expression of integrin and actin fibers. Cell spreading was promoted by coating the growth surface with collagen type IV and fibronectin. The growth of hMSC-TERT cells was similar in CDM and serum-containing medium whereas the lag phase of bm-hMSCs was prolonged in CDM. FGF-2 or surface coating with collagen type IV promoted the growth of bm-hMSCs, but laminin had no effect. All three cell types retained their trilineage differentiation capability in CDM and were detached by several enzymes (but not collagenase in the case of hMSC-TERT cells). The medium and coating did not affect detachment efficiency but influenced cell survival after detachment. CDM combined with cell-specific surface coatings and/or FGF-2 supplements is therefore as effective as serum-containing medium for the manufacture of different hMSC types. PMID:27006663

  8. Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming

    PubMed Central

    Moreau, Thomas; Evans, Amanda L.; Vasquez, Louella; Tijssen, Marloes R.; Yan, Ying; Trotter, Matthew W.; Howard, Daniel; Colzani, Maria; Arumugam, Meera; Wu, Wing Han; Dalby, Amanda; Lampela, Riina; Bouet, Guenaelle; Hobbs, Catherine M.; Pask, Dean C.; Payne, Holly; Ponomaryov, Tatyana; Brill, Alexander; Soranzo, Nicole; Ouwehand, Willem H.; Pedersen, Roger A.; Ghevaert, Cedric

    2016-01-01

    The production of megakaryocytes (MKs)—the precursors of blood platelets—from human pluripotent stem cells (hPSCs) offers exciting clinical opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chemically defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 105 mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufacturing practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro production of platelets for transfusion and basic research in MK and platelet biology. PMID:27052461

  9. Inhibitor of Clostridium perfringens Formed by Heating Sodium Nitrite in a Chemically Defined Medium

    PubMed Central

    Moran, Dennis M.; Tannenbaum, Steven R.; Archer, Michael C.

    1975-01-01

    An inhibitor of Clostridium perfringens formed when low levels of nitrite were autoclaved with a defined chemical medium. A systematic study of the medium revealed that only amino acids and mineral salts were involved in the production of this inhibitor, which was proven to be a toxic compound formed from cysteine, ferrous sulfate, and sodium nitrite. The inhibitor was compared to several known compounds. S-nitrosocysteine inhibited the test organism, but would not form in the test system in amounts large enough to explain the observed inhibition. Roussin red salt was unstable in the test system and therefore was not the inhibitor. Roussin black salt, which was also inhibitory, could form in sufficient amounts to explain the inhibition. A complex of cysteine, iron, and nitrie oxide was detected in the autoclaved solution of cysteine, ferrous sulfate, and sodium nitrite; this cysteine complex did not appear to be inhibitory, however, at levels which could form in the autoclaved medium. The observed inhibition may have been due to the combined effects of sublethal concentrations of each compound. PMID:173239

  10. Attachment, Growth, and Detachment of Human Mesenchymal Stem Cells in a Chemically Defined Medium.

    PubMed

    Salzig, Denise; Leber, Jasmin; Merkewitz, Katharina; Lange, Michaela C; Köster, Natascha; Czermak, Peter

    2016-01-01

    The manufacture of human mesenchymal stem cells (hMSCs) for clinical applications requires an appropriate growth surface and an optimized, preferably chemically defined medium (CDM) for expansion. We investigated a new protein/peptide-free CDM that supports the adhesion, growth, and detachment of an immortalized hMSC line (hMSC-TERT) as well as primary cells derived from bone marrow (bm-hMSCs) and adipose tissue (ad-hMSCs). We observed the rapid attachment and spreading of hMSC-TERT cells and ad-hMSCs in CDM concomitant with the expression of integrin and actin fibers. Cell spreading was promoted by coating the growth surface with collagen type IV and fibronectin. The growth of hMSC-TERT cells was similar in CDM and serum-containing medium whereas the lag phase of bm-hMSCs was prolonged in CDM. FGF-2 or surface coating with collagen type IV promoted the growth of bm-hMSCs, but laminin had no effect. All three cell types retained their trilineage differentiation capability in CDM and were detached by several enzymes (but not collagenase in the case of hMSC-TERT cells). The medium and coating did not affect detachment efficiency but influenced cell survival after detachment. CDM combined with cell-specific surface coatings and/or FGF-2 supplements is therefore as effective as serum-containing medium for the manufacture of different hMSC types. PMID:27006663

  11. Growth kinetics and yield study on Chlorella pyrenoidosa in chemically defined media

    SciTech Connect

    Joung, J.J.; Akin, C.

    1983-01-01

    A Chlorella culture free from heterotrophic bacteria was obtained by eliminating the bacteria with successive use of antibiotics and agar plants. The purified Chlorella was cultured in chemically defined media. Under a photon flux (16.7 mw/cmS) similar to insolation, both heterotrophic and mixotrophic cultures were luxurious but the growth rates of autotrophic cultures were reduced substantially. The Chlorella culture grew most rapidly at 30 C in the absence of heterotrophic bacteria, and the highest specific growth rates were 1.43 x 10 h and 0.46 x 10 h for mixotrophic and autotrophic cultures, respectively. The highest photosynthetic efficiency over its growth period was 2.9% for autotrophic cultures. Elimination of heterotrophic bacteria from Chlorella cultures improved the algal growth rate as well as biomass yield significantly. A parasite of 0.1- m size was identified. The motile microorganism played an important role in the growth of the Chlorella and appeared to be common to green algae. 16 references, 2 tables.

  12. Serum-Free and Xenobiotic-Free Preservation of Cultured Human Limbal Epithelial Cells

    PubMed Central

    Utheim, Oeygunn; Islam, Rakibul; Lyberg, Torstein; Roald, Borghild; Eidet, Jon Roger; de la Paz, Maria Fideliz; Dartt, Darlene A.; Raeder, Sten; Utheim, Tor Paaske

    2015-01-01

    Aim/Purpose of the Study To develop a one-week storage method, without serum and xenobiotics, that would maintain cell viability, morphology, and phenotype of cultured human limbal epithelial sheets. Materials and Methods Human limbal explants were cultured on intact human amniotic membranes for two weeks. The sheets were stored in a hermetically sealed container at 23°C in either a serum-free medium with selected animal serum-derived compounds (Quantum 286) or a xenobiotic-free medium (Minimal Essential Medium) for 4 and 7 days. Stored and non-stored cultures were analyzed for cell viability, amniotic membrane and epithelial sheet thickness, and a panel of immunohistochemical markers for immature cells (ΔNp63α, p63, Bmi-1, C/EBP∂, ABCG2 and K19), differentiated cells (K3 and Cx43), proliferation (PCNA), and apoptosis (Caspase-3). Results The cell viability of the cultures was 98 ± 1% and remained high after storage. Mean central thickness of non-stored limbal epithelial sheets was 23 ± 3 μm, and no substantial loss of cells was observed after storage. The non-stored epithelial sheets expressed a predominantly immature phenotype with ΔNp63α positivity of more than 3% in 9 of 13 cultures. After storage, the expression of ABCG2 and C/EBP∂ was reduced for the 7 day Quantum 286-storage group; (P = 0.04), and Bmi-1 was reduced after 4 day Quantum 286-storage; (P = 0.02). No other markers varied significantly. The expression of differentiation markers was unrelated to the thickness of the epithelia and amniotic membrane, apart from ABCG2, which correlated negatively with thickness of limbal epithelia (R = -0.69, P = 0.01) and ΔNp63α, which correlated negatively with amniotic membrane thickness (R = -0.59, P = 0.03). Conclusion Limbal epithelial cells cultured from explants on amniotic membrane can be stored at 23°C in both serum-free and xenobiotic-free media, with sustained cell viability, ultrastructure, and ΔNp63α-positivity after both 4 and 7 days

  13. Developmental competence and cryotolerance of caprine parthenogenetic embryos cultured in chemically defined media.

    PubMed

    Choi, Woo-Jae; Lee, Ji-Hyun; Lee, Sang-Hee; Yum, Soo-Young; Lee, Song-Jeon; Lim, Chae-Woong; Jang, Goo

    2016-07-15

    In animal reproduction technologies, the in vitro embryo culture system has advanced over the past few decades. However, in vitro cultured embryos still have reduced functional and physiological abilities compared with those from in vivo conditions, and many factors of oviduct and uterine environments have not yet been revealed. Here, we demonstrated the in vitro culture of domestic goat (Capra hircus) embryos using two types of culture media, modified synthetic oviductal fluid (mSOF) and a two-step chemically defined medium (DI/II). To obtain parthenogenetic goat embryos, oocytes were matured in vitro in tissue culture media-199 supplemented with 10% fetal bovine serum for 22 to 24 hours, and activated with 5 μM, Ca(2+) ionomycin for 4 minutes, followed by 1.9 mM, 6-dimethylaminopurine treatment for 4 hours. After 2 days of embryo culture in different culture media, there were no significant differences in cleavage rates (96.6% vs. 95.4% in mSOF vs. DI/II, respectively). However, the DI/II group showed improved development competence to blastocysts (64.6% vs. 82.3% in mSOF vs. DI/II, respectively) and the total cell number of blastocysts (144.3 ± 9.2 vs. 264.4 ± 15.2 in mSOF vs. DI/II, respectively) at Day 7. After the cryopreservation of early-stage blastocysts at Day 6 via the conventional slow-freezing procedure, the surviving embryos were analyzed. The re-expansion rate after freezing and thawing was significantly higher in DI/II (39.66% vs. 67.69% in mSOF vs. DI/II, respectively), but there were no statistical differences in total cell numbers (142.3 ± 12.1 vs. 172.1 ± 11.6 in mSOF vs. DI/II, respectively), apoptotic index (4.9 ± 0.8% vs. 3.8 ± 0.7 in mSOF vs. DI/II, respectively), and the gene expression levels (BAX, GLUT1, MnSOD, and OCT4) among the re-expanded blastocysts. Overall, our data reported that the defined in vitro culture media for goat embryos were established with high efficiency, which will be very useful for

  14. Measurement of membrane-bound human heme oxygenase-1 activity using a chemically defined assay system.

    PubMed

    Huber, Warren J; Marohnic, Christopher C; Peters, Michelle; Alam, Jawed; Reed, James R; Masters, Bettie Sue Siler; Backes, Wayne L

    2009-04-01

    Heme oxygenase (HO) catalyzes heme degradation in a reaction requiring NADPH-cytochrome P450 reductase (CPR). Although most studies with HO used a soluble 30-kDa form, lacking the C-terminal membrane-binding region, recent reports show that the catalytic behavior of this enzyme is very different if this domain is retained; the overall activity was elevated 5-fold, and the K(m) for CPR decreased approximately 50-fold. The goal of these studies was to accurately measure HO activity using a coupled assay containing purified biliverdin reductase (BVR). This allows measurement of bilirubin formation after incorporation of full-length CPR and heme oxygenase-1 (HO-1) into a membrane environment. When rat liver cytosol was used as the source of partially purified BVR, the reaction remained linear for 2 to 3 min; however, the reaction was only linear for 10 to 30 s when an equivalent amount of purified, human BVR (hBVR) was used. This lack of linearity was not observed with soluble HO-1. Optimal formation of bilirubin was achieved with concentrations of bovine serum albumin (0.25 mg/ml) and hBVR (0.025-0.05 microM), but neither supplement increased the time that the reaction remained linear. Various concentrations of superoxide dismutase had no effect on the reaction; however, when catalase was included, the reactions were linear for at least 4 to 5 min, even at high CPR levels. These results not only show that HO-1-generated hydrogen peroxide leads to a decrease in HO-1 activity but also provide for a chemically defined system to be used to examine the function of full-length HO-1 in a membrane environment. PMID:19131520

  15. Differential requirements of two insect cell lines for growth in serum-free medium.

    PubMed

    Vaughn, J L; Fan, F

    1997-06-01

    The development of a serum-free medium that supports the growth of cells from a Spodoptera frugiperda and a Lymantria dispar cell line is reported. A yeast hydrolysate provided the B-vitamin complex, and a combination of a meat hydrolysate and tryptose provided most of the free amino acids required for cell growth. Supplemental cystine and methionine were required to achieve maximum cell growth. The serum or serum replacements used in earlier formulations were replaced with commercial lipid preparations and increased levels of iron salts. Although the cell growth cycle had a somewhat extended lag phase and the population doubling time of the S. frugiperda cells was longer than on serum-containing medium, the saturation densities were much higher. Spodoptera cells grown in this medium replicated the Autographa californica nuclear polyhedrosis virus well, producing 8.71 x 10(6) TCID50 extracellular virus and 4.4 x 10(6) polyhedra/ml culture. The specific activity of the polyhedra was somewhat less than that of polyhedra produced in insects. PMID:9201517

  16. Development of dengue virus plaques under serum-free overlay medium.

    PubMed Central

    Malewicz, B; Jenkin, H M

    1979-01-01

    An improved plaque assay for dengue virus was developed utilizing baby hamster kidney (BHK-21) cells initially grown in shaker culture. Different media preparations were tested for uniform and fast formation of BHK-21 cell sheets. Several overlay formulas were tested to develop a rapid plaque assay in 6- and 24-well plastic plates. The best results were obtained utilizing Eagle minimal essential medium (pH 7.2 to 7.4) supplemented with 1 mg of NaHCO3 per ml and 5% newborn calf serum for the formation of cell monolayers after 8 to 24 h of incubation at 37 degrees C. Serum-free Eagle minimal essential medium supplemented with 1% methylcellulose and buffered with 10 mM N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid (pH 7.4 to 7.6) was used as an overlay medium. This system allowed for plaque formation after 3 days of incubation of dengue type 2 virus and after 4 days for dengue type 1 and 4 viruses. Images PMID:39085

  17. Misleading results from immunoassays of serum free thyroxine in the presence of rheumatoid factor.

    PubMed

    Norden, A G; Jackson, R A; Norden, L E; Griffin, A J; Barnes, M A; Little, J A

    1997-06-01

    A novel interference with measurements of serum free thyroxine (FT4) caused by rheumatoid factor (RhF) is described. We found misleading, sometimes gross, increases of FT4 results in 5 clinically euthyroid elderly female patients with high RhF concentrations. All 5 patients had high FT4 on Abbott AxSYM or IMx analyzers. "NETRIA" immunoassays gave misleading results in 4 of the 5 patients; Amerlex-MAB in 2 of 4 patients; AutoDELFIA in 2 of the 5; and Corning ACS-180 and Bayer Diagnostics Immuno 1 in 1 of the 5. BM-ES700 system results for FT4 in these women remained within the reference range. Results for serum T4, thyroid-stimulating hormone, free triiodothyronine, thyroid-hormone-binding globulin, and FT4 measured by equilibrium dialysis were normal in all 5 patients. Drugs, albumin-binding variants, and anti-thyroid-hormone antibodies were excluded as interferences. Addition to normal serum of the RhF isolated from each of the 5 patients increased the apparent FT4 (Abbott AxSYM). Screening of 83 unselected patients demonstrated a highly significant positive correlation between FT4 (Abbott AxSYM) and RhF concentrations. Discrepant, apparently increased FT4 with a normal result for thyroid-stimulating hormone should lead to measurement of the patient's RhF concentration. PMID:9191546

  18. Serum-free culture alters the quantity and protein composition of neuroblastoma-derived extracellular vesicles

    PubMed Central

    Li, Jinghuan; Lee, Yi; Johansson, Henrik J.; Mäger, Imre; Vader, Pieter; Nordin, Joel Z.; Wiklander, Oscar P. B.; Lehtiö, Janne; Wood, Matthew J. A.; Andaloussi, Samir EL

    2015-01-01

    Extracellular vesicles (EVs) play a significant role in cell–cell communication in numerous physiological processes and pathological conditions, and offer promise as novel biomarkers and therapeutic agents for genetic diseases. Many recent studies have described different molecular mechanisms that contribute to EV biogenesis and release from cells. However, little is known about how external stimuli such as cell culture conditions can affect the quantity and content of EVs. While N2a neuroblastoma cells cultured in serum-free (OptiMEM) conditions did not result in EVs with significant biophysical or size differences compared with cells cultured in serum-containing (pre-spun) conditions, the quantity of isolated EVs was greatly increased. Moreover, the expression levels of certain vesicular proteins (e.g. small GTPases, G-protein complexes, mRNA processing proteins and splicing factors), some of which were previously reported to be involved in EV biogenesis, were found to be differentially expressed in EVs under different culture conditions. These data, therefore, contribute to the understanding of how extracellular factors and intracellular molecular pathways affect the composition and release of EVs. PMID:26022510

  19. Performance of serum-free broth media for growth of Renibacterium salmoninarum

    USGS Publications Warehouse

    Starliper, C.E.; Schill, W.B.; Mathias, J.

    1998-01-01

    Growth of Renibacterium salmoninarum was compared in 14 different broth media; 13 serum-free, and 1 that contained newborn calf serum, KDM2+M. Supplementation with 1% v/v R. salmoninarum MCO4M metabolite was evaluated for 6 of the media that do not utilize it as part of their ingredients. Viable cells were enumerated on Days 10, 20, and 30 post inoculation to evaluate performance. The experiment was repeated 3 times using high, low, and medium (trials 1 to 3, respectively) cell concentrations as inoculum. In general there was no optimal medium and all performed well. The choice of which to employ depends on the ease of preparation and presence of certain ingredients that might affect subsequent assays. In trials 2 and 3, the pH was estimated using test papers at the same time as cells were counted. Maximum pH increase occurred with KDM2+M and those media containing charcoal. For most media, a simple pH determination could be used as a means to check that growth has occurred in a culture, particularly if charcoal was added directly to the media and a visual inspection could not be made to detect growth.

  20. Performance of serum-free broth media for growth of Renibacterium salmoninarum.

    PubMed

    Starliper, C E; Schill, W B; Mathias, J

    1998-09-11

    Growth of Renibacterium salmoninarum was compared in 14 different broth media; 13 serum-free, and 1 that contained newborn calf serum, KDM2+M. Supplementation with 1% v/v R. salmoninarum MCO4M metabolite was evaluated for 6 of the media that do not utilize it as part of their ingredients. Viable cells were enumerated on Days 10, 20, and 30 post inoculation to evaluate performance. The experiment was repeated 3 times using high, low, and medium (trials 1 to 3, respectively) cell concentrations as inoculum. In general there was no optimal medium and all performed well. The choice of which to employ depends on the ease of preparation and presence of certain ingredients that might affect subsequent assays. In trials 2 and 3, the pH was estimated using test papers at the same time as cells were counted. Maximum pH increase occurred with KDM2+M and those media containing charcoal. For most media, a simple pH determination could be used as a means to check that growth has occurred in a culture, particularly if charcoal was added directly to the media and a visual inspection could not be made to detect growth. PMID:9789976

  1. Rakkyo fructan as a cryoprotectant for serum-free cryopreservation of mammalian cells.

    PubMed

    Ogawa, Akiko; Mizui, Shinya; Chida, Yasuhito; Shimizu, Masafumi; Terada, Satoshi; Ohura, Takeshi; Kobayashi, Kyo-Ichi; Yasukawa, Saori; Moriyama, Nobuyuki

    2014-07-01

    Cryopreservation refers to the long-term storage of mammalian cells. Mammalian serum is generally used as a cryoprotectant, but is associated with problems including the risk of contamination by pathogens and quality control issues. Therefore, a serum-free cryopreservation method needs to be established. In this study, we focused on rakkyo fructan, a fructose polymer, derived from the Japanese shallot as an alternative factor to serum. Fructan contributes to tolerance to frost and dehydration in plants by stabilizing the plant membrane. However, whether fructan protects mammalian cells against freezing stress remains unknown. The ability of rakkyo fructan to be an alternative cryoprotectant to fetal bovine serum (FBS) was examined in the present study. 2E3-O, a mouse hybridoma, was preserved in rakkyo fructan, was highly viable after being defrosted, and then proliferated rapidly. When rakkyo fructan was combined with dimethylsulfoxide (DMSO), its ability to protect the hybridoma against freezing stress was improved. The rakkyo fructan and DMSO mixture was used in the cryopreservation of the mammalian cell lines CHO-DP12, a producer of recombinant antibodies, and HepG2, human hepatoma cells frequently tested in bio-artificial livers. Following the freezing and thawing processes, CHO-DP12 cells retained their ability to produce recombinant antibodies and as did HepG2 cells for albumin and mRNA expression of cytochrome P450 enzymes. These results indicate that rakkyo fructan is a promising cryoprotectant that prevents mammalian cells from freezing stress similar to FBS. PMID:24485744

  2. Monitoring the biology stability of human umbilical cord-derived mesenchymal stem cells during long-term culture in serum-free medium.

    PubMed

    Chen, Gecai; Yue, Aihuan; Ruan, Zhongbao; Yin, Yigang; Wang, Ruzhu; Ren, Yin; Zhu, Li

    2014-12-01

    Mesenchymal stem cells (MSCs) are multipotent adult stem cells that have an immunosuppressive effect. The biological stability of MSCs in serum-free medium during long-term culture in vitro has not been elucidated clearly. The morphology, immunophenotype and multi-lineage potential were analyzed at passages 3, 5, 10, 15, 20, and 25 (P3, P5, P10, P15, P20, and P25, respectively). The cell cycle distribution, apoptosis, and karyotype of human umbilical cord-derived (hUC)-MSCs were analyzed at P3, P5, P10, P15, P20, and P25. From P3 to P25, the three defining biological properties of hUC-MSCs [adherence to plastic, specific surface antigen expression, multipotent differentiation potential] met the standards proposed by the International Society for Cellular Therapy for definition of MSCs. The cell cycle distribution analysis at the P25 showed that the percentage of cells at G0/G1 was increased, compared with the cells at P3 (P < 0.05). Cells at P25 displayed an increase in the apoptosis rate (to 183 %), compared to those at P3 (P < 0.01). Within subculture generations 3-20 (P3-P20), the differences between the cell apoptotic rates were not statistically significant (P > 0.05). There were no detectable chromosome eliminations, displacements, or chromosomal imbalances, as assessed by the karyotyping guidelines of the International System for Human Cytogenetic Nomenclature (ISCN, 2009). Long-term culture affects the biological stability of MSCs in serum-free MesenCult-XF medium. MSCs can be expanded up to the 25th passage without chromosomal changes by G-band. The best biological activity period and stability appeared between the third to 20th generations. PMID:24407613

  3. Scalable cultivation of human pluripotent stem cells on chemically-defined surfaces

    NASA Astrophysics Data System (ADS)

    Hsiung, Michael Chi-Wei

    Human stem cells (SCs) are classified as self-renewing cells possessing great ability in therapeutic applications due of their ability to differentiate along any major cell lineage in the human body. Despite their restorative potential, widespread use of SCs is hampered by strenuous control issues. Along with the need for strict xeno-free environments to sustain growth in culture, current methods for growing human pluripotent stem cells (hPSCs) rely on platforms which impede large-scale cultivation and therapeutic delivery. Hence, any progress towards development of large-scale culture systems is severely hindered. In a concentrated effort to develop a scheme that can serve as a model precursor for large scale SC propagation in clinical use, we have explored methods for cultivating hPSCs on completely defined surfaces. We discuss novel approaches with the potential to go beyond the limitations presented by current methods. In particular, we studied the cultivation of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) on surface which underwent synthetic or chemical modification. Current methods for hPSCs rely on animal-based extracellular matrices (ECMs) such as mouse embryonic fibroblasts (MEFs) or feeders and murine sacoma cell-derived substrates to facilitate their growth. While these layers or coatings can be used to maximize the output of hPSC production, they cannot be considered for clinical use because they risk introducing foreign pathogens into culture. We have identified and developed conditions for a completely defined xeno-free substrate used for culturing hPSCs. By utilizing coupling chemistry, we can functionalize ester groups on a given surface and conjugate synthetic peptides containing the arginine-glycine-aspartic acid (RGD) motif, known for their role in cell adhesion. This method offers advantages over traditional hPSC culture by keeping the modified substrata free of xenogenic response and can be scaled up in

  4. Protocol optimization for long-term liquid storage of goat semen in a chemically defined extender.

    PubMed

    Zhao, B-T; Han, D; Xu, C-L; Luo, M-J; Chang, Z-L; Tan, J-H

    2009-12-01

    A specific problem in the preservation of goat semen has been the detrimental effect of seminal plasma on the viability of spermatozoa in extenders containing egg yolk or milk. The use of chemically defined extenders will have obvious advantages in liquid storage of buck semen. Our previous study showed that the self-made mZAP extender performed better than commercial extenders, and maintained a sperm motility of 34% for 9 days and a fertilizing potential for successful pregnancies for 7 days. The aim of this study was to extend the viability and fertilizing potential of liquid-stored goat spermatozoa by optimizing procedures for semen processing and storage in the mZAP extender. Semen samples collected from five goat bucks of the Lubei White and Boer breeds were diluted with the extender, cooled and stored at 5 degrees C. Stored semen was evaluated for sperm viability parameters, every 48 h of storage. Data from three ejaculates of different bucks were analysed for each treatment. The percentage data were arcsine-transformed before being analysed with anova and Duncan's multiple comparison test. While cooling at the rate of 0.1-0.25 degrees C/min did not affect sperm viability parameters, doing so at the rate of 0.6 degrees C/min from 30 to 15 degrees C reduced goat sperm motility and membrane integrity. Sperm motility and membrane integrity were significantly higher in semen coated with the extender containing 20% egg yolk than in non-coated semen. Sperm motility, membrane integrity and acrosomal intactness were significantly higher when coated semen was 21-fold diluted than when it was 11- or 51-fold diluted and when extender was renewed at 48-h intervals than when it was not renewed during storage. When goat semen coated with the egg yolk-containing extender was 21-fold diluted, cooled at the rate of 0.07-0.25 degrees C/min, stored at 5 degrees C and the extender renewed every 48 h, a sperm motility of 48% was maintained for 13 days, and an in vitro

  5. Pellet-freezing of Damascus goat semen in a chemically defined extender.

    PubMed

    Khalifa, T A A; El-Saidy, B E

    2006-07-01

    During the breeding season of goats (12 bucks and 64 does) in Egypt, five experiments were conducted using a chemically defined cryoextender (CDE) to investigate: (1) the influence of rates of semen dilution (1:2, 1:4 and 1:19) and methods of thawing of frozen semen pellets (dry thawing versus wet thawing) on sperm progressive motility (SPM), sperm acrosome abnormalities (SAA) and rate of lipid peroxidation in semen as measured by malonaldehyde (MAL) production, and (2) the effect of insemination of does in natural (n = 38) and cloprostenol-synchronized (n = 26) estrus with frozen semen on their kidding rates and prolificacy. Semen (two successive ejaculates/buck) was collected twice a week via an AV and only ejaculates of >2500 x 10(6) sperm/ml and 70% SPM were diluted in one step at 30 degrees C with the CDE, cooled to 5 degrees C over a 4h-period, frozen in the form of 0.30 ml pellets and stored in liquid nitrogen for 72 h. The results revealed that post-thaw SPM of semen diluted at a rate of 1:4 was significantly (P < 0.01) higher than that of semen diluted at the other rates. Dilution of semen at a rate of 1:19 (< or =151 x 10(6) sperm/ml) not only minimized (P < 0.01) pre-freeze and post-thaw SPM, but also augmented (P < 0.01) pre-freeze and post-thaw rates of lipid peroxidation as evidenced by the high level of MAL production and the ability of antioxidants (1mg/ml EDTA, 200 U/ml bovine liver catalase, 0.61 mg/ml reduced glutathione and 0.11 mg/ml sodium pyruvate) to restore (P < 0.01) pre-freeze and post-thaw SPM. Frozen semen pellets exposed to dry thawing had a greater percentage of SPM (P < 0.01) as well as lower values of SAA and MAL (P < 0.01) than those exposed to wet thawing. Although the kidding rates did not vary significantly among does in natural (55.26%) and synchronized (53.85%) estrus, a higher (P < 0.05) prolificacy was obtained after their insemination in natural (1.81+/-0.16) rather than in synchronized (1.22+/-0.11) estrus. PMID:16169690

  6. Canine and Equine Mesenchymal Stem Cells Grown in Serum Free Media Have Altered Immunophenotype.

    PubMed

    Clark, Kaitlin C; Kol, Amir; Shahbenderian, Salpi; Granick, Jennifer L; Walker, Naomi J; Borjesson, Dori L

    2016-04-01

    Mesenchymal stem cell (MSC) therapy is being increasingly used to treat dogs and horses with naturally-occurring diseases. However these animals also serve as critical large animal models for ongoing translation of cell therapy products to the human market. MSC manufacture for clinical use mandates improvement in cell culture systems to meet demands for higher MSC numbers and removal of xeno-proteins (i.e. fetal bovine serum, FBS). While serum-free media (SFM) is commercially available, its affects on MSC phenotype and immunomodulatory functions are not fully known. The objective of this study was to determine if specific MSC culture conditions, MSC expansion in HYPERFlasks® or MSC expansion in a commercially available SFM, would alter MSC proliferation, phenotype or immunomodulatory properties in vitro. MSCs cultured in HYPERFlasks® were similar in phenotype, proliferative capacity and immunomodulatory functions to MSCs grown in standard flasks however MSC yield was markedly increased. HYPERFlasks® therefore provide a viable option to generate greater cell numbers in a streamlined manner. Canine and equine MSCs expanded in SFM displayed similar proliferation, surface phenotype and inhibitory effect on lymphocyte proliferation in vitro. However, MSCs cultured in the absence of FBS secreted significantly less PGE2, and were significantly less able to inhibit IFNγ secretion by activated T-cells. Immunomodulatory functions altered by expansion in SFM were species dependent. Unlike equine MSCs, in canine adipose-derived MSCs, the inhibition of lymphocyte proliferation was not principally modulated by PGE2. The removal of FBS from both canine and equine MSC culture systems resulted in altered immunomodulatory properties in vitro and warrants further investigation prior to moving towards FBS-free culture conditions. PMID:26638159

  7. Prognostic Significance of Serum Free Light Chains in Chronic Lymphocytic Leukemia

    PubMed Central

    Sarris, Katerina; Koulieris, Efstathios; Bartzis, Vassiliki; Tzenou, Tatiana; Sachanas, Sotirios; Nikolaou, Eftychia; Efthymiou, Anna; Bitsani, Katerina; Dimou, Maria; Vassilakopoulos, Theodoros P.; Angelopoulou, Maria K.; Kontopidou, Flora; Tsaftaridis, Panagiotis; Kafasi, Nikolitsa; Pangalis, Gerasimos A.; Panayiotidis, Panayiotis P.; Harding, Stephen

    2013-01-01

    Background. Serum free light chains (sFLC), the most commonly detected paraprotein in CLL, were recently proposed as useful tools for the prognostication of CLL patients. Objective. To investigate the prognostic implication of sFLC and the summated FLC-kappa plus FLC-lambda in a CLL patients' series. Patients and Methods. We studied 143 CLL patients of which 18 were symptomatic and needed treatment, while 37 became symptomatic during follow-up. Seventy-two percent, 18%, and 10% were in Binet stage A, B and C, respectively. Median patients' followup was 32 months (range 4–228). Results. Increased involved (restricted) sFLC (iFLC) was found in 42% of patients, while the summated FLC-kappa plus FLC-lambda was above 60 mg/dL in 14%. Increased sFLC values as well as those of summated FLC above 60 were related to shorter time to treatment (P = 0.0005 and P = 0.000003, resp.) and overall survival (P = 0.05 and P = 0.003, resp.). They also correlated with β2-microglobulin (P = 0.009 and P = 0.03, resp.), serum albumin (P = 0.009 for summated sFLC), hemoglobin (P < 0.001), abnormal LDH (P = 0.037 and P = 0.001, resp.), Binet stage (P < 0.05) and with the presence of beta symptoms (P = 0.004 for summated sFLC). Conclusion. We confirmed the prognostic significance of sFLC in CLL regarding both time to treatment and survival and showed their relationship with other parameters. PMID:24288537

  8. Acidification in the Adirondacks: Defining the Biota in trophic Levels of 30 Chemically Diverse Acid-Impacted Lakes

    EPA Science Inventory

    The Adirondack Mountains in New York State have a varied surficial geology and chemically diverse surface waters that are among the most impacted by acid deposition in the U.S. No single Adirondack investigation has been comprehensive in defining the effects of acidification on ...

  9. Maximizing productivity of CHO cell-based fed-batch culture using chemically defined media conditions and typical manufacturing equipment.

    PubMed

    Huang, Yao-Ming; Hu, WeiWei; Rustandi, Eddie; Chang, Kevin; Yusuf-Makagiansar, Helena; Ryll, Thomas

    2010-01-01

    A highly productive chemically defined fed-batch process was developed to maximize titer and volumetric productivity for Chinese hamster ovary cell-based recombinant protein manufacturing. Two cell lines producing a recombinant antibody (cell line A) and an Fc-fusion protein (cell line B) were used for development. Both processes achieved product titers of 10 g/L on day 18 under chemically defined conditions. For cell line B, the use of plant derived hydrolysates combined with the optimized chemically defined medium increased the titer to 13 g/L. Volumetric productivities were increased from a base line of about 200 mg/L/d to about 500 mg/L/d under chemically defined conditions and as high as 700 mg/L/d with cell line B using plant derived hydrolysates. Peak cell densities reached greater than 20E6 vc/mL, and cell viabilities were maintained above 80% on day 18 without the use of antiapoptotic genes or temperature shift. A rapid compound screening method was developed to effectively test positive factors within 72 h. Peak volumetric oxygen uptake rates (OUR) more than tripled from the baseline condition. Oxygen demand continued to increase after maximum cell density was reached with a maximal OUR of 3.7 mmol/L/h. The new process format was scaled up and verified at 100 L pilot scale using reactor equipment of similar configuration as used at manufacturing scale. PMID:20945494

  10. Collagen nanofibres are a biomimetic substrate for the serum-free osteogenic differentiation of human adipose stem cells

    PubMed Central

    Sefcik, Lauren S.; Neal, Rebekah A.; Kaszuba, Stephanie N.; Parker, Anna M.; Katz, Adam J.; Ogle, Roy C.; Botchwey, Edward A.

    2011-01-01

    Electrospinning has recently gained widespread attention as a process capable of producing nanoscale fibres that mimic native extracellular matrix. In this study, we compared the osteogenic differentiation behaviour of human adipose stem cells (ASCs) on a 3D nanofibre matrix of type I rat tail collagen (RTC) and a 2D RTC collagen-coated substrate, using a novel serum-free osteogenic medium. The serum-free medium significantly enhanced the numbers of proliferating cells in culture, compared to ASCs in traditional basal medium containing 10% animal serum, highlighting a potential clinical role for in vitro stem cell expansion. Osteogenic differentiation behaviour was assessed at days 7, 14 and 21 using quantitative real-time RT–PCR analysis of the osteogenic genes collagen I (Coll I), alkaline phosphatase (ALP), osteopontin (OP), osteonectin (ON), osteocalcin (OC) and core-binding factor-α (cbfa1). All genes were upregulated (>one-fold) in ASCs cultured on nanofibre scaffolds over 2D collagen coatings by day 21. Synthesis of mineralized extracellular matrix on the scaffolds was assessed on day 21 with Alizarin red staining. These studies demonstrate that 3D nanoscale morphology plays a critical role in regulating cell fate processes and in vitro osteogenic differentiation of ASCs under serum-free conditions. PMID:18493910

  11. Development of Chemically Defined Media to Express Trp-Analog-Labeled Proteins in a Lactococcus lactis Trp Auxotroph.

    PubMed

    Shao, Jinfeng; Marcondes, Marcelo F M; Oliveira, Vitor; Broos, Jaap

    2016-01-01

    Chemically defined media for growth of Lactococcus lactis strains contain about 50 components, making them laborious and expensive growth media. However, they are crucial for metabolism studies as well as for expression of heterologous proteins labeled with unnatural amino acids. In particular, the L. lactis Trp auxotroph PA1002, overexpressing the tryptophanyl tRNA synthetase enzyme of L. lactis, is very suitable for the biosynthetic incorporation of Trp analogs in proteins because of its most relaxed substrate specificity reported towards Trp analogs. Here we present two much simpler defined media for L. lactis, which consist of only 24 or 31 components, respectively, and with which the L. lactis Trp auxotroph shows similar growth characteristics as with a 50-component chemically defined medium. Importantly, the expression levels of two recombinant proteins used for evaluation were up to 2-3 times higher in these new media than in the 50-component medium, without affecting the Trp analog incorporation efficiency. Taken together, the simplest chemically defined media reported so far for L. lactis are presented. Since L. lactis also shows auxotrophy for Arg, His, Ile, Leu Val, and Met, our simplified media may also be useful for the biosynthetic incorporation of analogs of these five amino acids. PMID:27172771

  12. Development of Chemically Defined Media Supporting High-Cell-Density Growth of Lactococci, Enterococci, and Streptococci▿ †

    PubMed Central

    Zhang, Guiying; Mills, David A.; Block, David E.

    2009-01-01

    Lactococcus lactis IL1403 was used as an experimental strain to develop a chemically defined medium for study of the physiology and metabolic pathways of lactococci. An experimental leave-one-out technique was employed to determine the necessity of each of the 57 chemical components used in medium development. A statistical experimental design approach including three fractional factorial designs and a central composite design was used to optimize the fermentation process with 21 variables composed of 19 nutritional factors grouped from the 57 components and two environmental factors (initial pH and temperature). For L. lactis IL1403, the maximum biomass concentrations obtained with the two optimal chemically defined media developed in this study (ZMB1 and ZMB2) were generally 3.5- to 4-fold higher than the maximum biomass concentrations obtained with the previously described best synthetic media (SA) and 50% to 68% higher than the maximum biomass concentrations obtained with M17, a complex medium commonly used for lactococci. The new chemically defined media support high-cell-density growth of numerous strains of L. lactis, Enterococcus faecalis, and Streptococcus thermophilus. PMID:19074601

  13. Maturation and fertilisation of sheep oocytes cultured in serum-free medium containing silk protein sericin.

    PubMed

    Yasmin, Cut; Otoi, Takeshige; Setiadi, Mohamad Agus; Karja, Ni Wayan Kurniani

    2015-03-01

    Sericin is a water-soluble component of silk and has been used as a biomaterial due to its antibacterial and ultraviolet radiation-resistant properties. This study was designed to evaluate the effect of sericin supplementation in a maturation medium on the meiotic competence and fertilisability of sheep oocytes. Cumulus-oocyte complexes (COCs) were cultured in TCM199 supplemented with sericin at various concentrations of 0 (control), 0.1, 0.25 and 0.5%, either with or without bovine serum albumin (BSA). When the COCs were matured without BSA, the supplementation of 0.1% sericin significantly increased the rates of maturation to metaphase II and the total fertilisation of oocytes compared with the other concentrations of sericin. When the COCs were matured with BSA, the beneficial effects of 0.1% sericin supplementation on the maturation and fertilisation of oocytes were not observed. Our findings indicate that supplementation with 0.1% sericin during maturation culture may improve the nuclear maturation and fertilisability of sheep oocytes. Moreover, it may be possible to replace BSA with sericin in chemically defined media without the risk of disease transmission. PMID:25655418

  14. Chemical Atmosphere-Snow-Sea Ice Interactions: defining future research in the field, lab and modeling

    NASA Astrophysics Data System (ADS)

    Frey, Markus

    2015-04-01

    The air-snow-sea ice system plays an important role in the global cycling of nitrogen, halogens, trace metals or carbon, including greenhouse gases (e.g. CO2 air-sea flux), and therefore influences also climate. Its impact on atmospheric composition is illustrated for example by dramatic ozone and mercury depletion events which occur within or close to the sea ice zone (SIZ) mostly during polar spring and are catalysed by halogens released from SIZ ice, snow or aerosol. Recent field campaigns in the high Arctic (e.g. BROMEX, OASIS) and Antarctic (Weddell sea cruises) highlight the importance of snow on sea ice as a chemical reservoir and reactor, even during polar night. However, many processes, participating chemical species and their interactions are still poorly understood and/or lack any representation in current models. Furthermore, recent lab studies provide a lot of detail on the chemical environment and processes but need to be integrated much better to improve our understanding of a rapidly changing natural environment. During a 3-day workshop held in Cambridge/UK in October 2013 more than 60 scientists from 15 countries who work on the physics, chemistry or biology of the atmosphere-snow-sea ice system discussed research status and challenges, which need to be addressed in the near future. In this presentation I will give a summary of the main research questions identified during this workshop as well as ways forward to answer them through a community-based interdisciplinary approach.

  15. Chemistry as the defining science: discipline and training in nineteenth-century chemical laboratories.

    PubMed

    Jackson, Catherine M

    2011-06-01

    The institutional revolution has become a major landmark of late-nineteenth century science, marking the rapid construction of large, institutional laboratories which transformed scientific training and practice. Although it has served historians of physics well, the institutional revolution has proved much more contentious in the case of chemistry. I use published sources, mainly written by chemists and largely focused on laboratories built in German-speaking lands between about 1865 and 1900, to show that chemical laboratory design was inextricably linked to productive practice, large-scale pedagogy and disciplinary management. I argue that effective management of the novel risks inherent in teaching and doing organic synthesis was significant in driving and shaping the construction of late-nineteenth century institutional chemical laboratories, and that these laboratories were essential to the disciplinary development of chemistry. Seen in this way, the laboratory necessarily becomes part of the material culture of late-nineteenth century chemistry, and I show how this view leads not only to a revision of what is usually known as the laboratory revolution in chemistry but also to a new interpretation of the institutional revolution in physics. PMID:21722963

  16. Physico-chemical gradients within the hydrothermal chimney Roane define sharp boundaries for microbial community ecology

    NASA Astrophysics Data System (ADS)

    Frank, K. L.; Kelley, D. S.; Girguis, P. R.

    2011-12-01

    The unique physico-chemical gradients characteristic of hydrothermal vents provide diverse niches for prokaryotic communities. To date, our knowledge of environmental constraints on microbial colonization and metabolic activity within active sulfide structures has been limited by the lack of co-registered in situ chemistry and appropriate, taxonomic and metabolic genetic markers. Here we characterize de novo endolithic microbial colonization using a sulfide microbial incubator within the hydrothermal vent Roane during a one-year deployment, with co-registered temperature, fluid chemistry and mineralogy. Taxanomic assessment of phylogenetic diversity via 16S rDNA extracted from the outer (40-70°C) and middle (150-240°C) chambers of the incubator revealed patterns of distribution comparable to previously published observations. However, quantitative and statistical analyses of 16S rDNA sequences from two chambers revealed very distinct communities, with less than 5% of the identified operational taxonomic units common to both chambers. Analyses of metagenomic data suggest an elevated potential for motility and select biosynthetic pathways in the outer chamber community. In contrast, the middle chamber community exhibits a greater potential for quorum sensing, biofilm formation and archaeal lipid biosynthesis. Striking differences in metabolic potential were also apparent. These data suggest that the distribution, abundance and physiological capacity of these communities is strongly governed by chemical and physical variability of the environment.

  17. Large-scale expansion of pre-isolated bone marrow mesenchymal stromal cells in serum-free conditions.

    PubMed

    Gottipamula, Sanjay; Muttigi, Manjunatha S; Chaansa, S; Ashwin, K M; Priya, Nancy; Kolkundkar, Udaykumar; SundarRaj, Swathi; Majumdar, Anish Sen; Seetharam, Raviraja N

    2016-02-01

    The regenerative potential of mesenchymal stromal or stem cells (MSCs) has generated tremendous interest for treating various degenerative diseases. Regulatory preference is to use a culture medium that is devoid of bovine components for stem cell expansion intended for therapeutic applications. However, a clear choice an alternative to fetal bovine serum (FBS) has not yet emerged. We have screened five different commercially available serum-free media (SFM) for their ability to support the growth and expansion of pre-isolated undifferentiated bone marrow-derived MSCs (BM-MSCs) and compared the results with cells grown in standard FBS-containing medium as control. In addition, based on initial screening results, BD Mosaic™ Mesenchymal Stem Cell Serum-free (BD-SFM) medium was evaluated in large-scale cultures for the performance and culture characteristics of BM-MSCs. Of the five different serum-free media, BD-SFM enhanced BM-MSCs growth and expansion in Cell STACK (CS), but the cell yield per CS-10 was less when compared to the control medium. The characteristics of MSCs were measured in terms of population doubling time (PDT), cell yield and expression of MSC-specific markers. Significant differences were observed between BD-SFM and control medium in terms of population doublings (PDs), cell yield, CFU-F and morphological features, whereas surface phenotype and differentiation potentials were comparable. The BD-SFM-cultured MSCs were also found to retain the differentiation potential, immune-privileged status and immunosuppressive properties inherent to MSCs. Our results suggest that BD-SFM supports large-scale expansion of BM-MSCs for therapeutic use. PMID:23495227

  18. Tenascin C Promotes Hematoendothelial Development and T Lymphoid Commitment from Human Pluripotent Stem Cells in Chemically Defined Conditions

    PubMed Central

    Uenishi, Gene; Theisen, Derek; Lee, Jeong-Hee; Kumar, Akhilesh; Raymond, Matt; Vodyanik, Maxim; Swanson, Scott; Stewart, Ron; Thomson, James; Slukvin, Igor

    2014-01-01

    Summary The recent identification of hemogenic endothelium (HE) in human pluripotent stem cell (hPSC) cultures presents opportunities to investigate signaling pathways that are essential for blood development from endothelium and provides an exploratory platform for de novo generation of hematopoietic stem cells (HSCs). However, the use of poorly defined human or animal components limits the utility of the current differentiation systems for studying specific growth factors required for HE induction and manufacturing clinical-grade therapeutic blood cells. Here, we identified chemically defined conditions required to produce HE from hPSCs growing in Essential 8 (E8) medium and showed that Tenascin C (TenC), an extracellular matrix protein associated with HSC niches, strongly promotes HE and definitive hematopoiesis in this system. hPSCs differentiated in chemically defined conditions undergo stages of development similar to those previously described in hPSCs cocultured on OP9 feeders, including the formation of VE-Cadherin+CD73−CD235a/CD43− HE and hematopoietic progenitors with myeloid and T lymphoid potential. PMID:25448067

  19. Fluoroquinolones as chemical tools to define a strategy for photogenotoxicity in vitro assessment.

    PubMed

    Marrot, L; Belaidi, J P; Chaubo, C; Meunier, J R; Perez, P; Agapakis-Causse, C

    2001-04-01

    Today's lifestyle is often associated with frequent exposure to sunlight, but some xenobiotics used in drugs, cosmetics or food chemicals can produce adverse biological effects when irradiated. In particular, they can increase the risk of photogenotoxicity already due to UV radiation itself. There is thus a need to design appropriate approaches in order to obtain relevant data at the molecular and cellular level in this field. For ethical and practical reasons, in vitro models can be very convenient at least for first evaluation tests. Here, we propose a strategy based on complementary experiments to study the photogenotoxic potential of a compound. The fluoroquinolones BAYy3118 and lomefloxacin were used as standards to demonstrate the performance of each test: photoinduced interaction with supercoiled circular DNA, photomutagenicity in the yeast Saccharomyces cerevisae, induction of DNA photodamage in cultured human skin cells as revealed by comet assay, and finally induction of specific phototoxic stress responses such as p53 activation or melanogenesis stimulation. Such a strategy should help to ensure the safety of products likely to undergo environmental sunlight exposure. PMID:11287172

  20. A Molecular and Chemical Perspective in Defining Melatonin Receptor Subtype Selectivity

    PubMed Central

    Chan, King Hang; Wong, Yung Hou

    2013-01-01

    Melatonin is primarily synthesized and secreted by the pineal gland during darkness in a normal diurnal cycle. In addition to its intrinsic antioxidant property, the neurohormone has renowned regulatory roles in the control of circadian rhythm and exerts its physiological actions primarily by interacting with the G protein-coupled MT1 and MT2 transmembrane receptors. The two melatonin receptor subtypes display identical ligand binding characteristics and mediate a myriad of signaling pathways, including adenylyl cyclase inhibition, phospholipase C stimulation and the regulation of other effector molecules. Both MT1 and MT2 receptors are widely expressed in the central nervous system as well as many peripheral tissues, but each receptor subtype can be linked to specific functional responses at the target tissue. Given the broad therapeutic implications of melatonin receptors in chronobiology, immunomodulation, endocrine regulation, reproductive functions and cancer development, drug discovery and development programs have been directed at identifying chemical molecules that bind to the two melatonin receptor subtypes. However, all of the melatoninergics in the market act on both subtypes of melatonin receptors without significant selectivity. To facilitate the design and development of novel therapeutic agents, it is necessary to understand the intrinsic differences between MT1 and MT2 that determine ligand binding, functional efficacy, and signaling specificity. This review summarizes our current knowledge in differentiating MT1 and MT2 receptors and their signaling capacities. The use of homology modeling in the mapping of the ligand-binding pocket will be described. Identification of conserved and distinct residues will be tremendously useful in the design of highly selective ligands. PMID:24018885

  1. Rate and yield relationships in the production of xanthan gum by batch fermentations using complex and chemically defined growth media

    SciTech Connect

    Pinches, A.; Pallent, L.J.

    1986-10-01

    Rate and yield information relating to biomass and product formation and to nitrogen, glucose and oxygen consumption are described for xanthan gum batch fermentations in which both chemically defined (glutamate nitrogen) and complex (peptone nitrogen) media are employed. Simple growth and product models are used for data interpretation. For both nitrogen sources, rate and yield parameter estimates are shown to be independent of initial nitrogen concentrations. For stationary phases, specific rates of gum production are shown to be independent of nitrogen source but dependent on initial nitrogen concentration. The latter is modeled empirically and suggests caution in applying simple product models to xanthan gum fermentations. 13 references.

  2. An inverse small molecule screen to design a chemically defined medium supporting long-term growth of Drosophila cell lines

    PubMed Central

    Burnette, M.; Brito-Robinson, T.; Li, J.; Zartman, J.

    2015-01-01

    Drosophila cell culture is used as a model system with multiple applications including the identification of new therapeutic targets in screens, the study of conserved signal transduction pathway mechanisms, and as an expression system for recombinant proteins. However, in vitro methods for Drosophila cell and organ culture are relatively undeveloped. To characterize the minimal requirements for long-term maintenance of Drosophila cell lines, we developed an inverse screening strategy to identify small molecules and synergies stimulating proliferation in a chemically defined medium. In this chemical-genetics approach, a compound-protein interaction database is used to systematically score genetic targets on a screen-wide scale to extract further information about cell growth. In the pilot screen, we focused on two well-characterized cell lines, Clone 8 (Cl.8) and Schneider 2 (S2). Validated factors were investigated for their ability to maintain cell growth over multiple passages in the chemically defined medium (CDM). The polyamine spermidine proved to be the critical component that enables the CDM to support the long-term maintenance of Cl.8 cells. Spermidine supplementation upregulates DNA synthesis for Cl.8 and S2 cells and increases MAPK signaling for Cl.8 cells. The CDM also supports the long-term growth of Kc167 cells. Our target scoring approach validated the importance of polyamines, with enrichment for multiple polyamine ontologies found for both cell lines. Future iterations of the screen will enable the identification of compound combinations optimized for specific applications—maintenance and generation of new cell lines or the production and purification of recombinant proteins— thus increasing the versatility of Drosophila cell culture as both a genetic and biochemical model system. Our cumulative target scoring approach improves on traditional chemical-genetics methods and is extensible to biological processes in other species. PMID:25096480

  3. Transient transfection of serum-free suspension HEK 293 cell culture for efficient production of human rFVIII

    PubMed Central

    2011-01-01

    Background Hemophilia A is a bleeding disorder caused by deficiency in coagulation factor VIII. Recombinant factor VIII (rFVIII) is an alternative to plasma-derived FVIII for the treatment of hemophilia A. However, commercial manufacturing of rFVIII products is inefficient and costly and is associated to high prices and product shortage, even in economically privileged countries. This situation may be solved by adopting more efficient production methods. Here, we evaluated the potential of transient transfection in producing rFVIII in serum-free suspension HEK 293 cell cultures and investigated the effects of different DNA concentration (0.4, 0.6 and 0.8 μg/106 cells) and repeated transfections done at 34° and 37°C. Results We observed a decrease in cell growth when high DNA concentrations were used, but no significant differences in transfection efficiency and in the biological activity of the rFVIII were noticed. The best condition for rFVIII production was obtained with repeated transfections at 34°C using 0.4 μg DNA/106 cells through which almost 50 IU of active rFVIII was produced six days post-transfection. Conclusion Serum-free suspension transient transfection is thus a viable option for high-yield-rFVIII production. Work is in progress to further optimize the process and validate its scalability. PMID:22115125

  4. Serum free light chain assays not total light chain assays are the standard of care to assess Monoclonal Gammopathies.

    PubMed

    Tietsche de Moraes Hungria, Vania; Allen, Syreeta; Kampanis, Petros; Soares, Elyara Maria

    2016-01-01

    The diagnosis of Multiple Myeloma is a challenge to the physician due to the non-specific symptoms (anemia, bone pain and recurrent infections) that are commonplace in the elderly population. However, early diagnosis is associated with less severe disease, including fewer patients presenting with acute renal injury, pathological fractures and severe anemia. Since 2006, the serum free light chain test Freelite(®) has been included alongside standard laboratory tests (serum and urine protein electrophoresis, and serum and urine immunofixation) as an aid in the identification of monoclonal proteins, which are a cornerstone for the diagnosis of Multiple Myeloma. The serum free light chain assay recognizes the light chain component of the immunoglobulin in its free form with high sensitivity. Other assays that measure light chains in the free and intact immunoglobulin forms are sensitive, but unfortunately, due to the nomenclature used, these assays (total light chains) are sometimes used in place of the free light chain assay. This paper reviews the available literature comparing the two assays and tries to clarify hypothetical limitations of the total assay to detect Multiple Myeloma. Furthermore, we elaborate on our study comparing the two assays used in 11 Light Chain Multiple Myeloma patients at presentation and 103 patients taken through the course of their disease. The aim of this article is to provide a clear discrimination between the two assays and to provide information to physicians and laboratory technicians so that they can utilize the International Myeloma Working Group guidelines. PMID:26969773

  5. Serum free light chain assays not total light chain assays are the standard of care to assess Monoclonal Gammopathies

    PubMed Central

    Tietsche de Moraes Hungria, Vania; Allen, Syreeta; Kampanis, Petros; Soares, Elyara Maria

    2016-01-01

    The diagnosis of Multiple Myeloma is a challenge to the physician due to the non-specific symptoms (anemia, bone pain and recurrent infections) that are commonplace in the elderly population. However, early diagnosis is associated with less severe disease, including fewer patients presenting with acute renal injury, pathological fractures and severe anemia. Since 2006, the serum free light chain test Freelite® has been included alongside standard laboratory tests (serum and urine protein electrophoresis, and serum and urine immunofixation) as an aid in the identification of monoclonal proteins, which are a cornerstone for the diagnosis of Multiple Myeloma. The serum free light chain assay recognizes the light chain component of the immunoglobulin in its free form with high sensitivity. Other assays that measure light chains in the free and intact immunoglobulin forms are sensitive, but unfortunately, due to the nomenclature used, these assays (total light chains) are sometimes used in place of the free light chain assay. This paper reviews the available literature comparing the two assays and tries to clarify hypothetical limitations of the total assay to detect Multiple Myeloma. Furthermore, we elaborate on our study comparing the two assays used in 11 Light Chain Multiple Myeloma patients at presentation and 103 patients taken through the course of their disease. The aim of this article is to provide a clear discrimination between the two assays and to provide information to physicians and laboratory technicians so that they can utilize the International Myeloma Working Group guidelines. PMID:26969773

  6. Comparison of serum free light chain and urine electrophoresis for the detection of the light chain component of monoclonal immunoglobulins in light chain and intact immunoglobulin multiple myeloma

    PubMed Central

    Dejoie, Thomas; Attal, Michel; Moreau, Philippe; Harousseau, Jean-Luc; Avet-Loiseau, Herve

    2016-01-01

    Response criteria for multiple myeloma are based upon changes in monoclonal protein levels quantified using serum and/or urine protein electrophoresis. The latter lacks sensitivity at low monoclonal protein levels and since 2001, the serum free light chain test has been available and its clinical utility proven, yet guidelines have not recommended it as a replacement for urine assessment. Herein we evaluated responses using serum free light chain measurements and serum and urine electrophoresis after 2 and 4 cycles of therapy and after stem cell transplantation in 25 light chain and 157 intact immunoglobulin myeloma patients enrolled in the IFM 2007-02 MM trial. All 25 light chain patients had measurable disease by serum free light chain and urine methods at presentation. By contrast 98 out of 157 intact immunoglobulin patients had measurable disease by serum free light chain compared to 55 out of 157 by urine electrophoresis. In all patients there was substantial agreement between predicate (serum/urine protein electrophoresis) and test (serum protein electrophoresis and serum free light chain) methods for response assessment (Weighted Kappa=0.83). Urine immunofixation became negative in 47% light chain and 43% intact immunoglobulin patients after 2 cycles of therapy. At this time the serum free light chain ratio normalised in only 11% and 27% patients, respectively. In summary we found good agreement between methods for response assessment, but the serum free light chain test provided greater sensitivity than urine electrophoresis for monitoring. To our knowledge this is the first report comparing both methods for response assignment based on the International Myeloma Working Group guidelines. PMID:26635032

  7. Identification of Neisseria by electron capture gas-liquid chromatography of metabolites in a chemically defined growth medium.

    PubMed Central

    Morse, C D; Brooks, J B; Kellogg, D S

    1977-01-01

    A dual-purpose study was carried out in an attempt to develop a rapid, sensitive method to identify Neisseria species by gas chromatography and to learn more about the metabolism of these organisms. Sixty-nine isolates of Neisseria were grown in a chemically defined fluid medium; the spent medium was extracted sequentially at pH 2 with diethyl ether and at pH 10 with chloroform. The pH 10 extracts were derivatized with heptafluorobutyric anhydride and analyzed by electron capture gas-liquid chromatography. The resulting spent culture medium electron capture gas-liquid chromatography profiles showed several qualitative and significant quantitative differences among the Neisseria species potentially useful in separating and identifying these organisms. Putrescine and cadaverine which were present in the spent culture medium of some Neisseria, including N. gonorrhoeae, were tentatively identified. Substituting carbohydrates for the chemically defined medium containing glucose in the base medium produced altered profiles with increased quantitative and qualitative differences. PMID:21889

  8. Development of a chemically defined serum- and protein-free medium for growth of human peripheral lymphocytes.

    PubMed

    Shive, W; Pinkerton, F; Humphreys, J; Johnson, M M; Hamilton, W G; Matthews, K S

    1986-01-01

    A chemically defined, protein-free medium (designated CFBI 1000, where CFBI = Clayton Foundation Biochemical Institute) that supports human peripheral lymphocyte proliferation has been developed. This medium allows exploration of individual metabolic differences by varying the medium composition as well as providing a base to explore further the mechanisms of lymphocyte activation in a system initially free of added macromolecular species other than mitogen. The peripheral blood lymphocyte is an ideal system for metabolic studies because it is easily obtained, is a primary resting cell that can be activated to proliferate, and presumably reflects both the genetic makeup and biochemical environmental history of the individual at the time the cells were formed. Examination of the role of various factors in lymphocyte activation and subsequent events may be simplified by the utilization of a medium that is protein-free and chemically defined. The CFBI 1000 medium supports the growth response of human peripheral lymphocytes to mitogen as measured by [3H]thymidine incorporation to an extent comparable to other media used widely in assessment of lymphocyte proliferation. PMID:3079905

  9. A genotype of modified vaccinia Ankara (MVA) that facilitates replication in suspension cultures in chemically defined medium.

    PubMed

    Jordan, Ingo; Horn, Deborah; John, Katrin; Sandig, Volker

    2013-01-01

    While vectored vaccines, based on hyperattenuated viruses, may lead to new treatment options against infectious diseases and certain cancers, they are also complex products and sometimes difficult to provide in sufficient amount and purity. To facilitate vaccine programs utilizing host-restricted poxviruses, we established avian suspension cell lines (CR and CR.pIX) and developed a robust, chemically defined, culturing process for production of this class of vectors. For one prominent member, modified vaccinia Ankara (MVA), we now describe a new strain that appears to replicate to greater yields of infectious units, especially in the cell-free supernatant of cultures in chemically defined media. The new strain was obtained by repeated passaging in CR suspension cultures and, consistent with reports on the exceptional genetic stability of MVA, sequencing of 135 kb of the viral genomic DNA revealed that only three structural proteins (A3L, A9L and A34R) each carry a single amino acid exchange (H639Y, K75E and D86Y, respectively). Host restriction in a plaque-purified isolate of the new genotype appears to be maintained in cell culture. Processing towards an injectable vaccine preparation may be simplified with this strain as a complete lysate, containing the main burden of host cell contaminants, may not be required anymore to obtain adequate yields. PMID:23337383

  10. Effect of commercially available chemically defined liquid diets on the intestinal microflora and bacterial translocation from the gut.

    PubMed

    Alverdy, J C; Aoys, E; Moss, G S

    1990-01-01

    The effect of chemically defined liquid diets on the intestinal microflora and bacterial translocation from the gut was studied in the rat. Seventy-five female Fischer rats were randomized to five groups of 15 animals each. Group I was fed rat chow and water, group II was fed Vivonex TEN, group III was fed Ensure, group IV was fed Enrich, and group V was fed Ensure plus ground corn cobs, a crude fiber source. Animals were fed their respective diets ad libitum for 1 week and then killed. Quantitative culture of the mesenteric lymph nodes (MLN) and cecum was performed to determine bacterial translocation from the gut. A 66% translocation rate (10/15) of bacteria to MLN was observed in the animals fed Ensure and Enrich compared to 21% in the Vivonex TEN group (3/14) and 20% in the animals fed Ensure plus ground corn cobs (3/15). None of the animals in the control group eating their normal diets of rat chow and water developed positive MLN. Statistical significance (p less than 0.001) was achieved between the Ensure and Enrich groups when compared to controls but not between the Vivonex TEN and Ensure plus corn cobs. Cecal culture revealed a statistically significant rise in cecal bacteria in all groups when compared to the control group (group I). These results indicate that chemically defined liquid diets result in altered intestinal microflora and bacterial translocation from the gut. PMID:2325237

  11. Benefits and Limitations of Protein Hydrolysates as Components of Serum-Free Media for Animal Cell Culture Applications

    NASA Astrophysics Data System (ADS)

    Lobo-Alfonso, Juliet; Price, Paul; Jayme, David

    Increased understanding of influential factors for the cultivation of animal cells, combined with heightened regulatory concern over potential transmission of adventitious contaminants associated with serum and other animal-derived components, has elevated interest in using protein hydrolysates as serum replacements or nutrient supplements. This paper reviews the chemistry and biology of various hydrolysates derived from animal, plant and microbial sources. It provides specific examples of a beneficial selection of plant and yeast hydrolysates as ingredients of serum-free nutrient formulations for bioproduction applications of cultured mammalian and insect cells. Strategies for customizing and optimizing nutrients for specialized applications and general benefits and limitations of protein hydrolysates for biopharmaceutical production are also discussed.

  12. Characterization of a subpopulation of developing cortical interneurons from human iPSCs within serum-free embryoid bodies

    PubMed Central

    Jacob, Samson; Sun, Bruce; Prè, Deborah; Sproul, Andrew A.; Hong, Seong Im; Woodard, Chris; Zimmer, Matthew; Chinchalongporn, Vorapin; Arancio, Ottavio; Noggle, Scott A.

    2014-01-01

    Production and isolation of forebrain interneuron progenitors are essential for understanding cortical development and developing cell-based therapies for developmental and neurodegenerative disorders. We demonstrate production of a population of putative calretinin-positive bipolar interneurons that express markers consistent with caudal ganglionic eminence identities. Using serum-free embryoid bodies (SFEBs) generated from human inducible pluripotent stem cells (iPSCs), we demonstrate that these interneuron progenitors exhibit morphological, immunocytochemical, and electrophysiological hallmarks of developing cortical interneurons. Finally, we develop a fluorescence-activated cell-sorting strategy to isolate interneuron progenitors from SFEBs to allow development of a purified population of these cells. Identification of this critical neuronal cell type within iPSC-derived SFEBs is an important and novel step in describing cortical development in this iPSC preparation. PMID:25394470

  13. Inducible packaging cells for large-scale production of lentiviral vectors in serum-free suspension culture.

    PubMed

    Broussau, Sophie; Jabbour, Nadine; Lachapelle, Guillaume; Durocher, Yves; Tom, Rosanne; Transfiguracion, Julia; Gilbert, Rénald; Massie, Bernard

    2008-03-01

    We have developed new packaging cell lines (293SF-PacLV) that can produce lentiviral vectors (LVs) in serum-free suspension cultures. A cell line derived from 293SF cells, expressing the repressor (CymR) of the cumate switch and the reverse transactivator (rtTA2(S)-M2) of the tetracycline (Tet) switch, was established first. We next generated clones stably expressing the Gag/Pol and Rev genes of human immunodeficiency virus-1, and the glycoprotein of vesicular stomatitis virus (VSV-G). Expression of Rev and VSV-G was tightly regulated by the cumate and Tet switches. Our best packaging cells produced up to 2.6 x 10(7) transducing units (TU)/ml after transfection with the transfer vector. Up to 3.4 x 10(7) TU/ml were obtained using stable producers generated by transducing the packaging cells with conditional-SIN-LV. The 293SF-PacLV was stable, as shown by the fact that some producers maintained high-level LV production for 18 weeks without selective pressure. The utility of the 293SF-PacLV for scaling up production in serum-free medium was demonstrated in suspension cultures and in a 3.5-L bioreactor. In shake flasks, the best packaging cells produced between 3.0 and 8.0 x 10(6) TU/ml/day for 3 days, and the best producer cells, between 1.0 and 3.4 x 10(7) TU/ml/day for 5 days. In the bioreactor, 2.8 liters containing 2.0 x 10(6) TU/ml was obtained after 3 days of batch culture following the transfection of packaging cells. In summary, the 293SF-PacLV possesses all the attributes necessary to become a valuable tool for scaling up LV production for preclinical and clinical applications. PMID:18180776

  14. Immobilization of sericin molecules via amorphous carbon plasma modified-polystyrene dish for serum-free culture

    NASA Astrophysics Data System (ADS)

    Tunma, Somruthai; Song, Doo-Hoon; Kim, Si-Eun; Kim, Kyoung-Nam; Han, Jeon-Geon; Boonyawan, Dheerawan

    2013-10-01

    In this study, we focused on sericin hydrolysates, originating from silkworm used in serum-free human bone marrow-derived mesenchymal stem cells (hBM-MSCs) culture. We reported the effect of a covalent linkage between a bioactive protein molecule and polystyrene dish surface via a carbon intermediate layer which can slow down the release rate of protein compounds into the phosphate buffer saline (PBS) solution. Films of amorphous carbon (a-C) and functionalized-carbon were deposited on PS culture dish surfaces by using a DC magnetron sputtering system and RF PECVD system. We found that a-C based-films can increase the hydrophilicity and biocompatibility of polystyrene (PS) dishes, especially a-C films and a-C:N2 films showed good attachment of hBM-MSCs at 24 h. However, in the case of silica surface (a-C:SiOx films), the cells showed a ragged and unattached boundary resulting from the presence of surface silanol groups. For the UV-vis absorbance, all carbon modified-PS dishes showed a lower release rate of sericin molecules into PBS solution than PS control. This revealed that the functionalized carbon could be enhanced by specific binding properties with given molecules. The carbon-coated PS dishes grafting with sericin protein were used in a serum-free condition. We also found that hBM-MSCs have higher percentage of proliferated cells at day 7 for the modified dishes with carbon films and coated with sericin than the PS control coated with sericin. The physical film properties were measured by atomic force microscopy (AFM), scanning electron microscope (SEM) and contact angle measurement. The presence of sbnd NH2 groups of sericin compounds on the PS dish was revealed by Fourier transform infrared spectroscopy (FTIR). The stability of covalent bonds of sericin molecules after washing out ungrafted sericin was confirmed by X-ray photoelectron spectroscopy (XPS).

  15. Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins.

    PubMed

    Watanabe, Masaaki; Zemack, Helen; Johansson, Helene; Hagbard, Louise; Jorns, Carl; Li, Meng; Ellis, Ewa

    2016-01-01

    Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of heterotrimeric basement membrane adhesion proteins, are highly cell and tissue type specific components of the extracellular matrix and strongly influence the behavior and function of associated cells and/or tissues. However, detailed biological functions of many laminin isoforms are still to be evaluated. In this study, we determined the distribution of laminin isoforms in human liver tissue and isolated primary human hepatocytes by western blot analysis, and investigated the efficacy of different human recombinant laminin isoforms on hepatic functions during culture. Protein expressions of laminin-chain α2, α3, α4, β1, β3, γ1, and γ2 were detected in both isolated human hepatocytes and liver tissue. No α1 and α5 expression could be detected in liver tissue or hepatocytes. Hepatocytes were isolated from five different individual livers, and cultured on human recombinant laminin isoforms -111, -211, -221, -332, -411, -421, -511, and -521 (Biolamina AB), matrigel (extracted from Engelbreth-Holm-Swarm sarcoma), or collagen type IV (Collagen). Hepatocytes cultured on laminin showed characteristic hexagonal shape in a flat cell monolayer. Viability, double stranded DNA concentration, and Ki67 expression for hepatocytes cultured for six days on laminin were comparable to those cultured on EHS and Collagen. Hepatocytes cultured on laminin also displayed production of human albumin, alpha-1-antitrypsin, bile acids, and gene expression of liver-enriched factors, such as hepatocyte nuclear factor 4 alpha, glucose-6-phosphate, cytochrome P450 3A4, and multidrug resistance-associated protein 2. We conclude that all forms of human recombinant laminin tested maintain cell viability and liver-specific functions of primary human

  16. Acidification in the Adirondacks: defining the biota in trophic levels of 30 chemically diverse acid-impacted lakes.

    PubMed

    Nierzwicki-Bauer, Sandra A; Boylen, Charles W; Eichler, Lawrence W; Harrison, James P; Sutherland, James W; Shaw, William; Daniels, Robert A; Charles, Donald F; Acker, Frank W; Sullivan, Timothy J; Momen, Bahram; Bukaveckas, Paul

    2010-08-01

    The Adirondack Mountains in New York State have a varied surficial geology and chemically diverse surface waters that are among the most impacted by acid deposition in the U.S. No single Adirondack investigation has been comprehensive in defining the effects of acidification on species diversity, from bacteria through fish, essential for understanding the full impact of acidification on biota. Baseline midsummer chemistry and community composition are presented for a group of chemically diverse Adirondack lakes. Species richness of all trophic levels except bacteria is significantly correlated with lake acid-base chemistry. The loss of taxa observed per unit pH was similar: bacterial genera (2.50), bacterial classes (1.43), phytoplankton (3.97), rotifers (3.56), crustaceans (1.75), macrophytes (3.96), and fish (3.72). Specific pH criteria were applied to the communities to define and identify acid-tolerant (pH<5.0), acid-resistant (pH 5.0-5.6), and acid-sensitive (pH>5.6) species which could serve as indicators. Acid-tolerant and acid-sensitive categories are at end-points along the pH scale, significantly different at P<0.05; the acid-resistant category is the range of pH between these end-points, where community changes continually occur as the ecosystem moves in one direction or another. The biota acid tolerance classification (batc) system described herein provides a clear distinction between the taxonomic groups identified in these subcategories and can be used to evaluate the impact of acid deposition on different trophic levels of biological communities. PMID:20614900

  17. A chemically defined culture medium containing Rho kinase inhibitor Y-27632 for the fabrication of stratified squamous epithelial cell grafts

    SciTech Connect

    Aslanova, Afag; Takagi, Ryo; Yamato, Masayuki; Okano, Teruo; Yamamoto, Masakazu

    2015-05-01

    With the development of a culture method for stratified squamous epithelial cells, tissue-engineered epithelial cell sheets have been successfully applied as clinical cell grafts. However, the implementation of these cell sheets without the use of any animal-derived materials is highly desirable. In this study, Rho-associated protein kinase inhibitor Y-27632 was used to develop a chemically defined culture medium for the fabrication of stratified epithelial cell grafts consisting of human epidermal and oral keratinocytes, and the proliferation activity, cell morphology, and gene expressions of the keratinocytes were analyzed. The results of a colorimetric assay indicated that Y-27632 significantly promoted the proliferation of the keratinocytes in culture media both with and without fetal bovine serum (FBS), although there were no indications of Y-27632 efficacy on cell morphology and stratification of the keratinocytes in culture medium without any animal-derived materials. The results of quantitative RT-PCR revealed that gene expressions correlated with cell adhesion, cell–cell junction, proliferation markers, and stem/progenitor markers in cultured keratinocytes were not strongly affected by the addition of Y-27632 to the culture medium. Moreover, gene expressions of differentiation markers in stratified keratinocytes cultured in medium without FBS were nearly identical to those of keratinocytes co-cultured with 3T3 feeder cells. Interestingly, the expressions of differentiation markers in cultured stratified keratinocytes were suppressed by FBS, whereas they were reconstructed by either co-culture of a 3T3 feeder layer or addition of Y-27632 into the culture medium containing FBS. These findings indicate that Y-27632 is a useful supplement for the development of a chemically defined culture medium for fabrication of stratified epithelial cell grafts for clinical applications for the purpose of developing the culture medium with a lower risk of pathogen

  18. A phosphodiesterase type-5 inhibitor, sildenafil, induces sperm capacitation and penetration into porcine oocytes in a chemically defined medium.

    PubMed

    Ioki, Sumire; Wu, Qing-Shan; Takayama, Osamu; Motohashi, Hideyuki H; Wakai, Takuya; Funahashi, Hiroaki

    2016-02-01

    The present study was undertaken to determine the effect of a phosphodiesterase (PDE) type-5 (cyclic guanosine monophosphate-specific) inhibitor, sildenafil, on capacitation and penetration of boar spermatozoa in a basic chemically defined medium (adenosine- and theophylline-free PGM-tac4). When ejaculated spermatozoa were cultured for 90 minutes in the absence or presence of sildenafil at 2.5 mM, the inhibitor significantly increased the percentage of capacitated/acrosome-reacted spermatozoa, as a result of the chlortetracycline assay. When fresh spermatozoa were co-cultured with oocytes in the presence of sildenafil at a different concentration (0, 2.5, 25, or 250 μM), higher sildenafil concentrations (25 and 250 μM) significantly resulted in higher sperm penetration rates. When oocytes matured in vitro were co-cultured with spermatozoa in the presence of 25 μM sildenafil or 25 mM caffeine benzoate for 8 hours, the incidence of penetrated oocytes did not differ between two groups, whereas the incidence of monospermic oocytes in penetrated one was significantly higher in the presence of sildenafil. Immunocytochemical analysis reported the presence of PDE type-5 on the acrosome region of boar spermatozoa. These results report that regulation of cyclic guanosine monophosphate-specific PDE type-5 by sildenafil somehow can increase the penetrability of boar spermatozoa in vitro. PMID:26443234

  19. Growth enhancement of the halotolerant Brevibacterium sp. JCM 6894 by methionine externally added to a chemically defined medium.

    PubMed

    Mimura, Haruo

    2014-01-01

    We examined amino acid requirements for the growth of the halotolerant Brevibacterium sp. JCM 6894 in the absence and presence of 1.2 M NaCl in a chemically defined medium. The experiment was also carried out in the presence of 1.2 M KCl. As a result, growth was highly enhanced by methionine in the absence and presence of KCl as well as NaCl up to 1.2 M. However, growth in the presence of 150 mM methionine was repressed by leucine (up to 100 mM)and valine (up to 100 mM). Concentration-dependent growth inhibition was observed in the presence of isoleucine (up to 150 mM) and threonine (up to 300 mM). When the cells were incubated in the absence of externally added K+, growth was strongly repressed, even in the presence of 150 mM methionine. The growth, however, recovered drastically by the addition of 1 mM KCl, regardless of the presence and absence of 1.2 M NaCl. These results indicate that methionine, which seems to be symported into cytoplasm with K+, plays an important role in the growth of the strain under salt stress. PMID:25252648

  20. Silicon-on-glass pore network micromodels with oxygen-sensing fluorophore films for chemical imaging and defined spatial structure.

    PubMed

    Grate, Jay W; Kelly, Ryan T; Suter, Jonathan; Anheier, Norm C

    2012-11-21

    Pore network microfluidic models were fabricated by a silicon-on-glass technique that provides the precision advantage of dry etched silicon while creating a structure that is transparent across all microfluidic channels and pores, and can be imaged from either side. A silicon layer is bonded to an underlying borosilicate glass substrate and thinned to the desired height of the microfluidic channels and pores. The silicon is then patterned and through-etched by deep reactive ion etching (DRIE), with the underlying glass serving as an etch stop. After bonding on a transparent glass cover plate, one obtains a micromodel in oxygen impermeable materials with water-wet surfaces where the microfluidic channels are transparent and structural elements such as the pillars creating the pore network are opaque. The advantageous features of this approach in a chemical imaging application are demonstrated by incorporating a Pt porphyrin fluorophore in a PDMS film serving as the oxygen-sensing layer and a bonding surface, or in a polystyrene film coated with a PDMS layer for bonding. The sensing of a dissolved oxygen gradient was demonstrated using fluorescence lifetime imaging, and it is shown that different matrix polymers lead to optimal use in different ranges of oxygen concentration. Imaging with the opaque pillars in between the observation direction and the continuous fluorophore film yields images that retain defined spatial structure in the sensor image. PMID:22995983

  1. Label-retaining assay enriches tumor-initiating cells in glioblastoma spheres cultivated in serum-free medium

    PubMed Central

    Zeng, Lingcheng; Zhao, Yiqing; Ouyang, Taohui; Zhao, Tianyuan; Zhang, Suojun; Chen, Jian; Yu, Jiasheng; Lei, Ting

    2016-01-01

    Label-retaining cells, which are characterized by dormancy or slow cycling, may be identified in a number of human normal and cancer tissues, and these cells demonstrate stem cell potential. In glioblastoma, label-retaining assays to enrich glioma stem cells remain to be fully investigated. In the present study, glioblastoma sphere cells cultured in serum-free medium were initially stained with the cell membrane fluorescent marker DiI. The fluorescence intensity during cell proliferation and sphere reformation was observed. At 2 weeks, the DiI-retaining cells were screened by fluorescence-activated cell sorting and compared phenotypically with the DiI-negative cells in terms of in vitro proliferation, clonogenicity and multipotency and for in vivo tumorigenicity, as well as sensitivity to irradiation and temozolomide treatment. It was observed that DiI-retaining cells accounted for a small proportion, <10%, within the glioblastoma spheres and that DiI-retaining cells proliferated significantly more slowly compared with DiI-negative cells (P=0.011, P=0.035 and P=0.023 in the of NCH421k, NCH441 and NCH644 glioblastoma sphere cell lines). Significantly increased clonogenicity (P=0.002, P=0.034 and P=0.016 in the NCH441, NCH644 and NCH421k glioblastoma sphere cell lines) and three-lineage multipotency were observed in DiI-retaining cells in vitro compared with DiI-negative cells. As few as 100 DiI-retaining cells were able to effectively generate tumors in the immunocompromised mouse brain, whereas the same number of DiI-negative cells possessed no such ability, indicating the increased tumorigenicity of DiI-retaining cells compared with DiI-negative cells. Furthermore, DiI-retaining cells demonstrated significant resistance following irradiation (P=0.012, P=0.024 and P=0.036) and temozolomide (P=0.003, P=0.005 and P=0.029) compared with DiI-negative cells in the NCH421k, NCH441 and NCH644 glioblastoma sphere cell lines, respectively. It was concluded that label

  2. Application of a serum-free medium for the growth of Vero cells and the production of reovirus.

    PubMed

    Butler, M; Burgener, A; Patrick, M; Berry, M; Moffatt, D; Huzel, N; Barnabé, N; Coombs, K

    2000-01-01

    Two strains of reovirus (serotype 1 Lang/TIL and serotype 3 Dearing/T3D) were propagated in Vero cells grown in stationary or agitated cultures in a serum-free medium, M-VSFM. Solid microcarriers (Cytodex-1) were used to support cell growth in agitated cultures with a normal doubling time of 25 h. Cell yields of 1 x 10(6) cells/mL were obtained from an inoculum of 2 x 10(5) cells/mL in 4 days in microcarrier cultures. The growth profile and cell yield was not significantly different from serum-supplemented cultures. The virus titer increased by 3-4 orders of magnitude over a culture period of 150 h. The maximum virus titer in stationary cultures reached >1 x 10(9) pfu/mL for both strains of reovirus in M-VSFM. M-VSFM also supported high viral yields in microcarrier cultures. Both the specific productivity and final viral yield was higher in M-VSFM than serum-supplemented cultures. The high viral productivity suggests that this is a suitable system for the production of reovirus as an oncolytic agent for human therapeutic use. PMID:11027181

  3. IgD multiple myeloma: Clinical, biological features and prognostic value of the serum free light chain assay.

    PubMed

    Djidjik, R; Lounici, Y; Chergeulaïne, K; Berkouk, Y; Mouhoub, S; Chaib, S; Belhani, M; Ghaffor, M

    2015-09-01

    IgD multiple myeloma (MM) is a rare subtype of myeloma, it affects less than 2% of patients with MM. To evaluate the clinical and prognostic attributes of serum free light chains (sFLCs) analysis, we examined 17 cases of IgD MM. From 1998 to 2012, we obtained 1250 monoclonal gammapathies including 590 multiple myeloma and 17 patients had IgD MM. With preponderance of men patients with a mean age at diagnosis of: 59±12years. Patients with IgD MM have a short survival (Median survival=9months). The presenting features included: bone pain (75%), lymphadenopathy (16%), hepatomegaly (25%), splenomegaly (8%), associated AL amyloidosis (6%), renal impairment function (82%), infections (47%), hypercalcemia (37%) and anemia (93%). Serum electrophoresis showed a subtle M-spike (Mean=13.22±10g/L) in all patients associated to a hypogammaglobulinemia. There was an over-representation of Lambda light chain (65%); high serum β2-microglobulin in 91% and Bence Jones proteinuria was identified in 71%. The median rate of sFLCs κ was 19.05mg/L and 296.75mg/L for sFLCs λ. sFLCR was abnormal in 93% of patients and it showed concordance between baseline sFLCR and the survival (P=0.034). The contribution of FLC assay is crucial for the prognosis of patients with IgD MM. PMID:26294067

  4. Derivation of embryonic stem cells from Kunming mice IVF blastocyst in feeder- and serum-free condition.

    PubMed

    Liu, Xiaokun; Wei, Qiang; Zhang, Junhong; Yang, Wanli; Zhao, Xiaoe; Ma, Baohua

    2015-06-01

    Kunming mice are widely used in China; however, it is difficult to isolate embryonic stem cells (ESCs) in conventional derivation condition containing feeder cells and serum. 6-Bromoindirubin-3'-oxime (BIO), a glycogen synthase kinase 3 (GSK3) inhibitor, could facilitate the maintenance of pluripotency of ESCs. Therefore, BIO could be considered as a candidate to replace feeder cells and serum. On the other hand, in vitro fertilization (IVF) is an important technology in assisted reproduction. It is reported that there was some difference in gene expression between IVF and in vivo developed blastocyst. ESCs derived from IVF blastocyst could provide a valuable tool to research the effect of IVF on differentiation and development. In the present study, we established two novel ESC lines from IVF blastocyst of Kunming mice in a feeder- and serum-free condition containing 2.5 μM BIO. In this condition, expanded IVF blastocyst could spontaneously hatch from zonae pellucidae and attached to the gelatin-coated bottom of dishes. ESC-like outgrowth could be observed without overfull trophoblast cells. After further propagation, two Kunming mice ESC lines, designated as KMES1 and KMES2, were obtained. These two novel ESCs shared common morphological characteristics with other rodent ESCs, showed strong alkaline phosphatase activity, and expressed pluripotent markers, including Oct-4, Nanog, and SSEA-1. Embryoid body (EB) and teratoma test indicated that these ESCs could spontaneously differentiate into cells representative of all three embryonic germ layers. PMID:25592083

  5. Ameloblasts serum-free conditioned medium: bone morphogenic protein 4-induced odontogenic differentiation of mouse induced pluripotent stem cells.

    PubMed

    Liu, Li; Liu, Ying-Feng; Zhang, Jing; Duan, Yin-Zhong; Jin, Yan

    2016-06-01

    Induced pluripotent stem (iPS) cells possess the ability of self-renewal and can differentiate into cells of the three germ layers, both in vitro and in vivo. Here we report a new method to efficiently induce differentiation of mouse iPS cells into the odontogenic lineage. Using ameloblasts serum-free conditioned medium (ASF-CM), we successfully generated ameloblast-like cells from mouse iPS cells. Importantly, culturing mouse iPS cells in ASF-CM supplemented with BMP4 (ASF-BMP4) promoted odontogenic differentiation, which was evident by the upregulation of ameloblast-specific as well as odontoblast-specific genes. On the other hand, culturing mouse iPS cells in ASF-CM supplemented with noggin (ASF-noggin), an inhibitor of BMP4, abrogated this effect. These results suggest that mouse iPS cells can be induced by ASF-BMP4 to differentiate into ameloblast-like and odontoblast-like cells. The results of our study raise the possibility of using patient-specific iPS cells for tooth regeneration in the future. Copyright © 2016 John Wiley & Sons, Ltd. PMID:23606575

  6. Use of a stationary bed reactor and serum-free medium for the production of recombinant proteins in insect cells.

    PubMed

    Kompier, R; Kislev, N; Segal, I; Kadouri, A

    1991-10-01

    Insect cells (Spodoptera frugiperda) have been cultured in a stationary bed reactor, packed with a fibrous polyester carrier. When the bioreactor was perfused with serum-supplemented medium, a cell density of 6 x 10(6) cells ml-1 packed carrier was reached. Scanning electron microscopy investigations have shown that the insect cells grew along the three-dimensionally oriented fibers of the Fibra-cel carrier. After infection of the logarithmically growing cells with a recombinant baculovirus (Autographa californica) containing the gene coding for beta-galactosidase, the medium in the bioreactor was changed to serum-free medium. At day 13 postinfection (p.i.), a beta-galactosidase level of 320 microgram ml-1 and, at day 17 p.i., a virus titer of 2.1 x 10(8) TCID50 units ml-1 (day 17 p.i.) were reached. In another bioreactor, operated in a similar way but with serum-containing medium, a beta-galactosidase concentration of 360 microgram ml-1 and a virus titer of 2.3 x 10(8) TCID50 units ml-1 were obtained. These results indicate the potential use of this production system for the production of recombinant protein and baculovirus in insect cells. PMID:1367637

  7. Medications that distort in vitro tests of thyroid function, with particular reference to estimates of serum free thyroxine.

    PubMed

    Stockigt, Jim R; Lim, Chen-Fee

    2009-12-01

    The combination of serum thyroid-stimulating hormone (TSH) with measurement of circulating thyroid hormones greatly improves sensitivity and specificity of thyroid diagnosis, but these assays are not impeccable. Estimation of serum free T4 conveniently accommodates variations in the concentration of thyroxine-binding globulin (TBG), but no current technique reliably reflects the in vivo free T4 concentration in numerous other situations. The effect of circulating competitors that increase T4 and T3 in vivo, in particular, many medications, is under-estimated by current free hormone estimates that involve sample dilution. Non-esterified fatty acids generated during sample storage and incubation can spuriously increase the measured free T4 estimate, especially after in vivo treatment with heparin. These artefacts are unlikely to be overcome by current assay strategies. Total serum T4, corrected for alterations in TBG concentration, gives a more robust estimate of thyroxine concentration than current methods of free hormone estimation and should now be reintroduced as the 'gold standard'. PMID:19942151

  8. Native chemical ligation for conversion of sequence-defined oligomers into targeted pDNA and siRNA carriers.

    PubMed

    Zhang, Can Yang; Kos, Petra; Müller, Katharina; Schrimpf, Waldemar; Troiber, Christina; Lächelt, Ulrich; Scholz, Claudia; Lamb, Don C; Wagner, Ernst

    2014-04-28

    Native chemical ligation (NCL) was established for the conversion of sequence-defined oligomers of different topologies into targeted and PEG shielded pDNA and siRNA carriers. From an existing library of non-targeted oligoethanamino amides, six oligomers containing N-terminal cysteines were selected as cationic cores, to which monodisperse polyethylene glycol (PEG) containing terminal folic acid as targeting ligand (or terminal alanine as targeting negative control ligand) were attached by NCL. Ligated conjugates plus controls (in sum 18 oligomers) were evaluated for pDNA or siRNA gene delivery. Biophysical characteristics including nucleic acid binding in the absence or presence of serum, as well as biological activities in cellular uptake and gene transfer (or gene silencing, respectively) were determined. In most cases, the folic acid-PEG-ligated oligomers displayed a strongly improved cellular binding, uptake and gene transfer into receptor-positive KB cells as compared to the alanine-PEG controls. Changing the topological structures by increasing the number of cationic arms, adding tyrosine trimers as polyplex stabilizing domains, or histidines facilitating endosomal escape resulted in beneficial gene transfer characteristics. The screen revealed different requirements for pDNA and siRNA delivery. A folate-PEG ligated histidinylated four-arm oligomer was most effective for pDNA delivery but inactive for siRNA, whereas a folate-PEG-ligated three-arm oligomer with tyrosine trimer modifications was most effective in siRNA mediated gene silencing. The results demonstrate the site-selective NCL reaction as powerful method to modify existing oligomers. Thus multifunctional targeted carriers can be obtained with ease and used to identify lead structures for subsequent in vivo delivery. PMID:24566255

  9. Susceptibility of Microsporum canis arthrospores to a mixture of chemically defined essential oils: a perspective for environmental decontamination.

    PubMed

    Nardoni, Simona; Tortorano, Annamaria; Mugnaini, Linda; Profili, Greta; Pistelli, Luisa; Giovanelli, Silvia; Pisseri, Francesca; Papini, Roberto; Mancianti, Francesca

    2015-01-01

    The zoophilic dermatophyte Microsporum canis has cats as natural reservoir, but it is able to infect a wide range of hosts, including humans, where different clinical features of the so-called ringworm dermatophytosis have been described. Human infections are increasingly been reported in Mediterranean countries. A reliable control program against M. canis infection in cats should include an antifungal treatment of both the infected animals and their living environment. In this article, a herbal mixture composed of chemically defined essential oils (EOs) of Litsea cubeba (1%), Illicium verum, Foeniculum vulgare, and Pelargonium graveolens (0.5% each) was formulated and its antifungal activity assessed against M. canis arthrospores which represent the infective environmental stage of M. canis. Single compounds present in higher amounts in the mixture were also separately tested in vitro. Litsea cubeba and P. graveolens EOs were most effective (minimum inhibitory concentration (MIC) 0.5%), followed by EOs of I. verum (MIC 2%) and F. vulgare (MIC 2.5%). Minimum fungicidal concentrations (MFC) values were 0.75% (L. cubeba), 1.5% (P. graveolens), 2.5% (I. verum) and 3% (F. vulgare). MIC and MFC values of the mixture were 0.25% and 0.5%, respectively. The daily spray of the mixture (200 μL) directly onto infected hairs inhibited fungal growth from the fourth day onwards. The compounds present in higher amounts exhibited variable antimycotic activity, with MIC values ranging from >10% (limonene) to 0.1% (geranial and neral). Thus, the mixture showed a good antifungal activity against arthrospores present in infected hairs. These results are promising for a further application of the mixture as an alternative tool or as an adjuvant in the environmental control of feline microsporosis. PMID:25854840

  10. Human vascular model with defined stimulation medium - a characterization study.

    PubMed

    Huttala, Outi; Vuorenpää, Hanna; Toimela, Tarja; Uotila, Jukka; Kuokkanen, Hannu; Ylikomi, Timo; Sarkanen, Jertta-Riina; Heinonen, Tuula

    2015-01-01

    The formation of blood vessels is a vital process in embryonic development and in normal physiology. Current vascular modelling is mainly based on animal biology leading to species-to-species variation when extrapolating the results to humans. Although there are a few human cell based vascular models available these assays are insufficiently characterized in terms of culture conditions and developmental stage of vascular structures. Therefore, well characterized vascular models with human relevance are needed for basic research, embryotoxicity testing, development of therapeutic strategies and for tissue engineering. We have previously shown that the in vitro vascular model based on co-culture of human adipose stromal cells (hASC) and human umbilical vein endothelial cells (HUVEC) is able to induce an extensive vascular-like network with high reproducibility. In this work we developed a defined serum-free vascular stimulation medium (VSM) and performed further characterization in terms of cell identity, maturation and structure to obtain a thoroughly characterized in vitro vascular model to replace or reduce corresponding animal experiments. The results showed that the novel vascular stimulation medium induced intact and evenly distributed vascular-like network with morphology of mature vessels. Electron microscopic analysis assured the three-dimensional microstructure of the network containing lumen. Additionally, elevated expressions of the main human angiogenesis-related genes were detected. In conclusion, with the new defined medium the vascular model can be utilized as a characterized test system for chemical testing as well as in creating vascularized tissue models. PMID:25742497

  11. Serum free hemoglobin test

    MedlinePlus

    Blood hemoglobin; Serum hemoglobin ... Hemoglobin (Hb) is the main component of red blood cells. It is a protein that carries oxygen. ... people may contain up to 5 mg/dL hemoglobin. Normal value ranges may vary slightly among different ...

  12. Serum Free Light Chains

    MedlinePlus

    ... changes in the ratio of kappa and lambda production, which indicate an excess of one clone of ... test to detect abnormal monoclonal protein (M-protein) production and to calculate a kappa/lambda free light ...

  13. Development and quality of bovine morulae cultured in serum-free medium with specific retinoid receptor agonists.

    PubMed

    Gómez, Enrique; Rodríguez, Aida; Muñoz, Marta; Caamaño, José Néstor; Carrocera, Susana; Martín, David; Facal, Nieves; Díez, Carmen

    2008-01-01

    Retinoids regulate development and differentiation of the bovine blastocyst in vitro, although the underlying mechanisms remain to be clarified. A challenge in reproductive biotechnology is the identification of pathways that regulate early embryonic development and their influence on blastocyst differentiation, apoptosis and survival to cryopreservation as traits of embryo quality. The present paper analyses the effects of short-term exposure (24 h) to retinoids on in vitro-produced bovine morulae. Immature cumulus oocyte complexes were in vitro matured and fertilised. Presumptive zygotes were subsequently cultured in modified synthetic oviduct fluid up to Day 6, in which morulae were randomly allocated to the different experimental groups. The treatments consisted of 0.1 microM LG100268 (LG; a retinoid X receptor agonist), 0.7 microM all-trans retinoic acid (ATRA; a retinoic acid receptor agonist) or no additives. Day 8 blastocyst development was increased in the ATRA-treated group compared with the LG and untreated embryos. In Day 7 embryos, the number of total cells and cells allocated to the trophectoderm were higher in the ATRA-treated group compared with untreated embryos. Apoptosis in the inner cell mass increased after LG treatment, whereas ATRA had no effect. After vitrification and warming, survival and hatching rates of Day 7 blastocysts did not change with retinoid treatment. Within the LG-treated and untreated blastocyst groups, survival and hatching rates were higher for Day 7 than Day 8 embryos; however, Day 8 blastocysts treated with ATRA showed improved hatching rates. In conclusion, treatment of morulae with ATRA in serum-free medium improves embryo development and quality without increasing the incidence of apoptosis and necrosis. PMID:19007552

  14. Maternal serum free beta-hCG and PAPP-A in fetal sex chromosome defects in the first trimester.

    PubMed

    Spencer, K; Tul, N; Nicolaides, K H

    2000-05-01

    We have studied maternal serum free beta-hCG and PAPP-A, and fetal nuchal translucency (NT) in a series of 46 cases of fetal Turner's syndrome, 13 cases of other sex chromosomal anomalies and compared these with 947 control pregnancies in the first trimester. In cases of Turner's syndrome (45,X) the median fetal NT was significantly higher than in controls (4.76 MoM), the median PAPP-A was significantly lower (0.49 MoM), whilst the free beta-hCG was not significantly different (1.11 MoM). For NT, 93% (43/46) of cases were equal to or greater than the 95th centile of controls, for PAPP-A 35% (16/46) of cases were less than or equal to the 5th centile of controls and for free beta-hCG 15% (7/46) of cases were equal to or greater than the 95th centile of controls. For other sex chromosomal anomalies (47XXX, XXY, XYY) the median NT was increased (2.07 MoM) whilst PAPP-A was not significantly decreased (0.88 MoM) and free beta-hCG was not significantly different (1.07 MoM) from controls. Using a previously derived multivariate risk algorithm for trisomy 21, incorporating NT, PAPP-A, free beta-hCG and maternal age, 96% of the Turner's cases and 62% of the other sex chromosomal anomalies would have been identified. PMID:10820406

  15. Characterization of Vero cell growth and death in bioreactor with serum-containing and serum-free media.

    PubMed

    Quesney, S; Marvel, J; Marc, A; Gerdil, C; Meignier, B

    2001-03-01

    The density of viable cells in a culture results from a balance between cell proliferation and cell death. The aim of this study was to characterize and compare these two phenomena in Vero cell cultures in one serum containing medium (ScA) and one serum free medium (SfB) in bioreactors. Cell growth was evaluated by cell counting(after crystal violet staining) and cell cycle analysis. Necrosis and apoptosis were characterized and quantified by measuring the release of LDH, trypan blue exclusion,annex in V-FITC/PI staining and TUNEL assay. ScA supported a higher maximal viable-cell density(2.3 x 10(6) vs. 1.8 x 10(6) cells ml(-1)). However, cell cycle analysis showed that cell division was more active in SfB than in ScA. LDH release in the supernatant increased much earlier in SfB than in ScA (one vs. five days), but trypan blue counts showed no apparent difference in the viability of the cultures. Apoptosis, evidenced by annexin V-FITC/PI staining, could be detected in the population of suspension cells detached from microcarriers, but not among adherent cells; positivity of the TUNEL assay occurred later than that of the annexin V-FITC/PI staining. Our data indicate that the lower cell yield in SfB,compared with that in ScA, results from a higher cell death rate. Apparently, cells die from apoptosis followed by secondary necrosis. PMID:19003288

  16. An improvement in the attaching capability of cryopreserved human hepatocytes by a proteinaceous high molecule, sericin, in the serum-free solution.

    PubMed

    Miyamoto, Yoshitaka; Teramoto, Naozumi; Hayashi, Shuji; Enosawa, Shin

    2010-01-01

    The methodology of cryopreservation of human hepatocytes remains unsatisfactory. Even when the viability of thawed cells is tolerable, the cells often lose the attaching capability to a culture dish, resulting in the cells' inability to survive. Previously, we described the effectiveness of maltose on the attachment of hepatocytes. This article demonstrates that a silk-derived high molecular protein, sericin, improves the cell-attaching capability in the serum-free freezing medium. When human hepatocytes [initial viability: 60.9 ± 3.1% (mean ± SD, n = 3)] were frozen with serum-free Dulbecco's modified Eagle medium (DMEM) containing 10% dimethyl sulfoxide (DMSO), the viability was 29.4 ± 3.2% and the cell-attaching capability 20.4 ± 4.1%. On the other hand, DMEM containing 10% DMSO and 1% sericin increased the values to 45.0 ± 0.8% and 26.2 ± 3.2%. Moreover, the addition of 0.1 mol/L maltose to the sericin-containing medium improved to 42.2 ± 3.2% and 51.1 ± 1.0%, as we demonstrated in a previous report. The present results indicated that sericin combined with maltose is a novel additive in the serum-free freezing medium for human hepatocytes. PMID:20525438

  17. Peripheral insulin resistance in obese girls with hyperandrogenism is related to oxidative phosphorylation and elevated serum free fatty acids.

    PubMed

    Cree-Green, Melanie; Newcomer, Bradley R; Coe, Gregory; Newnes, Lindsey; Baumgartner, Amy; Brown, Mark S; Pyle, Laura; Reusch, Jane E; Nadeau, Kristen J

    2015-05-01

    Hyperandrogenic syndrome (HAS) is associated with insulin resistance (IR) and type 2 diabetes. Muscle IR in type 2 diabetes is linked with defects in mitochondrial oxidative capacity. In vivo muscle mitochondrial function has not been studied in HAS, especially in youth, who are early in the disease process. Our goal was to measure muscle mitochondrial oxidative function and peripheral IR in obese youth with HAS. Obese girls without HAS [n = 22, age 15(13,17) yr, BMI Z-score 2.05 ± 0.37] and with HAS [n = 35, age 15(14,16) yr, BMI Z-score 2.18 ± 0.30] were enrolled. Mitochondrial function was assessed with (31)phosphorus MR spectroscopy before, during, and after near-maximal isometric calf exercise, and peripheral IR was assessed with an 80 mU·m(-2)·min(-1) hyperinsulinemic euglycemic clamp. Girls with HAS had higher androgens [free androgen index 7.9(6.6,15.5) vs. 3.5(3.0,4.0), P < 0.01] and more IR [glucose infusion rate 9.4(7.0, 12,2) vs. 14.5(13.2,15.8) mg·kg lean(-1)·min(-1), P < 0.01]. HAS girls also had increased markers of inflammation including CRP, platelets, and white blood cell count and higher serum free fatty acids during hyperinsulinemia. Mitochondrial oxidative phosphorylation was lower in HAS [0.11(0.06,0.19) vs. 0.18(0.12,0.23) mmol/s, P < 0.05], although other spectroscopy markers of mitochondrial function were similar between groups. In multivariate analysis of the entire cohort, IR related to androgens, oxidative phosphorylation, and free fatty acid concentrations during hyperinsulinemia. These relationships were present in just the HAS cohort as well. Obese girls with HAS have significant peripheral IR, which is related to elevated androgens and free fatty acids and decreased mitochondrial oxidative phosphorylation. These may provide future options as targets for therapeutic intervention. PMID:25714677

  18. Production of Inactivated Influenza H5N1 Vaccines from MDCK Cells in Serum-Free Medium

    PubMed Central

    Hu, Alan Yung-Chih; Tseng, Yu-Fen; Weng, Tsai-Chuan; Liao, Chien-Chun; Wu, Johnson; Chou, Ai-Hsiang; Chao, Hsin-Ju; Gu, Anna; Chen, Janice; Lin, Su-Chen; Hsiao, Chia-Hsin; Wu, Suh-Chin; Chong, Pele

    2011-01-01

    Background Highly pathogenic influenza viruses pose a constant threat which could lead to a global pandemic. Vaccination remains the principal measure to reduce morbidity and mortality from such pandemics. The availability and surging demand for pandemic vaccines needs to be addressed in the preparedness plans. This study presents an improved high-yield manufacturing process for the inactivated influenza H5N1 vaccines using Madin-Darby canine kidney (MDCK) cells grown in a serum-free (SF) medium microcarrier cell culture system. Principal Finding The current study has evaluated the performance of cell adaptation switched from serum-containing (SC) medium to several commercial SF media. The selected SF medium was further evaluated in various bioreactor culture systems for process scale-up evaluation. No significant difference was found in the cell growth in different sizes of bioreactors studied. In the 7.5 L bioreactor runs, the cell concentration reached to 2.3×106 cells/mL after 5 days. The maximum virus titers of 1024 Hemagglutinin (HA) units/50 µL and 7.1±0.3×108 pfu/mL were obtained after 3 days infection. The concentration of HA antigen as determined by SRID was found to be 14.1 µg/mL which was higher than those obtained from the SC medium. A mouse immunogenicity study showed that the formalin-inactivated purified SF vaccine candidate formulated with alum adjuvant could induce protective level of virus neutralization titers similar to those obtained from the SC medium. In addition, the H5N1 viruses produced from either SC or SF media showed the same antigenic reactivity with the NIBRG14 standard antisera. Conclusions The advantages of this SF cell-based manufacturing process could reduce the animal serum contamination, the cost and lot-to-lot variation of SC medium production. This study provides useful information to manufacturers that are planning to use SF medium for cell-based influenza vaccine production. PMID:21283675

  19. Peripheral insulin resistance in obese girls with hyperandrogenism is related to oxidative phosphorylation and elevated serum free fatty acids

    PubMed Central

    Newcomer, Bradley R.; Coe, Gregory; Newnes, Lindsey; Baumgartner, Amy; Brown, Mark S.; Pyle, Laura; Reusch, Jane E.; Nadeau, Kristen J.

    2015-01-01

    Hyperandrogenic syndrome (HAS) is associated with insulin resistance (IR) and type 2 diabetes. Muscle IR in type 2 diabetes is linked with defects in mitochondrial oxidative capacity. In vivo muscle mitochondrial function has not been studied in HAS, especially in youth, who are early in the disease process. Our goal was to measure muscle mitochondrial oxidative function and peripheral IR in obese youth with HAS. Obese girls without HAS [n = 22, age 15(13,17) yr, BMI Z-score 2.05 ± 0.37] and with HAS [n = 35, age 15(14,16) yr, BMI Z-score 2.18 ± 0.30] were enrolled. Mitochondrial function was assessed with 31phosphorus MR spectroscopy before, during, and after near-maximal isometric calf exercise, and peripheral IR was assessed with an 80 mU·m−2·min−1 hyperinsulinemic euglycemic clamp. Girls with HAS had higher androgens [free androgen index 7.9(6.6,15.5) vs. 3.5(3.0,4.0), P < 0.01] and more IR [glucose infusion rate 9.4(7.0, 12,2) vs. 14.5(13.2,15.8) mg·kg lean−1·min−1, P < 0.01]. HAS girls also had increased markers of inflammation including CRP, platelets, and white blood cell count and higher serum free fatty acids during hyperinsulinemia. Mitochondrial oxidative phosphorylation was lower in HAS [0.11(0.06,0.19) vs. 0.18(0.12,0.23) mmol/s, P < 0.05], although other spectroscopy markers of mitochondrial function were similar between groups. In multivariate analysis of the entire cohort, IR related to androgens, oxidative phosphorylation, and free fatty acid concentrations during hyperinsulinemia. These relationships were present in just the HAS cohort as well. Obese girls with HAS have significant peripheral IR, which is related to elevated androgens and free fatty acids and decreased mitochondrial oxidative phosphorylation. These may provide future options as targets for therapeutic intervention. PMID:25714677

  20. Understanding the apothecaries within: the necessity of a systematic approach for defining the chemical output of the human microbiome.

    PubMed

    Beebe, Kirk; Sampey, Brante; Watkins, Steven M; Milburn, Michael; Eckhart, Andrea D

    2014-02-01

    The human microbiome harbors a massive diversity of microbes that effectively form an "organ" that strongly influences metabolism and immune function and hence, human health. Although the growing interest in the microbiome has chiefly arisen due to its impact on human physiology, the probable rules of operation are embedded in the roots of microbiology where chemical communication (i.e., with metabolites) is a dominant feature of coexistence. Indeed, recent examples in microbiome research offer the impression that the collective microbiome operates as an "apothecary," creating chemical concoctions that influence health and alter drug response. Although these principles are not unappreciated, the majority of emphasis is on metagenomics and research efforts often omit systematic efforts to interrogate the chemical component of the complex equation between microbial community and host phenotype. One of the reasons for this omission may be due to the inaccessibility to high-breadth, high-throughput, and scalable technologies. Since these technologies are now available, we propose that a more systematic effort to survey the host-microbiota chemical output be embedded into microbiome research as there is strong likelihood, and growing precedence, that this component may often be integral to developing our understanding of these ultimate apothecaries and how they impact human health. PMID:24422665

  1. An effective freezing/thawing method for human pluripotent stem cells cultured in chemically-defined and feeder-free conditions

    PubMed Central

    Nishishita, Naoki; Muramatsu, Marie; Kawamata, Shin

    2015-01-01

    Culturing human Pluripotent Stem Cells (hPSC)s in chemically defined medium and feeder-free condition can facilitate metabolome and proteome analysis of culturing cells and medium, and reduce regulatory concerns for clinical application of cells. And in addition, if hPSC are passaged and cryopreserved in single cells it also facilitates quality control of cells at single cell level. Here we report a robust single cell freezing and thawing method of hPSCs cultured in chemically-defined medium TeSRTM-E8TM and on cost-effective recombinant human Vitronectin-N (rhVTN-N)-coated dish. Cells are dissociated into single cells with recombinant TrypLETM Select and 0.5 mM EDTA/PBS (3:1 solution) in the presence of Rock inhibitor and cryopreserved with chemically defined CryoStemTM. Approximately 60% of cells were viable after dissociation. AggrewellTM 400 was used to form cell clumps of 500 cells after thaw in the presence of Rock inhibitor and cells were cultured for two days with TeSR-E8. Cells clumps were then seeded on rhVTN-N-coated dish and cultured with TeSR-E8 for two days prior to the first passage after thawing. Number of viable cells at the first passage increased around 10 times of that just before freezing. This robust single cell freezing method for hPSCs cultured in chemically defined medium will facilitate quality control of cultured cells at single cell level before cryopreservation and consequently assure the quality of cells in frozen vials for further manipulation after thawing. PMID:25973330

  2. An effective freezing/thawing method for human pluripotent stem cells cultured in chemically-defined and feeder-free conditions.

    PubMed

    Nishishita, Naoki; Muramatsu, Marie; Kawamata, Shin

    2015-01-01

    Culturing human Pluripotent Stem Cells (hPSC)s in chemically defined medium and feeder-free condition can facilitate metabolome and proteome analysis of culturing cells and medium, and reduce regulatory concerns for clinical application of cells. And in addition, if hPSC are passaged and cryopreserved in single cells it also facilitates quality control of cells at single cell level. Here we report a robust single cell freezing and thawing method of hPSCs cultured in chemically-defined medium TeSR(TM)-E8(TM) and on cost-effective recombinant human Vitronectin-N (rhVTN-N)-coated dish. Cells are dissociated into single cells with recombinant TrypLE(TM) Select and 0.5 mM EDTA/PBS (3:1 solution) in the presence of Rock inhibitor and cryopreserved with chemically defined CryoStem(TM). Approximately 60% of cells were viable after dissociation. Aggrewell(TM) 400 was used to form cell clumps of 500 cells after thaw in the presence of Rock inhibitor and cells were cultured for two days with TeSR-E8. Cells clumps were then seeded on rhVTN-N-coated dish and cultured with TeSR-E8 for two days prior to the first passage after thawing. Number of viable cells at the first passage increased around 10 times of that just before freezing. This robust single cell freezing method for hPSCs cultured in chemically defined medium will facilitate quality control of cultured cells at single cell level before cryopreservation and consequently assure the quality of cells in frozen vials for further manipulation after thawing. PMID:25973330

  3. Successful Non-Surgical Deep Uterine Transfer of Porcine Morulae after 24 Hour Culture in a Chemically Defined Medium

    PubMed Central

    Martinez, Emilio A.; Angel, Miguel Angel; Cuello, Cristina; Sanchez-Osorio, Jonatan; Gomis, Jesus; Parrilla, Inmaculada; Vila, Jordi; Colina, Ignaci; Diaz, Marta; Reixach, Josep; Vazquez, Jose Luis; Vazquez, Juan Maria; Roca, Jordi; Gil, Maria Antonia

    2014-01-01

    Excellent fertility and prolificacy have been reported after non-surgical deep uterine transfers of fresh in vivo-derived porcine embryos. Unfortunately, when this technology is used with vitrified embryos, the reproductive performance of recipients is low. For this reason and because the embryos must be stored until they are transferred to the recipient farms, we evaluated the potential application of non-surgical deep uterine transfers with in vivo-derived morulae cultured for 24 h in liquid stage. In Experiment 1, two temperatures (25°C and 37°C) and two media (one fully defined and one semi-defined) were assessed. Morulae cultured in culture medium supplemented with bovine serum albumin and fetal calf serum at 38.5°C in 5% CO2 in air were used as controls. Irrespective of medium, the embryo viability after 24 h of culture was negatively affected (P<0.05) at 25°C but not at 37°C compared with the controls. Embryo development was delayed in all experimental groups compared with the control group (P<0.001). Most of the embryos (95.7%) cultured at 37°C achieved the full or expanded blastocyst stage, and unlike the controls, none of them hatched at the end of culture. In Experiment 2, 785 morulae were cultured in the defined medium at 37°C for 24 h, and the resulting blastocysts were transferred to the recipients (n = 24). Uncultured embryos collected at the blastocyst stage (n = 750) were directly transferred to the recipients and used as controls (n = 25). No differences in farrowing rates (91.7% and 92.0%) or litter sizes (9.0±0.6 and 9.4±0.8) were observed between the groups. This study demonstrated, for the first time, that high reproductive performance can be achieved after non-surgical deep uterine transfers with short-term cultured morulae in a defined medium, which opens new possibilities for the sanitary, safe national and international trade of porcine embryos and the commercial use of embryo transfer in pigs. PMID:25118944

  4. Separation of Scaptotrigona postica workers into defined task groups by the chemical profile on their epicuticle wax layer.

    PubMed

    Poiani, Silvana B; Morgan, E David; Drijfhout, Falko P; da Cruz-Landim, Carminda

    2014-04-01

    During evolution, the cuticle surface of insects acquired functions in communication, such as inter- and intra-specific recognition, identification of gender, physiological state, and fertility. In eusocial bees, the information in the cuticular surface is important not only to discriminate nestmates from non-nestmates but also to identify an individual's class, life phase or task. A comparative study of the cuticular surface chemical profile of workers of Scaptotrigona postica in different phases of life, i.e., newly emerged workers (NE), brood comb area workers (CA), and forager workers (FO) was undertaken by gas chromatography linked to mass spectrometry. Multivariate statistical analysis was performed to verify how workers are grouped according to their chemical profile and to determine which compounds are responsible for separating them into groups. The cuticle surface of workers contains mainly hydrocarbons and a small amount of oxygenated compounds. Multivariate statistical analysis showed qualitative and quantitative variation in relation to the life phases/tasks performed, and all groups were distinct. The most abundant compound found in NE and CA was n-heptacosane, while in FO, it was (Z)-9-heptacosene. The compounds that differentiate NE from other groups are n-tricosane and n-hexacosane. A (Z)-X-octacosene and n-nonacosane are the chemicals that distinguish CA from NE and FO, while 11- and 13-methylpentacosane, (Z)-X-hexacosene, and (Z)-9-heptacosene characterize FO as distinct from NE and CA. The probable function of alkenes is nestmate recognition, mainly in FO. The results show that the cuticle surfaces of workers are characteristic of the phase of life/task performed by workers, allowing intra-colonial recognition. PMID:24752855

  5. Label-free quantitative proteomic analysis of benzo(a)pyrene-transformed 16HBE cells serum-free culture supernatant and xenografted nude mice sera.

    PubMed

    Zhao, Peng; Fu, Juanling; Yao, Biyun; Jia, Yongrui; Zhang, Hongtao; Li, Xuehui; Dong, Lisha; Gao, Ya; Liu, Wenli; Chen, Wen; Zhou, Zongcan

    2016-02-01

    To screen potential biomarkers of benzo(a)pyrene (BaP)-induced lung cancer, the proteomic profiles of BaP-transformed 16HBE cell line T-16HBE-C1 cells serum-free culture supernatant and xenografted nude mice sera were compared with those of 16HBE group by utilizing label-free quantitative proteomic strategy. By employing nano-LC-MS/MS technology followed by MaxQuant and Perseus processing, 489 differentially expressed proteins were identified between T-16HBE-C1 and 16HBE cells serum-free culture supernatant, and 49 significantly up-regulated proteins were identified in T-16HBE-C1 xenografted nude mice sera. Three proteins neuropilin-2 (NRP2), clusterin (CLU) and A-kinase anchor protein 12 (AKAP12) were up-regulated in the serum-free culture supernatant of T-16HBE-C1 cells. These 3 human proteins were present in the sera of nude mice xenografted with T-16HBE-C1 cells, but were undetectable in mice xenografted with 16HBE cells. The proteomic results of NRP2 and AKAP12 were confirmed by Western blotting and enzyme-linked immunosorbent assays, respectively. Moreover, the serum NRP2 levels were significantly elevated at the 4th day after tumor cell implantation and showed good positive correlation with tumor growth characterized by tumor volume. In conclusion, serum NRP2, CLU and AKAP12 could be potential biomarkers of BaP-induced lung cancer. The proteomic results will gain deeper insights into the mechanisms of BaP-induced carcinogenesis. PMID:26748308

  6. Isolation and identification of cancer stem cells from human osteosarcom by serum-free three-dimensional culture combined with anticancer drugs.

    PubMed

    Zhou, Song; Li, Feng; Xiao, Jun; Xiong, Wei; Fang, Zhong; Chen, Wenjian; Niu, Pengyan

    2010-02-01

    The cancer stem cells (CSCs) from human osteosarcoma by serum-free three-dimensional culture combined with anticancer drugs were isolated and identified. The primary cells derived from human osteosarcoma were digested by trypsin to prepare a single-cell suspension, and mixed homogeneously into 1.2% alginate gel. Single-cell alginate gel was cultured with serum-free DMEM/F12 medium. Epirubicin (0.8 microg/mL) was added to the medium to enrich CSCs. After cultured conventionally for 7 to 10 days, most of cells suspended in alginate gel were killed by epirubicin. But few cells survived and some single-cell cloning spheres formed. Immunofluorescent staining for Oct3/4 and Nanog was implemented to find cells with properties of self-renewal and multi-potential differentiation. Cells from cloning spheres were transplanted into BALB/c mice to detect the tumorigenicity in vivo. The results showed that some cells positive for Oct3/4 (TRITC) and Nanog (TRITC) were found in single-cell cloning spheres, and most of positive cells were concentrated in the core of sphere. Cells from spheres could form osteosarcoma in the body of mice. It was concluded that cells from single-cell cloning spheres had the properties of the expression of parts of stem cell genes (Oct3/4 and Nanog), resisting anti-cancer drugs, and tumorigenicity in vivo. To sum up, it is believed that cells obtained from osteosarcoma by serum-free three-dimensional culture combined with anticancer drugs are cancer stem cells. PMID:20155460

  7. Development of serum-free media for the cultivation and recovery of Acholeplasma laidlawii used for challenge testing sterilizing-grade filters in biopharmaceutical applications.

    PubMed

    Bates, Shawn; Nguyen, Nhung; Lentine, Kerry Roche

    2008-01-01

    A serum-free cultivation broth and recovery agar for use in 0.1 micron-rated filter characterization and validation studies for biopharmaceutical applications were developed using Acholeplasma laidlawii as the test microorganism. Selection criteria were (A) a single-stage, serum-free broth medium to support high-titer cell growth and yield a cellular morphology suitable for bacterial challenge testing within 24 h and (B) a serum-free agar growth medium that can recover cells using conventional enumeration techniques. Different formulation components at various concentrations were screened for their effects on growth, cell size, and recovery on membrane filters. An optimized broth, Glucose Hydrolysate Broth, containing glucose, polypeptone, bovine serum albumin, 2-Amino-2-(hydroxymethyl)-1,3-propanediol (Tris base), oleic acid, and palmitic acid produced small 370 X 406 nanometer monodisperse cells at titers in excess of 1 x 10(9) colony-forming units per milliliter (cfu mL(-1)) within 20-24 h of incubation. Glucose Mycoplasma Agar, containing glucose, mycoplasma broth base, bovine serum albumin, Tris base, oleic acid, and palmitic acid, produced colonies approximately 1 mm in size and facilitated recovery on 0.2-microm polyvinylidene fluoride membrane filters. A comparison of recovery of A. laidlawii on Glucose Mycoplasma Agar pour-plate versus membrane filters resulted in an 89 +/- 4% recovery. Linearity across the target recovery range was 0.9992 (r2). Presence/absence tests of filtrate from each 0.2-microm assay membrane resulted in absence of growth as compared to controls, thus demonstrating the suitability of using 0.2-microm membrane filters for recovery of A. laidlawii. PMID:19634344

  8. Using chemical and physical parameters to define the quality of Pardo River water (Botucatu-SP-Brazil).

    PubMed

    Da Silva, A M; Sacomani, L B

    2001-04-01

    This paper aims to study the ecological system of the Pardo River, at the source and lower-order passages, which are in the Botucatu area, São Paulo State, Brazil. This study was carried out to determine water quality with some chemical-physical indicators, coliforms, and chemical species of samples taken monthly, 1995/02-1996/01, from eight sampling stations sited along the Pardo River. The results in the river monitoring are discussed based on annual averages, analysis of variance, and compared to Tukey's Studentized Range--HSD, and principal component analysis (PCA) was applied to normalize data to assess association between variables. We can conclude that the variables used are very efficient for identifying and that the dry season shows the worst water quality. These were caused by organic matter, nutrients (originate) from anthropogenic sources (spatial sources) and mainly municipal wastewater, affecting the quality and hydrochemistry of the river water, which have been differentiated and assigned to polluting sources. Meanwhile, the degree of degradation of the Pardo River is low (sewage treatment carried out by the city of Pardinho is efficient), leaving the water of the river suitable for use by the population of Botucatu, after conventional treatment (Conama, Resolução No. 20, CONAMA, Brazilia DF, 09-23, 1986--the water of the Pardo river is classified as level 03). PMID:11317910

  9. What Kind of Signaling Maintains Pluripotency and Viability in Human-Induced Pluripotent Stem Cells Cultured on Laminin-511 with Serum-Free Medium?

    PubMed Central

    Nakashima, Yoshiki; Omasa, Takeshi

    2016-01-01

    Abstract Xeno-free medium contains no animal-derived components, but is composed of minimal growth factors and is serum free; the medium may be supplemented with insulin, transferrin, and selenium (ITS medium). Serum-free and xeno-free culture of human-induced pluripotent stem cells (hiPSCs) uses a variety of components based on ITS medium and Dulbecco's modified Eagle's medium/Ham's nutrient mixture F12 (DMEM/F12) that contain high levels of iron salt and glucose. Culture of hiPSCs also requires scaffolding materials, such as extracellular matrix, collagen, fibronectin, laminin, proteoglycan, and vitronectin. The scaffolding component laminin-511, which is composed of α5, β1, and γ1 chains, binds to α3β1, α6β1, and α6β4 integrins on the cell membrane to induce activation of the PI3K/AKT- and Ras/MAPK-dependent signaling pathways. In hiPSCs, the interaction of laminin-511/α6β1 integrin with the cell–cell adhesion molecule E-cadherin confers protection against apoptosis through the Ras homolog gene family member A (RhoA)/Rho kinase (ROCK) signaling pathway (the major pathways for cell death) and the proto-oncogene tyrosine-protein kinase Fyn (Fyn)-RhoA-ROCK signaling pathway. The expression levels of α6β1 integrin and E-cadherin on cell membranes are controlled through the activation of insulin receptor/insulin, FGF receptor/FGF2, or activin-like kinase 5 (ALK5)-dependent TGF-β signaling. A combination of growth factors, medium constituents, cell membrane-located E-cadherin, and α6β1 integrin-induced signaling is required for pluripotent cell proliferation and for optimal cell survival on a laminin-511 scaffold. In this review, we discuss and explore the influence of growth factors on the cadherin and integrin signaling pathways in serum-free and xeno-free cultures of hiPSCs during the preparation of products for regenerative medicinal therapies. In addition, we suggest the optimum serum-free medium components for use with laminin-511, a new

  10. Silicon-on-glass pore network micromodels with oxygen-sensing fluorophore films for chemical imaging and defined spatial structure

    SciTech Connect

    Grate, Jay W.; Kelly, Ryan T.; Suter, Jonathan D.; Anheier, Norman C.

    2012-11-21

    Pore network microfluidic models were fabricated by a silicon-on-glass technique that provides the precision advantage of dry etched silicon while creating a structure that is transparent across all microfluidic channels and pores, and can be imaged from either side. A silicon layer is bonded to an underlying borosilicate glass substrate and thinned to the desired height of the microfluidic channels and pores. The silicon is then patterned and through-etched by deep reactive ion etching (DRIE), with the underlying glass serving as an etch stop. After bonding on a transparent glass cover plate, one obtains a micromodel in oxygen impermeable materials with water wet surfaces where the microfluidic channels are transparent and structural elements such as the pillars creating the pore network are opaque. The micromodel can be imaged from either side. The advantageous features of this approach in a chemical imaging application are demonstrated by incorporating a Pt porphyrin fluorophore in a PDMS film serving as the oxygen sensing layer and a bonding surface, or in a polystyrene film coated with a PDMS layer for bonding. The sensing of a dissolved oxygen gradient was demonstrated using fluorescence lifetime imaging, and it is shown that different matrix polymers lead to optimal use in different ranges dissolved oxygen concentration. Imaging with the opaque pillars in between the observation direction and the continuous fluorophore film yields images that retain spatial information in the sensor image.

  11. An improved method for western blotting when extracting proteins from mammalian cells cultured on a collagen gel under serum-free conditions.

    PubMed

    Ishihara, Seiichiro; Mizutani, Takeomi; Kawabata, Kazushige; Haga, Hisashi

    2016-01-01

    Western blotting is a widely used method for detection and quantification of specific proteins extracted from mammalian cells. In the conventional method of protein extraction, we found that collagen-containing gels interfered with detection of the p65 protein (one of the subunits in the NF-κB family of proteins) in human lung adenocarcinoma A549 cells cultured on a collagen gel containing serum. In contrast, the collagen gels did not affect detection of the GAPDH protein. Then, we established an improved method for preparation of protein extracts (using trichloroacetic acid fixation and collagenase treatment) from the cells cultured on the collagen gel. Using the improved method, we were able to detect p65 proteins without loss in A549 cells cultured on a collagen gel under serum-free conditions, but we could not detect the proteins if serum was present in cell culture. Thus, using western blotting and serum-free culture conditions, we succeeded in comparing the p65 expression between the cells grown in a plastic dish and cells grown on a collagen gel. PMID:25005915

  12. Yeast hydrolysate as a low-cost additive to serum-free medium for the production of human thrombopoietin in suspension cultures of Chinese hamster ovary cells.

    PubMed

    Sung, Y H; Lim, S W; Chung, J Y; Lee, G M

    2004-02-01

    To enhance the performance of a serum-free medium (SFM) for human thrombopoietin (hTPO) production in suspension cultures of recombinant Chinese hamster ovary (rCHO) cells, several low-cost hydrolysates such as yeast hydrolysate (YH), soy hydrolysate, wheat gluten hydrolysate and rice hydrolysate were tested as medium additives. Among various hydrolysates tested, the positive effect of YH on hTPO production was most significant. When 5 g l(-1) YH was added to SFM, the maximum hTPO concentration in batch culture was 40.41 microg ml(-1), which is 11.5 times higher than that in SFM without YH supplementation. This enhanced hTPO production in YH-supplemented SFM was obtained by the combined effect of enhanced q(hTPO) (the specific rate of hTPO production). The supplementation of YH in SFM increased q(hTPO) by 294% and extended culture longevity by >2 days if the culture was terminated at a cell viability of 50%. Furthermore, cell viability throughout the culture using YH-supplemented SFM was higher than that using any other hydrolysate-supplemented SFM tested, thereby minimizing degradation of hTPO susceptible to proteolytic degradation. In addition, YH supplementation did not affect in vivo biological activity of hTPO. Taken together, the results obtained demonstrate the potential of YH as a medium additive for hTPO production in serum-free suspension cultures of rCHO cells. PMID:12856163

  13. Use of multiple chemical tracers to define habitat use of Indo-Pacific mangrove crab, Scylla serrata (Decapoda: Portunidae)

    USGS Publications Warehouse

    Demopoulos, A.W.J.; Cormier, N.; Ewel, K.C.; Fry, B.

    2008-01-01

    The mangrove or mud crab, Scylla serrata, is an important component of mangrove fisheries throughout the Indo-Pacific. Understanding crab diets and habitat use should assist in managing these fisheries and could provide additional justification for conservation of the mangrove ecosystem itself. We used multiple chemical tracers to test whether crab movements were restricted to local mangrove forests, or extended to include adjacent seagrass beds and reef flats. We sampled three mangrove forests on the island of Kosrae in the Federated States of Micronesia at Lelu Harbor, Okat River, and Utwe tidal channel. Samples of S. serrata and likely food sources were analyzed for stable carbon (??13C), nitrogen (??15N), and sulfur (??34S) isotopes. Scylla serrata tissues also were analyzed for phosphorus (P), cations (K, Ca, Mg, Na), and trace elements (Mn, Fe, Cu, Zn, and B). Discriminant analysis indicated that at least 87% of the crabs remain in each site as distinct populations. Crab stable isotope values indicated potential differences in habitat use within estuaries. Values for ??13C and ??34S in crabs from Okat and Utwe were low and similar to values expected from animals feeding within mangrove forests, e.g., feeding on infauna that had average ??13C values near -26.5???. In contrast, crabs from Lelu had higher ?? 13C and ??34S values, with average values of -21.8 and 7.8???, respectively. These higher isotope values are consistent with increased crab foraging on reef flats and seagrasses. Given that S. serrata have been observed feeding on adjacent reef and seagrass environments on Kosrae, it is likely that they move in and out of the mangroves for feeding. Isotope mixing model results support these conclusions, with the greatest mangrove ecosystem contribution to S. serrata diet occurring in the largest mangrove forests. Conserving larger island mangrove forests (> 1 km deep) appears to support crab foraging activities. ?? 2007 Coastal and Estuarine Research

  14. Human Induced Pluripotent Stem Cell Derived Neuronal Cells Cultured on Chemically-Defined Hydrogels for Sensitive In Vitro Detection of Botulinum Neurotoxin

    PubMed Central

    Pellett, Sabine; Schwartz, Michael P.; Tepp, William H.; Josephson, Richard; Scherf, Jacob M.; Pier, Christina L.; Thomson, James A.; Murphy, William L.; Johnson, Eric A.

    2015-01-01

    Botulinum neurotoxin (BoNT) detection provides a useful model for validating cell-based neurotoxicity screening approaches, as sensitivity is dependent on functionally competent neurons and clear quantitative endpoints are available for correlating results to approved animal testing protocols. Here, human induced pluripotent stem cell (iPSC)-derived neuronal cells were cultured on chemically-defined poly(ethylene glycol) (PEG) hydrogels formed by “thiol-ene” photopolymerization and tested as a cell-based neurotoxicity assay by determining sensitivity to active BoNT/A1. BoNT/A1 sensitivity was comparable to the approved in vivo mouse bioassay for human iPSC-derived neurons and neural stem cells (iPSC-NSCs) cultured on PEG hydrogels or treated tissue culture polystyrene (TCP) surfaces. However, maximum sensitivity for BoNT detection was achieved two weeks earlier for iPSC-NSCs that were differentiated and matured on PEG hydrogels compared to TCP. Therefore, chemically-defined synthetic hydrogels offer benefits over standard platforms when optimizing culture conditions for cell-based screening and achieve sensitivities comparable to an approved animal testing protocol. PMID:26411797

  15. Human Induced Pluripotent Stem Cell Derived Neuronal Cells Cultured on Chemically-Defined Hydrogels for Sensitive In Vitro Detection of Botulinum Neurotoxin.

    PubMed

    Pellett, Sabine; Schwartz, Michael P; Tepp, William H; Josephson, Richard; Scherf, Jacob M; Pier, Christina L; Thomson, James A; Murphy, William L; Johnson, Eric A

    2015-01-01

    Botulinum neurotoxin (BoNT) detection provides a useful model for validating cell-based neurotoxicity screening approaches, as sensitivity is dependent on functionally competent neurons and clear quantitative endpoints are available for correlating results to approved animal testing protocols. Here, human induced pluripotent stem cell (iPSC)-derived neuronal cells were cultured on chemically-defined poly(ethylene glycol) (PEG) hydrogels formed by "thiol-ene" photopolymerization and tested as a cell-based neurotoxicity assay by determining sensitivity to active BoNT/A1. BoNT/A1 sensitivity was comparable to the approved in vivo mouse bioassay for human iPSC-derived neurons and neural stem cells (iPSC-NSCs) cultured on PEG hydrogels or treated tissue culture polystyrene (TCP) surfaces. However, maximum sensitivity for BoNT detection was achieved two weeks earlier for iPSC-NSCs that were differentiated and matured on PEG hydrogels compared to TCP. Therefore, chemically-defined synthetic hydrogels offer benefits over standard platforms when optimizing culture conditions for cell-based screening and achieve sensitivities comparable to an approved animal testing protocol. PMID:26411797

  16. A novel chemical-defined medium with bFGF and N2B27 supplements supports undifferentiated growth in human embryonic stem cells

    SciTech Connect

    Liu Yanxia; Song Zhihua; Zhao Yang; Qin Han; Cai Jun; Zhang Hong; Yu Tianxin; Jiang Siming; Wang Guangwen; Ding Mingxiao; Deng Hongkui . E-mail: hongkui_deng@pku.edu.cn

    2006-07-21

    Traditionally, undifferentiated human embryonic stem cells (hESCs) are maintained on mouse embryonic fibroblast (MEF) cells or on matrigel with an MEF-conditioned medium (CM), which hampers the clinical applications of hESCs due to the contamination by animal pathogens. Here we report a novel chemical-defined medium using DMEM/F12 supplemented with N2, B27, and basic fibroblast growth factor (bFGF) [termed NBF]. This medium can support prolonged self-renewal of hESCs. hESCs cultured in NBF maintain an undifferentiated state and normal karyotype, are able to form embryoid bodies in vitro, and differentiate into three germ layers and extraembryonic cells. Furthermore, we find that hESCs cultured in NBF possess a low apoptosis rate and a high proliferation rate compared with those cultured in MEF-CM. Our findings provide a novel, simplified chemical-defined culture medium suitable for further therapeutic applications and developmental studies of hESCs.

  17. Monolayer culturing and cloning of human pluripotent stem cells on laminin-521-based matrices under xeno-free and chemically defined conditions.

    PubMed

    Rodin, Sergey; Antonsson, Liselotte; Hovatta, Outi; Tryggvason, Karl

    2014-10-01

    A robust method for culturing human pluripotent stem (hPS) cells under chemically defined and xeno-free conditions is an important tool for stem cell research and for the development of regenerative medicine. Here, we describe a protocol for monolayer culturing of Oct-4-positive hPS cells on a specific laminin-521 (LN-521) isoform, under xeno-free and chemically defined conditions. The cells are dispersed into single-cell suspension and then plated on LN-521 isoform at densities higher than 5,000 cells per cm², where they attach, migrate and survive by forming small monolayer cell groups. The cells avidly divide and expand horizontally until the entire dish is covered by a confluent monolayer. LN-521, in combination with E-cadherin, allows cloning of individual hPS cells in separate wells of 96-well plates without the presence of rho-associated protein kinase (ROCK) inhibitors or any other inhibitors of anoikis. Characterization of cells maintained for several months in culture reveals pluripotency with a minimal degree of genetic abnormalities. PMID:25211513

  18. Immunogenicity of an adeno-vector vaccine expressing the F protein of a respiratory syncytial virus manufactured from serum-free suspension culture.

    PubMed

    Shao, Hsiao-Yun; Hsu, Huai-Sheng; Yu, Shu-Ling; Wu, Shang-Rung; Hu, Kai-Chieh; Chang, Ching-Kun; Liu, Chia-Chyi; Chow, Yen-Hung

    2016-06-01

    We have developed an efficient cell culture process to scale up the production of a recombinant adenovirus that expresses the membrane-trunked fusion protein of respiratory syncytial virus (RSV; Ad-F0ΔTM). Adherent cells of human embryonic kidney (HEK) 293-derived cell, 293A, which supports the production of E1/E3-deleted Ad-F0ΔTM when cultured in the presence of fetal bovine serum (FBS), were adapted to suspension growth under serum-free medium. In doing so, we studied the immunogenicity of Ad-F0ΔTMsus, which propagated in a bioreactor that was cultured with serum-free suspension of 293A, in comparison with Ad-F0ΔTMadh, which was produced from parental 293A cells that were adherently cultured in medium containing FBS. The size and morphology of Ad-F0ΔTMsus and Ad-F0ΔTMadh virions were identical upon inspection with electron microscopy. The results showed that anti-F IgG and RSV-neutralizing titer were raised in the serum of both mice that were intranasally immunized twice with Ad-F0ΔTMsus or Ad-F0ΔTMadh at two-week injection intervals. Furthermore, the immune responses persisted for six months after vaccination. Activation of F protein-specific CD8(+) T cell's epitope associated IFN-ɣ and IL-4 was induced in both Ad-F0ΔTMsus- and Ad-F0ΔTMadh, but not in Ad-LacZsus, -immunized mouse splenocytes. No vaccine-enhanced lung inflammation, airway mucus occlusion or eosinophils infiltration were observed in Ad-immunized mice followed by RSV challenge; however, these symptoms were observed following immunization with formalin-inactivated RSV vaccine. These results indicate that the safety and potency of Ad-F0ΔTM produced from either adherent cells or suspension and serum-free cells are the same. PMID:27001351

  19. Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions.

    PubMed

    Slamecka, Jaroslav; Salimova, Lilia; McClellan, Steven; van Kelle, Mathieu; Kehl, Debora; Laurini, Javier; Cinelli, Paolo; Owen, Laurie; Hoerstrup, Simon P; Weber, Benedikt

    2016-01-01

    Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or β-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable. PMID:26654216

  20. Production of biogenic Mn oxides by Leptothrix discophora SS-1 in a chemically defined growth medium and evaluation of their Pb adsorption characteristics

    SciTech Connect

    Nelson, Y.M.; Lion, L.W.; Ghiorse, W.C.; Shuler, M.L.

    1999-01-01

    Biogenic Mn oxides were produced by the bacterium Leptothrix discophora SS-1 (= ATCC 3182) in a chemically defined mineral salts medium, and the Pb binding and specific surface area of these oxides were characterized. Growth of SS-1 in the defined medium with pyruvate as a carbon and energy source required the addition of vitamin B{sub 12}. Complete oxidation of Mn(II) within 60 h required the addition of {ge}0.1 {micro}M FeSO{sub 4}. Pb adsorption isotherms were determined for the biogenic Mn oxides (and associated cells with their extracellular polymer) and compared to the Pb adsorption isotherms of cells and exopolymer alone, as well as to abiotic Mn oxides. The Pb adsorption to cells and exopolymer with biogenic Mn oxides at pH 6.0 and 25 C was 2 orders of magnitude greater than the Pb adsorption to cells and exopolymer alone. The Pb adsorption to the biogenic Mn oxide was two to five times greater than the Pb adsorption to a chemically precipitated abiotic Mn oxide and several orders of magnitude greater than the Pb adsorption to two commercially available crystalline MnO{sub 2} minerals. The N{sub 2} Brunauer-Emmet-Teller specific surface areas of the biogenic Mn oxide and fresh Mn oxide precipitate were significantly greater than those of the commercial Mn oxide minerals. The Pb adsorption capacity of the biogenic Mn oxide also exceeded that of a chemically precipitated colloidal hydrous Fe oxide under similar solution conditions. These results show that amorphous biogenic Mn oxides similar to those produced by SS-1 may play a significant rule in the control of trace metal phase distribution in aquatic systems.

  1. Production of biogenic Mn oxides by leptothrix discophora SS-1 in a chemically defined growth medium and evaluation of their Pb adsorption characteristics

    PubMed

    Nelson; Lion; Ghiorse; Shuler

    1999-01-01

    Biogenic Mn oxides were produced by the bacterium Leptothrix discophora SS-1 (= ATCC 3182) in a chemically defined mineral salts medium, and the Pb binding and specific surface area of these oxides were characterized. Growth of SS-1 in the defined medium with pyruvate as a carbon and energy source required the addition of vitamin B12. Complete oxidation of Mn(II) within 60 h required the addition of >/=0.1 &mgr;M FeSO4. Pb adsorption isotherms were determined for the biogenic Mn oxides (and associated cells with their extracellular polymer) and compared to the Pb adsorption isotherms of cells and exopolymer alone, as well as to abiotic Mn oxides. The Pb adsorption to cells and exopolymer with biogenic Mn oxides (0.8 mmol of Mn per g) at pH 6.0 and 25 degreesC was 2 orders of magnitude greater than the Pb adsorption to cells and exopolymer alone (on a dry weight basis). The Pb adsorption to the biogenic Mn oxide was two to five times greater than the Pb adsorption to a chemically precipitated abiotic Mn oxide and several orders of magnitude greater than the Pb adsorption to two commercially available crystalline MnO2 minerals. The N2 Brunauer-Emmet-Teller specific surface areas of the biogenic Mn oxide and fresh Mn oxide precipitate (224 and 58 m2/g, respectively) were significantly greater than those of the commercial Mn oxide minerals (0.048 and 4. 7 m2/g). The Pb adsorption capacity of the biogenic Mn oxide also exceeded that of a chemically precipitated colloidal hydrous Fe oxide under similar solution conditions. These results show that amorphous biogenic Mn oxides similar to those produced by SS-1 may play a significant role in the control of trace metal phase distribution in aquatic systems. PMID:9872777

  2. Production of Biogenic Mn Oxides by Leptothrix discophora SS-1 in a Chemically Defined Growth Medium and Evaluation of Their Pb Adsorption Characteristics

    PubMed Central

    Nelson, Yarrow M.; Lion, Leonard W.; Ghiorse, William C.; Shuler, Michael L.

    1999-01-01

    Biogenic Mn oxides were produced by the bacterium Leptothrix discophora SS-1 (= ATCC 3182) in a chemically defined mineral salts medium, and the Pb binding and specific surface area of these oxides were characterized. Growth of SS-1 in the defined medium with pyruvate as a carbon and energy source required the addition of vitamin B12. Complete oxidation of Mn(II) within 60 h required the addition of ≥0.1 μM FeSO4. Pb adsorption isotherms were determined for the biogenic Mn oxides (and associated cells with their extracellular polymer) and compared to the Pb adsorption isotherms of cells and exopolymer alone, as well as to abiotic Mn oxides. The Pb adsorption to cells and exopolymer with biogenic Mn oxides (0.8 mmol of Mn per g) at pH 6.0 and 25°C was 2 orders of magnitude greater than the Pb adsorption to cells and exopolymer alone (on a dry weight basis). The Pb adsorption to the biogenic Mn oxide was two to five times greater than the Pb adsorption to a chemically precipitated abiotic Mn oxide and several orders of magnitude greater than the Pb adsorption to two commercially available crystalline MnO2 minerals. The N2 Brunauer-Emmet-Teller specific surface areas of the biogenic Mn oxide and fresh Mn oxide precipitate (224 and 58 m2/g, respectively) were significantly greater than those of the commercial Mn oxide minerals (0.048 and 4.7 m2/g). The Pb adsorption capacity of the biogenic Mn oxide also exceeded that of a chemically precipitated colloidal hydrous Fe oxide under similar solution conditions. These results show that amorphous biogenic Mn oxides similar to those produced by SS-1 may play a significant role in the control of trace metal phase distribution in aquatic systems. PMID:9872777

  3. Use of cycloheximide to study independent lipid metabolism of Chlamydia trachomatis cultivated in mouse L cells grown in serum-free medium.

    PubMed Central

    Reed, S I; Anderson, L E; Jenkin, H M

    1981-01-01

    A system for measuring chlamydial lipid synthesis was developed with mouse L cells grown in serum-free modified Waymouth 752/l medium in a shaker culture. Host lipid synthesis was reduced approximately 90% when cells were incubated for 24 h in medium containing cycloheximide (2 micrograms/ml). Lipid metabolism was monitored by measuring the incorporation of [3H]isoleucine into the total lipid of normal and infected cells. The results suggested that lipid synthesis of Chlamydia trachomatis lymphogranuloma venereum (LGV-404L) was not inhibited by cycloheximide treatment when the chlamydiae were grown in L cells, whereas host lipid synthesis was inhibited. Chlamydial lipid metabolism began about 6 to 12 h after infection when the noninfectious reticulate body was found and continually increased until the beginning of the appearance of intracellular infectious elementary bodies at 24 to 30 h. PMID:7216466

  4. Measurement of Serum Free Thyroxine Index May Provide Additional Case Detection Compared to Free Thyroxine in the Diagnosis of Central Hypothyroidism

    PubMed Central

    Pantalone, Kevin M.; Hatipoglu, Betul; Gupta, Manjula K.; Kennedy, Laurence; Hamrahian, Amir H.

    2015-01-01

    The diagnosis of central hypothyroidism is often suspected in patients with hypothalamic/pituitary pathology, in the setting of low, normal, or even slightly elevated serum TSH and low free thyroxine (FT4). We present four cases of central hypothyroidism (three had known pituitary pathology) in whom central hypothyroidism was diagnosed after the serum free thyroxine index (FTI) was found to be low. All had normal range serum TSH and free thyroxine levels. This report illustrates that the assessment of the serum FTI may be helpful in making the diagnosis of central hypothyroidism in the appropriate clinical setting and when free T4 is in the low-normal range, particularly in patients with multiple anterior pituitary hormone deficiencies and/or with symptoms suggestive of hypothyroidism. PMID:26779356

  5. Maintenance of oestradiol production and expression of cytochrome P450 aromatase enzyme mRNA in long-term serum-free cultures of pig granulosa cells.

    PubMed

    Picton, H M; Campbell, B K; Hunter, M G

    1999-01-01

    Studies were carried out to investigate the conditions required for maintenance of aromatase activity and expression in long-term cultures of pig granulosa cells. Cells from large (> 2 mm) and small (< or = 2 mm) follicles were cultured at 37 degrees C with 5% CO2 in McCoys 5a medium supplemented with 0.1% (w/v) BSA, testosterone (100 micrograms l-1), insulin (10 micrograms l-1) and long R3 insulin-like growth factor I (IGF-I) (100 micrograms l-1). Cells were cultured with five concentrations of USDA pFSH-I-2 (0-100 micrograms l-1) for 48, 96 or 144 h with or without fetal calf serum (FCS). The number of cells and oestradiol, progesterone and inhibin production were measured. In marked contrast to oestradiol production from cells cultured in plates precoated with FCS, 1 microgram FSH l-1 was optimal for the maintenance of high oestradiol production by granulosa cells from large follicles after 144 h of serum-free culture. Culture with FCS promoted cell proliferation, reduced oestradiol production, and supported FSH-dependent (P < 0.01) increased progesterone and inhibin production indicating cellular luteinization. Northern blot analysis of total RNA from cells cultured with 1 microgram FSH l-1 detected 2.5 and 1.8 kb transcripts encoding aromatase cytochrome P450 (P450arom) and cholesterol side-chain cleavage cytochrome P450 (P450scc), respectively. Transcript expression was hormone sensitive, irrespective of the presence of FCS. High concentrations of FSH (100 micrograms l-1) stimulated expression of P450scc, but inhibited P450arom expression as the cells luteinized after 144 h of culture. This serum-free system, which maintains the aromatase enzyme complex, is fundamental if physiologically relevant observations are to be made of the mechanisms regulating follicle hierarchy development from long-term cultures of pig cells. PMID:10341724

  6. Protecting capacity against malaria of chemically defined tetramer forms based on the Plasmodium falciparum apical sushi protein as potential vaccine components.

    PubMed

    Vanegas, Magnolia; Bermúdez, Adriana; Guerrero, Yuly Andrea; Cortes-Vecino, Jesús Alfredo; Curtidor, Hernando; Patarroyo, Manuel Elkin; Lozano, José Manuel

    2014-08-15

    Developing novel generations of subunit-based antimalarial vaccines in the form of chemically-defined macromolecule systems for multiple antigen presentation represents a classical problem in the field of vaccine development. Many efforts involving synthesis strategies leading to macromolecule constructs have been based on dendrimer-like systems, the condensation of large building blocks and conventional asymmetric double dimer constructs, all based on lysine cores. This work describes novel symmetric double dimer and condensed linear constructs for presenting selected peptide multi-copies from the apical sushi protein expressed in Plasmodium falciparum. These molecules have been proved to be safe and innocuous, highly antigenic and have shown strong protective efficacy in rodents challenged with two Plasmodium species. Insights into systematic design, synthesis and characterisation have led to such novel antigen systems being used as potential platforms for developing new anti-malarial vaccine candidates. PMID:25063026

  7. Inverse Association Between Serum Free Thyroxine Levels and Hepatic Steatosis: Results from the Study of Health in Pomerania

    PubMed Central

    Haring, Robin; Wallaschofski, Henri; Baumeister, Sebastian E.; Nauck, Matthias; Dörr, Marcus; Lerch, Markus; Meyer zu Schwabedissen, Henriette E.; Rosskopf, Dieter; Völzke, Henry

    2012-01-01

    Background Associations between thyroid function and hepatic steatosis defined by enzymatic and sonographic criteria are largely unknown in the general population. Thus, the aim of the present study was to investigate the association between thyroid function tests and sonographic as well as enzymatic criteria of liver status in a large population-based study, the Study of Health in Germany (SHIP). Methods Data from 3661 SHIP participants without a self-reported history of thyroid or liver disease were analyzed. Hepatic steatosis was defined as the presence of a hyperechogenic ultrasound pattern of the liver and increased serum alanine transferase concentrations. Serum thyroid-stimulating hormone (TSH), free triiodothyronine (FT3), and free thyroxine (FT4) concentrations were associated with hepatic steatosis using multinomial regression models adjusted for sex, age, physical activity, alcohol consumption, waist circumference, and food intake pattern. Results We detected no consistent association of serum TSH and FT3 concentrations with hepatic steatosis. In contrast, serum FT4 concentrations were inversely associated with hepatic steatosis in men (odds ratio (OR)=0.04 [95% confidence interval (CI)=0.01; 0.17]) and women (OR=0.06 [95% CI=0.01; 0.42]). Conclusions Results from the present cross-sectional study suggest that low FT4 concentrations are associated with hepatic steatosis. Longitudinal and intervention studies are warranted to investigate whether hypothyroidism increases the risk of hepatic steatosis or vice versa. PMID:22574630

  8. An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery.

    PubMed

    Sailer, Martin H M; Sarvepalli, Durga; Brégère, Catherine; Fisch, Urs; Guentchev, Marin; Weller, Michael; Guzman, Raphael; Bettler, Bernhard; Ghosh, Arkasubhra; Hutter, Gregor

    2016-01-01

    Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGFβ-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGFβ-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGFβ-interaction. The results point to a network of FGF-, BMP- and TGFβ-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT. PMID:27583933

  9. Diverse in- and output polarities and high complexity of local synaptic and non-synaptic signaling within a chemically defined class of peptidergic Drosophila neurons

    PubMed Central

    Karsai, Gergely; Pollák, Edit; Wacker, Matthias; Vömel, Matthias; Selcho, Mareike; Berta, Gergely; Nachman, Ronald J.; Isaac, R. Elwyn; Molnár, László; Wegener, Christian

    2013-01-01

    Peptidergic neurons are not easily integrated into current connectomics concepts, since their peptide messages can be distributed via non-synaptic paracrine signaling or volume transmission. Moreover, the polarity of peptidergic interneurons in terms of in- and out-put sites can be hard to predict and is very little explored. We describe in detail the morphology and the subcellular distribution of fluorescent vesicle/dendrite markers in CCAP neurons (NCCAP), a well defined set of peptidergic neurons in the Drosophila larva. NCCAP can be divided into five morphologically distinct subsets. In contrast to other subsets, serial homologous interneurons in the ventral ganglion show a mixed localization of in- and output markers along ventral neurites that defy a classification as dendritic or axonal compartments. Ultrastructurally, these neurites contain both pre- and postsynaptic sites preferably at varicosities. A significant portion of the synaptic events are due to reciprocal synapses. Peptides are mostly non-synaptically or parasynaptically released, and dense-core vesicles and synaptic vesicle pools are typically well separated. The responsiveness of the NCCAP to ecdysis-triggering hormone may be at least partly dependent on a tonic synaptic inhibition, and is independent of ecdysteroids. Our results reveal a remarkable variety and complexity of local synaptic circuitry within a chemically defined set of peptidergic neurons. Synaptic transmitter signaling as well as peptidergic paracrine signaling and volume transmission from varicosities can be main signaling modes of peptidergic interneurons depending on the subcellular region. The possibility of region-specific variable signaling modes should be taken into account in connectomic studies that aim to dissect the circuitry underlying insect behavior and physiology, in which peptidergic neurons act as important regulators. PMID:23914156

  10. Differential regulation of pig theca cell steroidogenesis by LH, insulin-like growth factor I and granulosa cells in serum-free culture.

    PubMed

    Shores, E M; Picton, H M; Hunter, M G

    2000-03-01

    The regulation of pig theca cell steroidogenesis was studied by the development of a physiological serum-free culture system, which was subsequently extended to investigate potential theca-granulosa cell interactions. Theca cells were isolated from antral follicles 6-9 mm in diameter and the effects of plating density (50-150x10(3) viable cells per well), LH (0.01-1.0 ng ml(-1)), Long R3 insulin-like growth factor I (IGF-I) (10, 100 ng ml(-1)) and insulin (1, 10 ng ml(-1)) on the number of cells and steroidogenesis were examined. The purity of the theca cell preparation was verified biochemically and histologically. Co-cultures contained 50x10(3) viable cells per well in granulosa to theca cell ratio of 4:1. Wells containing granulosa cells only were supplemented with 'physiological' doses of androstenedione or 100 ng ml(-1). Oestradiol production by co-cultures was compared with the sum of the oestradiol synthesized by granulosa and theca cells cultured separately. Oestradiol and androstenedione production continued throughout culture. High plating density decreased steroid production (P < 0.01). LH increased androstenedione (P < 0.001) and oestradiol (P < 0.05) synthesis and the sensitivity of the cells increased with time in culture. Oestradiol production was increased by 10 ng IGF-I ml(-1) (P < 0.001) but androstenedione required 100 ng ml(-1) (P < 0.001). Co-cultures produced more oestradiol than the sum of oestradiol synthesized by theca and granulosa cells cultured separately (P < 0. 001), irrespective of the androstenedione dose. This serum-free culture system for pig theca cells maintained in vivo steroidogenesis and gonadotrophin responsiveness. Thecal androstenedione and oestradiol production were differentially regulated and were primarily stimulated by LH and IGF-I, respectively. Theca-granulosa cell interactions stimulated oestradiol synthesis and this interaction was mediated by factors additional to the provision of thecal androgen substrate to

  11. Defining "Development".

    PubMed

    Pradeu, Thomas; Laplane, Lucie; Prévot, Karine; Hoquet, Thierry; Reynaud, Valentine; Fusco, Giuseppe; Minelli, Alessandro; Orgogozo, Virginie; Vervoort, Michel

    2016-01-01

    Is it possible, and in the first place is it even desirable, to define what "development" means and to determine the scope of the field called "developmental biology"? Though these questions appeared crucial for the founders of "developmental biology" in the 1950s, there seems to be no consensus today about the need to address them. Here, in a combined biological, philosophical, and historical approach, we ask whether it is possible and useful to define biological development, and, if such a definition is indeed possible and useful, which definition(s) can be considered as the most satisfactory. PMID:26969977

  12. Characterization of the Pivotal Carbon Metabolism of Streptococcus suis Serotype 2 under ex Vivo and Chemically Defined in Vitro Conditions by Isotopologue Profiling*

    PubMed Central

    Willenborg, Jörg; Huber, Claudia; Koczula, Anna; Lange, Birgit; Eisenreich, Wolfgang; Valentin-Weigand, Peter; Goethe, Ralph

    2015-01-01

    Streptococcus suis is a neglected zoonotic pathogen that has to adapt to the nutritional requirements in the different host niches encountered during infection and establishment of invasive diseases. To dissect the central metabolic activity of S. suis under different conditions of nutrient availability, we performed labeling experiments starting from [13C]glucose specimens and analyzed the resulting isotopologue patterns in amino acids of S. suis grown under in vitro and ex vivo conditions. In combination with classical growth experiments, we found that S. suis is auxotrophic for Arg, Gln/Glu, His, Leu, and Trp in chemically defined medium. De novo biosynthesis was shown for Ala, Asp, Ser, and Thr at high rates and for Gly, Lys, Phe, Tyr, and Val at moderate or low rates, respectively. Glucose degradation occurred mainly by glycolysis and to a minor extent by the pentose phosphate pathway. Furthermore, the exclusive formation of oxaloacetate by phosphoenolpyruvate (PEP) carboxylation became evident from the patterns in de novo synthesized amino acids. Labeling experiments with S. suis grown ex vivo in blood or cerebrospinal fluid reflected the metabolic adaptation to these host niches with different nutrient availability; however, similar key metabolic activities were identified under these conditions. This points at the robustness of the core metabolic pathways in S. suis during the infection process. The crucial role of PEP carboxylation for growth of S. suis in the host was supported by experiments with a PEP carboxylase-deficient mutant strain in blood and cerebrospinal fluid. PMID:25575595

  13. Characterization of the pivotal carbon metabolism of Streptococcus suis serotype 2 under ex vivo and chemically defined in vitro conditions by isotopologue profiling.

    PubMed

    Willenborg, Jörg; Huber, Claudia; Koczula, Anna; Lange, Birgit; Eisenreich, Wolfgang; Valentin-Weigand, Peter; Goethe, Ralph

    2015-02-27

    Streptococcus suis is a neglected zoonotic pathogen that has to adapt to the nutritional requirements in the different host niches encountered during infection and establishment of invasive diseases. To dissect the central metabolic activity of S. suis under different conditions of nutrient availability, we performed labeling experiments starting from [(13)C]glucose specimens and analyzed the resulting isotopologue patterns in amino acids of S. suis grown under in vitro and ex vivo conditions. In combination with classical growth experiments, we found that S. suis is auxotrophic for Arg, Gln/Glu, His, Leu, and Trp in chemically defined medium. De novo biosynthesis was shown for Ala, Asp, Ser, and Thr at high rates and for Gly, Lys, Phe, Tyr, and Val at moderate or low rates, respectively. Glucose degradation occurred mainly by glycolysis and to a minor extent by the pentose phosphate pathway. Furthermore, the exclusive formation of oxaloacetate by phosphoenolpyruvate (PEP) carboxylation became evident from the patterns in de novo synthesized amino acids. Labeling experiments with S. suis grown ex vivo in blood or cerebrospinal fluid reflected the metabolic adaptation to these host niches with different nutrient availability; however, similar key metabolic activities were identified under these conditions. This points at the robustness of the core metabolic pathways in S. suis during the infection process. The crucial role of PEP carboxylation for growth of S. suis in the host was supported by experiments with a PEP carboxylase-deficient mutant strain in blood and cerebrospinal fluid. PMID:25575595

  14. When Good Intentions Go Awry: Modification of a Recombinant Monoclonal Antibody in Chemically Defined Cell Culture by Xylosone, an Oxidative Product of Ascorbic Acid.

    PubMed

    Chumsae, Chris; Hossler, Patrick; Raharimampionona, Haly; Zhou, Yu; McDermott, Sean; Racicot, Chris; Radziejewski, Czeslaw; Zhou, Zhaohui Sunny

    2015-08-01

    With the advent of new initiatives to develop chemically defined media, cell culture scientists screen many additives to improve cell growth and productivity. However, the introduction or increase of supplements, typically considered beneficial or protective on their own, to the basal media or feed stream may cause unexpected detrimental consequences to product quality. For instance, because cultured cells are constantly under oxidative stress, ascorbic acid (vitamin C, a potent natural reducing agent) is a common additive to cell culture media. However, as reported herein, a recombinant monoclonal antibody (adalimumab) in cell culture was covalently modified by xylosone (molecular weight 148), an oxidative product of ascorbate. Containing reactive carbonyl groups, xylosone modifies various amines (e.g., the N-termini of the heavy and light chains and susceptible lysines), forming either hemiaminal (+148 Da) or Schiff base (imine, +130 Da) products. Our findings show, for the first time, that ascorbate-derived xylosone can contribute to an increase in molecular heterogeneity, such as acidic species. Our work serves as a reminder that additives to cell culture and their metabolites may become reactive and negatively impact the overall product quality and should be carefully monitored with any changes in cell culture conditions. PMID:26151084

  15. Stepwise differentiation of pluripotent stem cells into osteoblasts using four small molecules under serum-free and feeder-free conditions.

    PubMed

    Kanke, Kosuke; Masaki, Hideki; Saito, Taku; Komiyama, Yuske; Hojo, Hironori; Nakauchi, Hiromitsu; Lichtler, Alexander C; Takato, Tsuyoshi; Chung, Ung-Il; Ohba, Shinsuke

    2014-06-01

    Pluripotent stem cells are a promising tool for mechanistic studies of tissue development, drug screening, and cell-based therapies. Here, we report an effective and mass-producing strategy for the stepwise differentiation of mouse embryonic stem cells (mESCs) and mouse and human induced pluripotent stem cells (miPSCs and hiPSCs, respectively) into osteoblasts using four small molecules (CHIR99021 [CHIR], cyclopamine [Cyc], smoothened agonist [SAG], and a helioxanthin-derivative 4-(4-methoxyphenyl)pyrido[4',3':4,5]thieno[2,3-b]pyridine-2-carboxamide [TH]) under serum-free and feeder-free conditions. The strategy, which consists of mesoderm induction, osteoblast induction, and osteoblast maturation phases, significantly induced expressions of osteoblast-related genes and proteins in mESCs, miPSCs, and hiPSCs. In addition, when mESCs defective in runt-related transcription factor 2 (Runx2), a master regulator of osteogenesis, were cultured by the strategy, they molecularly recapitulated osteoblast phenotypes of Runx2 null mice. The present strategy will be a platform for biological and pathological studies of osteoblast development, screening of bone-augmentation drugs, and skeletal regeneration. PMID:24936463

  16. Transient Gene Expression in Serum-Free Suspension-Growing Mammalian Cells for the Production of Foot-and-Mouth Disease Virus Empty Capsids

    PubMed Central

    Mignaqui, Ana Clara; Ruiz, Vanesa; Perret, Sylvie; St-Laurent, Gilles; Singh Chahal, Parminder; Transfiguracion, Julia; Sammarruco, Ayelén; Gnazzo, Victoria; Durocher, Yves; Wigdorovitz, Andrés

    2013-01-01

    Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. It produces severe economic losses in the livestock industry. Currently available vaccines are based on inactivated FMD virus (FMDV). The use of empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production and conserves the conformational epitopes of the virus. In this report, we explored transient gene expression (TGE) in serum-free suspension-growing mammalian cells for the production of FMDV recombinant empty capsids as a subunit vaccine. The recombinant proteins produced, assembled into empty capsids and induced protective immune response against viral challenge in mice. Furthermore, they were recognized by anti-FMDV bovine sera. By using this technology, we were able to achieve expression levels that are compatible with the development of a vaccine. Thus, TGE of mammalian cells is an easy to perform, scalable and cost-effective technology for the production of a recombinant subunit vaccine against FMDV. PMID:23977353

  17. Comparison of the Freelite serum free light chain (SFLC) assay with serum and urine electrophoresis/immunofixation and the N Latex FLC assay.

    PubMed

    Sasson, S C; McGill, K; Wienholt, L; Carr, A; Brown, D A; Kelleher, A D; Breit, S N; Sewell, W A

    2015-10-01

    Few reports have compared available serum free light chain (SFLC) assays. Here, a retrospective audit of the Freelite SFLC assay compared results to electrophoresis (EP)/immunofixation (IFX) and the N Latex FLC assay.A total of 244 samples collected over 3.5 months were studied using the Freelite and N Latex FLC nephelometry assays. Results were compared with serum and/or urine EP/IFX. The precision and linearity of the N Latex FLC assay was examined.Detectable paraprotein by serum or urine EP/IFX was present in 94% of samples with kappa and 100% with lambda FLC restriction. The correlation between the assays was higher for kappa (rho = 0.97) than lambda (rho = 0.89) especially when lambda results were above the upper limit of normal (rho = 0.62). Agreement in the categorical diagnosis as measured by the Cohen's kappa statistic was good (0.70). The N Latex FLC assay displayed good precision and linearity. In discordant samples the Freelite and N Latex FLC assays had equivalent agreement with IFX.Traditional methods of EP/IFX detected paraproteins in the majority of cases. Correlation between the Freelite and N Latex FLC assay is better for kappa than lambda FLC. The two assays are not entirely equivalent. Care should be taken by interpreting physicians and laboratories considering switching assays. PMID:26352111

  18. Comparison of growth and drug response of human tumor cells in serum-free and serum-supplemented media in human tumor-clonogenic assay.

    PubMed

    Zirvi, K A; Hill, G J

    1988-06-01

    A comparison was made of growth and drug-response of five human tumor cell lines (HT-29, colon carcinoma; TWI, melanoma; A-549, lung carcinoma; Panc-1, pancreatic carcinoma; and EJ, bladder carcinoma) in serum-free media (SFM) and in serum-supplemented media (SSM) using the human tumor-clonogenic assay (HTCA) system. HT-29 cells, which had the highest plating efficiency in both SFM and SSM, were used to obtain dose-response curves for four drugs (adriamycin, 5-fluorouracil, cisplatin, and BCNU) in the HTCA. Three of the drugs (adriamycin, 5-fluorouracil, and cisplatin) produced identical drug-response curves in both SFM and SSM. These results suggest that, for some chemotherapeutic agents, results comparable to those obtained with SSM in the HTCA can be achieved using SFM. Step-by-step addition of growth factors and hormones to SFM may be a useful technique to improve some of the technical and logistic problems associated with the HTCA. PMID:3379971

  19. Influence of exogenous ATP on blood sugar, serum insulin and serum free fatty acids in short-term experimental hyperthyroid dogs and in euthyroid controls.

    PubMed

    Renauld, A; Sverdlik, R C

    1989-01-01

    We studied the influence of ATP administration on blood sugar (BS), serum immunoreactive insulin (IRI) and serum free fatty acid (FFA) responses to glucose-induced hyperglycemia in short-term experimental hyperthyroid (STEH) dogs and in euthyroid controls. Hyperthyroidism was induced by a 10-day s.c. treatment with 100 micrograms/kg body weight 1-thyroxine. Glucose challenge was performed by i.v. infusion (700 mg/kg body weight as a priming dose, followed by 20 mg/kg body weight/min for 60 min). ATP (1 mg/kg body weight/min) was infused for 100 min 40 min before and 60 min during glucose administration). Glucose-induced hyperglycemia was more prolonged in the hyperthyroid group. ATP treatment did not affect the BS profile of controls but raised that of STEH animals. In the normal controls the insulinemic response to hyperglycemia was enhanced during ATP infusion. By contrast, in the STEH dogs insulin levels during glucose infusion was lower than in controls and did not significantly increase when ATP was added. ATP infusion induced a significant elevation of serum FFA, which was more pronounced in hyperthyroid animals with a greater fall during glucose administration and a marked increase during the period of recovery from hyperglycemia. In conclusion, we postulate that short-term hyperthyroidism in dogs may inhibit adenylate cyclase function in pancreatic B-cells and chiefly stimulate the action of cAMP-phosphodiesterase activity, thereby affecting insulin secretion. PMID:2698040

  20. Wnt3a Protein Reduces Growth Factor-Driven Expansion of Human Hematopoietic Stem and Progenitor Cells in Serum-Free Cultures

    PubMed Central

    Duinhouwer, Lucia E.; Tüysüz, Nesrin; Rombouts, Elwin W. J. C.; ter Borg, Mariette N. D.; Mastrobattista, Enrico; Spanholtz, Jan; Cornelissen, Jan J.; ten Berge, Derk; Braakman, Eric

    2015-01-01

    Ex vivo expansion of hematopoietic stem and progenitor cells (HSPC) is a promising approach to improve insufficient engraftment after umbilical cord blood stem cell transplantation (UCB-SCT). Although culturing HSPC with hematopoietic cytokines results in robust proliferation, it is accompanied with extensive differentiation and loss of self-renewal capacity. Wnt signaling has been implicated in regulating HSPC fate decisions in vivo and in promoting HSPC self-renewal by inhibition of differentiation, but the effects of Wnt on the ex vivo expansion of HSPC are controversial. Here, we demonstrate that exogenous Wnt3a protein suppresses rather than promotes the expansion of UCB-derived CD34+ cells in serum free expansion cultures. The reduced expansion was also observed in cultures initiated with Lin-CD34+CD38lowCD45RA-CD90+ cells which are highly enriched in HSC and was also observed in response to activation of beta-catenin signaling by GSK3 inhibition. The presence of Wnt3a protein during the culture reduced the frequency of multilineage CFU-GEMM and the long-term repopulation ability of the expanded HSPC. These data suggest that Wnt signaling reduces expansion of human HSPC in growth factor-driven expansion cultures by promoting differentiation of HSPC. PMID:25807521

  1. Evaluation of serum markers in the LRF CLL4 trial: β2-microglobulin but not serum free light chains, is an independent marker of overall survival.

    PubMed

    Pratt, Guy; Thomas, Peter; Marden, Nicola; Alexander, Denis; Davis, Zadie; Hussey, David; Parry, Helen; Harding, Stephen; Catovsky, Daniel; Begley, Joe; Oscier, David

    2016-10-01

    Chronic lymphocytic leukemia (CLL) is characterized by heterogeneous clinical behavior and there is a need for improved biomarkers. The current study evaluated the prognostic significance of serum free light chains (sFLC, kappa, and lambda) and other serum markers (bar, serum thymidine kinase (sTK), soluble CD23, and LDH) together with established biomarkers in 289 patients enrolled into the LRF CLL4 trial. In a multivariable analysis of serum markers alone, higher big and kappa light chains were statistically significant in predicting disease progression and higher blg, and sTK in predicting mortality. In multivariable analysis for overall survival the following were independently significant: β2M levels, immunoglobulin gene (IGHV) mutational status (>98% homology), age, 17p13 deletions (>10%), and CD38 expression. β2M is the only serum marker that retained clear independent value as a biomarker in the LRF CLL4 trial and remains powerfully prognostic requiring evaluation in any future method of risk stratifying patients. PMID:26732125

  2. A new mammalian host cell with enhanced survival enables completely serum-free development of high-level protein production cell lines.

    PubMed

    Rossi, Diane L; Rossi, Edmund A; Goldenberg, David M; Chang, Chien-Hsing

    2011-01-01

    With over 25 monoclonal antibodies (mAbs) currently approved and many more in development, there is considerable interest in gaining improved productivity by increasing cell density and enhancing cell survival of production cell lines. In addition, high costs and growing safety concerns with use of animal products have made the availability of serum-free cell lines more appealing. We elected to transfect the myeloma cell line Sp2/0-Ag14 with Bcl2-EEE, the constitutively active phosphomimetic mutant of Bcl2, for extended cell survival. After adaptation of the initial transfectants to serum-independent growth, a clone with superior growth properties, referred to as SpESF, was isolated and further subjected to iterative rounds of stressful growth over a period of 4 months. The effort resulted in the selection of a promising clone, designated SpESFX-10, which was shown to exhibit robust growth and resist apoptosis induced by sodium butyrate or glutamine deprivation. The advantage of SpESFX-10 as a host for generating mAb-production cell lines was demonstrated by its increased transfection efficiency, culture longevity, and mAb productivity, as well as by the feasibility of accomplishing the entire cell line development process, including transfection, subcloning, and cryopreservation, in the complete absence of serum. PMID:21473000

  3. Defining Infertility

    MedlinePlus

    ... of the American Society for Reproductive Medicine Defining infertility What is infertility? Infertility is “the inability to conceive after 12 months ... to conceive after 6 months is generally considered infertility. How common is it? Infertility affects 10%-15% ...

  4. Defining Risk.

    ERIC Educational Resources Information Center

    Tholkes, Ben F.

    1998-01-01

    Defines camping risks and lists types and examples: (1) objective risk beyond control; (2) calculated risk based on personal choice; (3) perceived risk; and (4) reckless risk. Describes campers to watch ("immortals" and abdicators), and several "treatments" of risk: avoidance, safety procedures and well-trained staff, adequate insurance, and a…

  5. Defining cure.

    PubMed

    Hilton, Paul; Robinson, Dudley

    2011-06-01

    This paper is a summary of the presentations made as Proposal 2-"Defining cure" to the 2nd Annual meeting of the ICI-Research Society, in Bristol, 16th June 2010. It reviews definitions of 'cure' and 'outcome', and considers the impact that varying definition may have on prevalence studies and cure rates. The difference between subjective and objective outcomes is considered, and the significance that these different outcomes may have for different stakeholders (e.g. clinicians, patients, carers, industry etc.) is discussed. The development of patient reported outcome measures and patient defined goals is reviewed, and consideration given to the use of composite end-points. A series of proposals are made by authors and discussants as to how currently validated outcomes should be applied, and where our future research activity in this area might be directed. PMID:21661023

  6. Ca, Sr, O and D isotope approach to defining the chemical evolution of hydrothermal fluids: example from Long Valley, CA, USA

    USGS Publications Warehouse

    Brown, Shaun T.; Kennedy, B. Mack; DePaolo, Donald J.; Hurwitz, Shaul; Evans, William C.

    2013-01-01

    We present chemical and isotopic data for fluids, minerals and rocks from the Long Valley meteoric-hydrothermal system. The samples encompass the presumed hydrothermal upwelling zone in the west moat of the caldera, the Casa Diablo geothermal field, and a series of wells defining a nearly linear, ∼16 km long, west-to-east trend along the likely fluid flow path. Fluid samples were analyzed for the isotopes of water, Sr, and Ca, the concentrations of major cations and anions, alkalinity, and total CO2. Water isotope data conform to trends documented in earlier studies, interpreted as indicating a single hydrothermal fluid mixing with local groundwater. Sr isotopes show subtle changes along the flow path, which requires rapid fluid flow and minimal reaction between the channelized fluids and the wallrocks. Sr and O isotopes are used to calculate fracture spacing using a dual porosity model. Calculated fracture spacing and temperature data for hydrothermal fluids indicate the system is (approximately) at steady-state. Correlated variations among total CO2, and the concentration and isotopic composition of Ca suggest progressive fluid degassing (loss of CO2), which drives calcite precipitation as the fluid flows west-to-east and cools. The shifts in Ca isotopes require that calcite precipitated at temperatures of 150–180 °C is fractionated by ca. −0.3‰ to −0.5‰ relative to aqueous species. Our data are the first evidence that Ca isotopes undergo kinetic fractionation at high temperatures (>100 °C) and can be used to trace calcite precipitation along hydrothermal fluid flow paths.

  7. Conscious worst case definition for risk assessment, part I: a knowledge mapping approach for defining most critical risk factors in integrative risk management of chemicals and nanomaterials.

    PubMed

    Sørensen, Peter B; Thomsen, Marianne; Assmuth, Timo; Grieger, Khara D; Baun, Anders

    2010-08-15

    This paper helps bridge the gap between scientists and other stakeholders in the areas of human and environmental risk management of chemicals and engineered nanomaterials. This connection is needed due to the evolution of stakeholder awareness and scientific progress related to human and environmental health which involves complex methodological demands on risk management. At the same time, the available scientific knowledge is also becoming more scattered across multiple scientific disciplines. Hence, the understanding of potentially risky situations is increasingly multifaceted, which again challenges risk assessors in terms of giving the 'right' relative priority to the multitude of contributing risk factors. A critical issue is therefore to develop procedures that can identify and evaluate worst case risk conditions which may be input to risk level predictions. Therefore, this paper suggests a conceptual modelling procedure that is able to define appropriate worst case conditions in complex risk management. The result of the analysis is an assembly of system models, denoted the Worst Case Definition (WCD) model, to set up and evaluate the conditions of multi-dimensional risk identification and risk quantification. The model can help optimize risk assessment planning by initial screening level analyses and guiding quantitative assessment in relation to knowledge needs for better decision support concerning environmental and human health protection or risk reduction. The WCD model facilitates the evaluation of fundamental uncertainty using knowledge mapping principles and techniques in a way that can improve a complete uncertainty analysis. Ultimately, the WCD is applicable for describing risk contributing factors in relation to many different types of risk management problems since it transparently and effectively handles assumptions and definitions and allows the integration of different forms of knowledge, thereby supporting the inclusion of multifaceted risk

  8. Functional Langerinhigh-Expressing Langerhans-like Cells Can Arise from CD14highCD16- Human Blood Monocytes in Serum-Free Condition.

    PubMed

    Picarda, Gaëlle; Chéneau, Coraline; Humbert, Jean-Marc; Bériou, Gaëlle; Pilet, Paul; Martin, Jérôme; Duteille, Franck; Perrot, Pierre; Bellier-Waast, Frédérique; Heslan, Michèle; Haspot, Fabienne; Guillon, Fabien; Josien, Regis; Halary, Franck Albert

    2016-05-01

    Langerhans cells (LCs) are epithelial APCs that sense danger signals and in turn trigger specific immune responses. In steady-state, they participate in the maintenance of peripheral tolerance to self-antigens whereas under inflammation LCs efficiently trigger immune responses in secondary lymphoid organs. It has been demonstrated in mice that LC-deprived epithelia are rapidly replenished by short half-life langerin-expressing monocyte-derived LCs (MDLCs). These surrogate LCs are thought to be progressively replaced by langerin(high) LCs arising from self-renewing epithelial precursors of hematopoietic origin. How LCs arise from blood monocytes is not fully understood. Hence, we sought to characterize key factors that induce differentiation of langerin(high)-expressing monocyte-derived Langerhans-like cells. We identified GM-CSF and TGF-β1 as key cytokines to generate langerin(high)-expressing cells but only in serum-free conditions. These cells were shown to express the LC-specific TROP-2 and Axl surface markers and contained Birbeck granules. Surprisingly, E-cadherin was not spontaneously expressed by these cells but required a direct contact with keratinocytes to be stably induced. MDLCs induced stronger allogeneic T cell proliferations but released low amounts of inflammatory cytokines upon TLR stimulation compared with donor-paired monocyte-derived dendritic cells. Immature langerin(high) MDLCs were responsive to MIP-3β/CCL20 and CTAC/CCL27 chemokine stimulations. Finally, we demonstrated that those cells behaved as bona fide LCs when inserted in a three-dimensional rebuilt epithelium by becoming activated upon TLR or UV light stimulations. Collectively, these results prompt us to propose these langerin(high) MDLCs as a relevant model to address LC biology-related questions. PMID:27016604

  9. Iron absorption by intestinal epithelial cells: 1. CaCo2 cells cultivated in serum-free medium, on polyethyleneterephthalate microporous membranes, as an in vitro model.

    PubMed

    Halleux, C; Schneider, Y J

    1991-04-01

    Iron absorption by intestinal epithelial cells, passage onto plasmatic apotransferrin, and regulation of the process remain largely misunderstood. To investigate this problem, we have set up an in vitro model, consisting in CaCo2 cells (a human colon adenocarcinoma line, which upon cultivation displays numerous differentiation criteria of small intestine epithelial cells). Cells are cultivated in a serum-free medium, containing 1 microgram/ml insulin, 1 ng/ml epidermal growth factor, 10 micrograms/ml albumin-linoleic acid, 100 nM hydrocortisone, and 2 nM T3 on new, transparent, Cyclopore polyethyleneterephthalate microporous membranes coated with type I collagen. Cells rapidly adhere, grow, and form confluent monolayers; after 15 days, scanning electron microscopy reveals numerous uniform microvilli. Domes, which develop on nonporous substrata, are absent on high porosity membranes. Culture medium from upper and lower compartments of microplate inserts and cell lysates were immunoprecipitated after labeling with [3H]glucosamine and leucine; analysis was done by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by autoradiography. [3H]transferrin is found mainly in the lower compartment and in cells; [3H]apolipoprotein B is released in both compartments, and fibronectin almost entirely recovered in the lower compartment; [3H]transferrin receptors and ferritin are only present in cell lysates. Binding experiments also show that transferrin receptors are accessible from the lower compartment. These results suggest that CaCo2 cells, cultivated in synthetic medium on membranes of appropriate porosity, could provide an in vitro model of the intestinal barrier, with the upper compartment of the culture insert corresponding to the apical pole facing the intestinal lumen and the lower one to the basal pole in contact with blood. PMID:1830303

  10. Inactivated Enterovirus 71 Vaccine Produced by 200-L Scale Serum-Free Microcarrier Bioreactor System Provides Cross-Protective Efficacy in Human SCARB2 Transgenic Mouse

    PubMed Central

    Wu, Chia-Ying; Lin, Yi-Wen; Kuo, Chia-Ho; Liu, Wan-Hsin; Tai, Hsiu-Fen; Pan, Chien-Hung; Chen, Yung-Tsung; Hsiao, Pei-Wen; Chan, Chi-Hsien; Chang, Ching-Chuan; Liu, Chung-Cheng; Chow, Yen-Hung; Chen, Juine-Ruey

    2015-01-01

    Epidemics and outbreaks caused by infections of several subgenotypes of EV71 and other serotypes of coxsackie A viruses have raised serious public health concerns in the Asia-Pacific region. These concerns highlight the urgent need to develop a scalable manufacturing platform for producing an effective and sufficient quantity of vaccines against deadly enteroviruses. In this report, we present a platform for the large-scale production of a vaccine based on the inactivated EV71(E59-B4) virus. The viruses were produced in Vero cells in a 200 L bioreactor with serum-free medium, and the viral titer reached 107 TCID50/mL 10 days after infection when using an MOI of 10−4. The EV71 virus particles were harvested and purified by sucrose density gradient centrifugation. Fractions containing viral particles were pooled based on ELISA and SDS-PAGE. TEM was used to characterize the morphologies of the viral particles. To evaluate the cross-protective efficacy of the EV71 vaccine, the pooled antigens were combined with squalene-based adjuvant (AddaVAX) or aluminum phosphate (AlPO4) and tested in human SCARB2 transgenic (Tg) mice. The Tg mice immunized with either the AddaVAX- or AlPO4-adjuvanted EV71 vaccine were fully protected from challenges by the subgenotype C2 and C4 viruses, and surviving animals did not show any degree of neurological paralysis symptoms or muscle damage. Vaccine treatments significantly reduced virus antigen presented in the central nervous system of Tg mice and alleviated the virus-associated inflammatory response. These results strongly suggest that this preparation results in an efficacious vaccine and that the microcarrier/bioreactor platform offers a superior alternative to the previously described roller-bottle system. PMID:26287531

  11. A robust and reproducible animal serum-free culture method for clinical-grade bone marrow-derived mesenchymal stromal cells.

    PubMed

    Laitinen, Anita; Oja, Sofia; Kilpinen, Lotta; Kaartinen, Tanja; Möller, Johanna; Laitinen, Saara; Korhonen, Matti; Nystedt, Johanna

    2016-08-01

    Efficient xenofree expansion methods to replace fetal bovine serum (FBS)-based culture methods are strongly encouraged by the regulators and are needed to facilitate the adoption of mesenchymal stromal cell (MSC)-based therapies. In the current study we established a clinically-compliant and reproducible animal serum-free culture protocol for bone marrow-(BM-) MSCs based on an optimized platelet-derived supplement. Our study compared two different platelet-derived supplements, platelet lysate PL1 versus PL2, produced by two different methods and lysed with different amounts of freeze-thaw cycles. Our study also explored the effect of a low oxygen concentration on BM-MSCs. FBS-supplemented BM-MSC culture served as control. Growth kinetics, differentiation and immunomodulatory potential, morphology, karyotype and immunophenotype was analysed. Growth kinetics in long-term culture was also studied. Based on the initial results, we chose to further process develop the PL1-supplemented culture protocol at 20 % oxygen. The results from 11 individual BM-MSC batches expanded in the chosen condition were consistent, yielding 6.60 × 10(9) ± 4.74 × 10(9) cells from only 20 ml of bone marrow. The cells suppressed T-cell proliferation, displayed normal karyotype and typical MSC differentiation potential and phenotype. The BM-MSCs were, however, consistently HLA-DR positive when cultured in platelet lysate (7.5-66.1 %). We additionally show that culture media antibiotics and sterile filtration of the platelet lysate can be successfully omitted. We present a robust and reproducible clinically-compliant culture method for BM-MSCs based on platelet lysate, which enables high quantities of HLA-DR positive MSCs at a low passage number (p2) and suitable for clinical use. PMID:25777046

  12. Inactivated Enterovirus 71 Vaccine Produced by 200-L Scale Serum-Free Microcarrier Bioreactor System Provides Cross-Protective Efficacy in Human SCARB2 Transgenic Mouse.

    PubMed

    Wu, Chia-Ying; Lin, Yi-Wen; Kuo, Chia-Ho; Liu, Wan-Hsin; Tai, Hsiu-Fen; Pan, Chien-Hung; Chen, Yung-Tsung; Hsiao, Pei-Wen; Chan, Chi-Hsien; Chang, Ching-Chuan; Liu, Chung-Cheng; Chow, Yen-Hung; Chen, Juine-Ruey

    2015-01-01

    Epidemics and outbreaks caused by infections of several subgenotypes of EV71 and other serotypes of coxsackie A viruses have raised serious public health concerns in the Asia-Pacific region. These concerns highlight the urgent need to develop a scalable manufacturing platform for producing an effective and sufficient quantity of vaccines against deadly enteroviruses. In this report, we present a platform for the large-scale production of a vaccine based on the inactivated EV71(E59-B4) virus. The viruses were produced in Vero cells in a 200 L bioreactor with serum-free medium, and the viral titer reached 10(7) TCID50/mL 10 days after infection when using an MOI of 10(-4). The EV71 virus particles were harvested and purified by sucrose density gradient centrifugation. Fractions containing viral particles were pooled based on ELISA and SDS-PAGE. TEM was used to characterize the morphologies of the viral particles. To evaluate the cross-protective efficacy of the EV71 vaccine, the pooled antigens were combined with squalene-based adjuvant (AddaVAX) or aluminum phosphate (AlPO4) and tested in human SCARB2 transgenic (Tg) mice. The Tg mice immunized with either the AddaVAX- or AlPO4-adjuvanted EV71 vaccine were fully protected from challenges by the subgenotype C2 and C4 viruses, and surviving animals did not show any degree of neurological paralysis symptoms or muscle damage. Vaccine treatments significantly reduced virus antigen presented in the central nervous system of Tg mice and alleviated the virus-associated inflammatory response. These results strongly suggest that this preparation results in an efficacious vaccine and that the microcarrier/bioreactor platform offers a superior alternative to the previously described roller-bottle system. PMID:26287531

  13. Defining Life

    PubMed Central

    2010-01-01

    Abstract Any definition is intricately connected to a theory that gives it meaning. Accordingly, this article discusses various definitions of life held in the astrobiology community by considering their connected “theories of life.” These include certain “list” definitions and a popular definition that holds that life is a “self-sustaining chemical system capable of Darwinian evolution.” We then act as “anthropologists,” studying what scientists do to determine which definition-theories of life they constructively hold as they design missions to seek non-terran life. We also look at how constructive beliefs about biosignatures change as observational data accumulate. And we consider how a definition centered on Darwinian evolution might itself be forced to change as supra-Darwinian species emerge, including in our descendents, and consider the chances of our encountering supra-Darwinian species in our exploration of the Cosmos. Last, we ask what chemical structures might support Darwinian evolution universally; these structures might be universal biosignatures. Key Words: Evolution—Life—Life detection—Biosignatures. Astrobiology 10, 1021–1030. PMID:21162682

  14. Defining chaos

    SciTech Connect

    Hunt, Brian R.; Ott, Edward

    2015-09-15

    In this paper, we propose, discuss, and illustrate a computationally feasible definition of chaos which can be applied very generally to situations that are commonly encountered, including attractors, repellers, and non-periodically forced systems. This definition is based on an entropy-like quantity, which we call “expansion entropy,” and we define chaos as occurring when this quantity is positive. We relate and compare expansion entropy to the well-known concept of topological entropy to which it is equivalent under appropriate conditions. We also present example illustrations, discuss computational implementations, and point out issues arising from attempts at giving definitions of chaos that are not entropy-based.

  15. Culturing in serum-free culture medium on collagen type-I-coated plate increases expression of CD133 and retains original phenotype of HT-29 cancer stem cell

    PubMed Central

    Arab-Bafrani, Zahra; Shahbazi-Gahrouei, Daryoush; Abbasian, Mehdi; Saberi, Alihossein; Fesharaki, Mehrafarin; Hejazi, Seyed Hossein; Manshaee, Samira

    2016-01-01

    Background: A sub-population of tumor cells termed cancer stem cells (CSCs) has an important role in tumor initiation, progression, and recurrence. Selecting a suitable procedure for isolation and enrichment of CSCs is the biggest challenge in the study of CSCs. In the present study, the role of the combination of stem cell culture medium and collagen type-I was evaluated for successful isolation and enrichment of HT-29 CSCs. Materials and Methods: HT-29 cells were cultured in serum-containing medium (parental culture medium: Medium + 10% fetal bovine serum) and serum-free medium (stem cell culture medium); both on collagen-coated plates. Spheres forming ability and CD133 expression, as a potential marker of colorectal CSCs, were evaluated in two culture mediums. Results: The results show spheroids usually give rise completely within 15 days in the stem cell culture medium on the collagen-coated plate. CD133 expression in spheroid cells (84%) is extensively higher than in parental cells (25%). Moreover, relative to parental cells, spheroid cells were more radioresistance. Conclusion: Finding of this study suggested that CSCs derived from colon cancer cell line (HT-29) can be propagated and form colonospheres in serum-free culture medium on collagen type-I. According to maintenance of their original phenotype in these conditions, it seems serum-free culture medium on collagen type-I is a suitable way to drug screening of HT-29 CSCs. PMID:27135028

  16. Monoclonal and polyclonal gammopathy measured by serum free light chain and immunofixation subdivide the clinical outcomes of diffuse large B-cell lymphoma according to molecular classification.

    PubMed

    Kim, Yu Ri; Kim, Soo-Jeong; Cheong, June-Won; Kim, Yundeok; Jang, Ji Eun; Lee, Jung Yeon; Min, Yoo Hong; Song, Jae-Woo; Yang, Woo Ick; Kim, Jin Seok

    2014-11-01

    Elevated serum free light chain (FLC) is known to be an adverse prognostic factor for diffuse large B-cell lymphoma (DLBCL). We hypothesized that monoclonal gammopathy (MG; elevated kappa [κ] or lambda [λ] FLC with an abnormal κ/λ ratio or a positive IF [immunofixation]) and polyclonal gammopathy (PG; elevated κ and/or λ FLC with a normal κ/λ ratio and a negative IF) would have different clinical outcome according to the molecular classification of DLBCL. In addition, MG would be a poor prognostic factor in patients with activated B-cell like type of DLBCL. Molecular classification of DLBCL, such as germinal center B-cell (GCB) type and non-GCB type, was performed according to the Hans algorithm. Among 175 newly diagnosed DLBCL patients, 96 (54.9 %) patients had an elevated FLC. MG and PG were observed in 34 and 68 patients, respectively. The 2-year overall survival (OS) and event-free survival (EFS) rates were 79.0 % and 71.6 %, respectively. In multivariate analysis, high-intermediate/high International Prognostic Index score and elevated FLC were significant for the OS (P = 0.002, P = 0.005, respectively) and EFS (P < 0.002, P = 0.010, respectively). MG and PG were also associated with inferior OS (P = 0.002, P = 0.011, respectively) and EFS (P = 0.002, P = 0.013, respectively). Ninety-six patients from a total 133 evaluable patients were classified to the non-GCB type. Patients with PG showed inferior clinical outcome for OS and EFS in patients with the GCB type (P = 0.006, P = 0.035, respectively). MG was a significant poor prognostic factor for OS and EFS in patients with the non-GCB type (P = 0.017, P = 0.004, respectively). MG was a poor prognostic maker in patients with the non-GCB type and PG was a poor prognostic indicator for the GCB type of DLBCL who were treated with R-CHOP. PMID:24947797

  17. Zeta potential of transfection complexes formed in serum-free medium can predict in vitro gene transfer efficiency of transfection reagent.

    PubMed

    Son, K K; Tkach, D; Patel, D H

    2000-09-29

    We have tested the zeta potential (zeta, the surface charge density) of transfection complexes formed in serum-free medium as a rapid and reliable technique for screening transfection efficiency of a new reagent or formulation. The complexes of CAT plasmid DNA (1 microgram) and DC-chol/DOPE liposomes (3-20 nmol) were largely negatively charged (zeta=-15 to -21 mV), which became neutral or positive as 0.5 microgram or a higher amount of poly-L-lysine (PLL, MW 29300 or MW 204000) was added (-3.16+/-3.47 to +6.04+/-2.23 mV). However, the complexes of CAT plasmid DNA (1 microgram) and PLL MW 29300 (0.5 microgram or higher) were neutral or positively charged (-3.22+/-2.3 to +6.55+/-0.64 mV), which remained the same as 6.6 nmol of the liposomes was added. The complexes formed between two positively charged compounds, PLL MW 29300 (0.5 microgram) and the liposomes (3-20 nmol), were as closely positively charged as DNA/PLL or DNA/liposomes/PLL complexes (+3.31+/-0.41 to 7.16+/-1.0 mV). These results indicate that PLL determined the overall charge of the DNA/liposome/PLL ternary complexes. The complexes formed with histone (0.75 microgram or higher) were also positively charged, whose transfection activity was as high as PLL MW 29300. However, the complexes formed with protamine or PLL MW 2400 remained negatively charged. These observations are in good agreement with the transfection activity of the formulation containing each polycationic polymer. The presence of PLL MW 29300 did not change the hydrodynamic diameter of DNA/liposome/PLL complexes (d(H)=275-312 nm). The complexes made of different sizes of PLL (MW 2400 and 204000) also did not significantly change their size. This suggests that DNA condensation may not be critical. Therefore, zeta of the transfection complex can predict the transfection efficiency of a new formulation or reagent. PMID:11018646

  18. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions

    PubMed Central

    Lian, Xiaojun; Zhang, Jianhua; Azarin, Samira M.; Zhu, Kexian; Hazeltine, Laurie B.; Bao, Xiaoping; Hsiao, Cheston; Kamp, Timothy J.; Palecek, Sean P.

    2013-01-01

    The protocols described here efficiently direct human pluripotent stem cells (hPSCs) to functional cardiomyocytes in a completely defined, serum-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate temporal application of Gsk3 inhibitor followed by expression of β-catenin shRNA or a chemical Wnt inhibitor is sufficient to produce a high yield (0.8–1.3 million cardiomyocytes/cm2) of virtually pure (80%–98%) functional cardiomyocytes from multiple hPSC lines without cell sorting or selection. Characterization of differentiated cells is performed in qualitative (immunostaining) and quantitative (flow cytometry) manners to assess expression of cardiac transcription factors and myofilament proteins. Flow cytometry of BrdU incorporation or Ki67 expression in conjuction with cardiac sarcomere myosin protein expression can be used to determine the proliferative capacity of hPSC-derived cardiomyocytes. Functional human cardiomyocytes differentiated via these protocols may constitute a potential cell source for heart disease modeling, drug screening, and cell-based therapeutic applications. PMID:23257984

  19. Quantitative and qualitative shifts in defensive metabolites define chemical defense investment during leaf development in Inga, a genus of tropical trees.

    PubMed

    Wiggins, Natasha L; Forrister, Dale L; Endara, María-José; Coley, Phyllis D; Kursar, Thomas A

    2016-01-01

    Selective pressures imposed by herbivores are often positively correlated with investments that plants make in defense. Research based on the framework of an evolutionary arms race has improved our understanding of why the amount and types of defenses differ between plant species. However, plant species are exposed to different selective pressures during the life of a leaf, such that expanding leaves suffer more damage from herbivores and pathogens than mature leaves. We hypothesize that this differential selective pressure may result in contrasting quantitative and qualitative defense investment in plants exposed to natural selective pressures in the field. To characterize shifts in chemical defenses, we chose six species of Inga, a speciose Neotropical tree genus. Focal species represent diverse chemical, morphological, and developmental defense traits and were collected from a single site in the Amazonian rainforest. Chemical defenses were measured gravimetrically and by characterizing the metabolome of expanding and mature leaves. Quantitative investment in phenolics plus saponins, the major classes of chemical defenses identified in Inga, was greater for expanding than mature leaves (46% and 24% of dry weight, respectively). This supports the theory that, because expanding leaves are under greater selective pressure from herbivores, they rely more upon chemical defense as an antiherbivore strategy than do mature leaves. Qualitatively, mature and expanding leaves were distinct and mature leaves contained more total and unique metabolites. Intraspecific variation was greater for mature leaves than expanding leaves, suggesting that leaf development is canalized. This study provides a snapshot of chemical defense investment in a speciose genus of tropical trees during the short, few-week period of leaf development. Exploring the metabolome through quantitative and qualitative profiling enables a more comprehensive examination of foliar chemical defense investment

  20. CYTOTOXICITY OF CHEMICAL CARCINOGENS TOWARDS HUMAN BRONCHIAL EPITHELIAL CELLS EVALUATED IN A CLONAL ASSAY

    EPA Science Inventory

    Survival of human bronchial epithelial cells after administration of four chemical carcinogens was measured in a clonal assay. Human bronchial epithelial cells were obtained from outgrowths of explanted tissue pieces. Serum-free medium was used for both explant culture and clonal...

  1. Fetal bovine serum and human constitutive androstane receptor: Evidence for activation of the SV23 splice variant by artemisinin, artemether, and arteether in a serum-free cell culture system

    SciTech Connect

    Lau, Aik Jiang; Chang, Thomas K.H.

    2014-06-01

    The naturally occurring SV23 splice variant of human constitutive androstane receptor (hCAR-SV23) is activated by di-(2-ethylhexyl)phthalate (DEHP), which is detected as a contaminant in fetal bovine serum (FBS). In our initial experiment, we compared the effect of dialyzed FBS, charcoal-stripped, dextran-treated FBS (CS-FBS), and regular FBS on the basal activity and ligand-activation of hCAR-SV23 in a cell-based reporter gene assay. In transfected HepG2 cells cultured in medium supplemented with 10% FBS, basal hCAR-SV23 activity varied with the type of FBS (regular > dialyzed > CS). DEHP increased hCAR-SV23 activity when 10% CS-FBS, but not regular FBS or dialyzed FBS, was used. With increasing concentrations (1–10%) of regular FBS or CS-FBS, hCAR-SV23 basal activity increased, whereas in DEHP-treated cells, hCAR-SV23 activity remained similar (regular FBS) or slightly increased (CS-FBS). Subsequent experiments identified a serum-free culture condition to detect DEHP activation of hCAR-SV23. Under this condition, artemisinin, artemether, and arteether increased hCAR-SV23 activity, whereas they decreased it in cells cultured in medium supplemented with 10% regular FBS. By comparison, FBS increased the basal activity of the wild-type isoform of hCAR (hCAR-WT), whereas it did not affect the basal activity of the SV24 splice variant (hCAR-SV24) or ligand activation of hCAR-SV24 and hCAR-WT by 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO). The use of serum-free culture condition was suitable for detecting CITCO activation of hCAR-WT and hCAR-SV24. In conclusion, FBS leads to erroneous classification of pharmacological ligands of hCAR-SV23 in cell-based assays, but investigations on functional ligands of hCAR isoforms can be conducted in serum-free culture condition. - Highlights: • FBS leads to erroneous pharmacological classification of hCAR-SV23 ligands. • Artemisinin, artemether, and arteether activate h

  2. Role of glass structure in defining the chemical dissolution behavior, bioactivity and antioxidant properties of zinc and strontium co-doped alkali-free phosphosilicate glasses.

    PubMed

    Kapoor, Saurabh; Goel, Ashutosh; Tilocca, Antonio; Dhuna, Vikram; Bhatia, Gaurav; Dhuna, Kshitija; Ferreira, José M F

    2014-07-01

    We investigated the structure-property relationships in a series of alkali-free phosphosilicate glass compositions co-doped with Zn(2+) and Sr(2+). The emphasis was laid on understanding the structural role of Sr(2+) and Zn(2+) co-doping on the chemical dissolution behavior of glasses and its impact on their in vitro bioactivity. The structure of glasses was studied using molecular dynamics simulations in combination with solid state nuclear magnetic resonance spectroscopy. The relevant structural properties are then linked to the observed degradation behavior, in vitro bioactivity, osteoblast proliferation and oxidative stress levels. The apatite-forming ability of glasses has been investigated by X-ray diffraction, infrared spectroscopy and scanning electron microscopy-energy-dispersive spectroscopy after immersion of glass powders/bulk in simulated body fluid (SBF) for time durations varying between 1h and 14 days, while their chemical degradation has been studied in Tris-HCl in accordance with ISO 10993-14. All the glasses exhibit hydroxyapatite formation on their surface within 1-3h of their immersion in SBF. The cellular responses were observed in vitro on bulk glass samples using human osteosarcoma MG63 cell line. The dose-dependent cytoprotective effect of glasses with respect to the concentration of zinc and strontium released from the glasses is also discussed. PMID:24709542

  3. Integration of multi-channel piezometry and electrical tomography to better define chemical heterogeneity in a landfill leachate plume within a sand aquifer.

    PubMed

    Acworth, R I; Jorstad, L B

    2006-02-10

    The Hanai-Bruggeman effective medium theory is used to relate bulk electrical conductivity, measured by surface and cross-borehole images, to fluid electrical conductivity, surface conductance, porosity and the geometry factor, in a medium- to fine-grained sand deposit. The change in bulk EC is caused by the presence of a landfill leachate plume. Repeated electrical images over a period of 16 months indicate that various segments of the plume are moving. The chemical constituents of the leachate plume have been determined by sampling from a bundled piezometer located in the electrical image field. Very close agreement is demonstrated between the fluid EC anomaly and the presence of elevated bulk EC indicating that the electrical images can be used to map the plume geometry and to monitor the movement of the plume segments. PMID:16412530

  4. The influence of fetal sex in screening for trisomy 21 by fetal nuchal translucency, maternal serum free beta-hCG and PAPP-A at 10-14 weeks of gestation.

    PubMed

    Spencer, K; Ong, C Y; Liao, A W; Papademetriou, D; Nicolaides, K H

    2000-08-01

    In a study of 2923 normal pregnancies and 203 pregnancies affected by trisomy 21 we have shown a significant difference in the median MoM of the markers: fetal nuchal translucency, maternal serum free beta-hCG and PAPP-A in the presence of a female fetus compared with a male fetus. For maternal serum free beta-hCG levels are higher by 15% if the fetus is chromosomally normal and by 11% if the fetus has trisomy 21. For maternal serum PAPP-A the levels in chromosomally normal fetuses are 10% higher in the presence of a female fetus and 13% higher if the fetus has trisomy 21. In contrast, fetal nuchal translucency is 3-4% lower in both chromosomally normal and trisomy 21 female fetuses. The consequence of such changes when screening for trisomy 21 will be a reduction in the detection rate in female fetuses by a factor of 1-2%. Correction of risk algorithms for fetal sex, however, is probably not feasible, since ultrasound detection of fetal sex is only 70-90% accurate in the 10-14 week period. PMID:10951481

  5. The importance of defining chemical potentials, substitution mechanisms and solubility in trace element diffusion studies: the case of Zr and Hf in olivine

    NASA Astrophysics Data System (ADS)

    Jollands, Michael C.; O'Neill, Hugh St. C.; Hermann, Jörg

    2014-09-01

    The diffusion, substitution mechanism and solubility limits of Zr and Hf in synthetic forsterite (Mg2SiO4) and San Carlos olivine (Mg0.9Fe0.1)2SiO4 have been investigated between 1,200 and 1,500 °C as a function of the chemical potentials of the components in the system MgO(FeO)-SiO2-ZrO2(HfO2). The effect of oxygen fugacity and crystallographic orientation were also investigated. The solubilities of Zr in forsterite are highest and diffusion fastest when the coexisting three-phase source assemblage includes ZrSiO4 (zircon) or HfSiO4 (hafnon), and lower and slower, respectively, when the source assemblage includes MgO (periclase). This indicates that Zr and Hf substitute on the octahedral sites in olivine, charge balanced by magnesium vacancies. Diffusion is anisotropic, with rates along the crystal axes increasing in the order a < b < c. The generalized diffusion relationship as a function of chemical activity (as ), orientation and temperature is: where the values of log D 0 are -3.8(±0.5), -3.4(±0.5) and -3.1(±0.5) along the a, b and c axes, respectively. Most experiments were conducted in air ( fO2 = 10-0.68 bars), but one at fO2 = 10-11.2 bars at 1,400 °C shows no resolvable effect of oxygen fugacity on Zr diffusion. Hf is slightly more soluble in olivine than Zr, but diffuses slightly slower. Diffusivities of Zr in experiments in San Carlos olivine at 1,400 °C, fO2 = 10-6.6 bars are similar to those in forsterite at the same conditions, showing that the controls on diffusivities are adequately captured by the simple system (nominally iron-free) experiments. Diffusivities are in good agreement with those measured by Spandler and O'Neill (Contrib Miner Petrol 159:791-818, 2010) in San Carlos olivine using silicate melt as the source at 1,300 °C, and fall within the range of most measurements of Fe-Mg inter-diffusion in olivine at this temperature. Forsterite-melt partitioning experiments in the CaO-MgO-Al2O3-SiO2-ZrO2/HfO2 show that the interface

  6. Effect of processing of rapeseed under defined conditions in a pilot plant on chemical composition and standardized ileal amino acid digestibility in rapeseed meal for pigs.

    PubMed

    Eklund, M; Sauer, N; Schöne, F; Messerschmidt, U; Rosenfelder, P; Htoo, J K; Mosenthin, R

    2015-06-01

    Five rapeseed meals (RSM) were produced from a single batch of rapeseed in a large-scale pilot plant under standardized conditions. The objective was to evaluate the effect of residence time in the desolventizer/toaster (DT) on chemical composition and standardized ileal digestibility (SID) of AA in RSM. Four RSM, with 48, 64, 76, and 93 min residence time and using unsaturated steam in the DT, referred to as RSM48, RSM64, RSM76, and RSM93, respectively, and 1 low-glucosinolate RSM, which was subjected to sequential treatment with unsaturated steam, saturated steam, and dry heat in the DT, referred to as low-GSL RSM, were assayed. Six barrows (average initial BW = 22 ± 1 kg) were surgically fitted with a T-cannula at the distal ileum. Pigs were allotted to a 5 × 6 row × column design with 5 diets and 5 periods. The 5 RSM were included in a cornstarch-casein-based basal diet. In addition, basal ileal endogenous losses and SID of AA originating from casein were determined at the conclusion of the experiment in 2 additional periods by means of the regression method and using 3 graded levels of casein. The SID of AA in the 5 RSM was determined in difference to SID of AA originating from casein. The glucosinolates (GSL) were efficiently reduced, whereas NDF, ADF, ADL, and NDIN contents increased and reactive Lys (rLys) and Lys:CP ratio decreased as the residence time in the DT was increased from 48 to 93 min. The SID of most AA in RSM linearly decreased (P < 0.05) as the residence time in the DT increased from 48 to 93 min. Moreover, there was a linear decrease (P < 0.05) in SID of AA with increasing NDF, ADF, ADL, and NDIN contents in these RSM, whereas SID of AA linearly decreased (P < 0.05) with decreasing levels of GSL and rLys and a decreasing Lys:CP ratio. The decrease (P < 0.05) in SID of AA amounted from 3 up to 6 (percentage units) for most AA, except for SID of Cys and Lys, which decreased by 10 and 11%-units (P < 0.05), respectively, as the residence time

  7. Influence of caffeine consumption on 7,12-dimethylbenz(a)anthracene-induced mammary gland tumorigenesis in female rats fed a chemically defined diet containing standard and high levels of unsaturated fat.

    PubMed

    Welsch, C W; DeHoog, J V

    1988-04-15

    The effect of caffeine (430-500 mg/liter of drinking water) on the initiation and promotion phases of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary gland tumorigenesis in female Sprague-Dawley rats fed a chemically defined diet containing standard (5%) or high (20%) levels of fat (corn oil) was examined. In the initiation studies, caffeine and the standard or high fat diet treatments were provided for 34 days, from 24-29 days of age to 58-63 days of age. Three days prior to termination of caffeine-fat diet treatments, each rat received a single dose of DMBA. In the promotion studies, caffeine and the standard or high fat diets were provided commencing 3 days after a single dose of DMBA (at 56-61 days of age) and until termination of the study. Caffeine consumption, during the initiation phase significantly (P less than 0.05) reduced mammary carcinoma multiplicity (number of tumors/rat), in rats fed either a standard or high fat diet. In the promotion studies, prolonged consumption of caffeine in rats fed either a standard or high fat diet did not significantly effect mammary carcinoma multiplicity. In the early stages of promotion, an apparent increase in mammary carcinoma multiplicity was observed; this increase in mammary carcinoma multiplicity did not, however, reach the 5% level of statistical probability. When caffeine was administered during both the initiation and promotion phases, no significant effect on mammary carcinoma multiplicity was observed. Treatment of rats during the initiation or promotion phases with caffeinated coffee (via drinking water) mimicked the mammary tumor modulating activities of caffeine. Decaffeinated coffee consumption did not effect either the initiation or promotion phases of this tumorigenic process. In both the initiation and promotion studies, caffeine and/or coffee consumption did not significantly affect the incidence of mammary carcinomas (percentage of rats bearing mammary carcinomas) or the mean latency period of

  8. Flt3/Flk-2-ligand in synergy with thrombopoietin delays megakaryocyte development and increases the numbers of megakaryocyte progenitor cells in serum-free cultures initiated with CD34+ cells.

    PubMed

    Sigurjónsson, Olafur Eysteinn; Gudmundsson, Kristbjörn Orri; Haraldsdóttir, Vilhelmína; Rafnar, Thorunn; Gudmundsson, Sveinn; Guomundsson, Kristbjörn Orri; Guomundsson, Sveinn

    2002-04-01

    Megakaryocytopoiesis involves proliferation and maturation of committed precursors that increase their size by polyploidy, a process that is believed to be critical for the efficient production and release of platelets. Thrombopoietin has been shown to act on proliferation, maturation, and survival pathways in megakaryocytopoiesis. Less is known about the role of Flt3/Flk-2-ligand in this development. Apoptosis has an important role in hematopoiesis in general. It has been shown to have an effect on senescent megakaryocytes but not megakaryocyte progenitor cells. In this study, a serum-free culture model was developed, differentiating bone marrow CD34(+) hematopoietic stem cells into megakaryocytes, using thrombopoietin and Flt3/Flk-2-ligand. The model was used to study the effect of these growth factors on expansion of megakaryocyte progenitor cells, differentiation of megakaryocytes, and ploidy. Our results demonstrate that bone marrow CD34(+) cells cultured with thrombopoietin and Flt3/Flk-2-ligand show a lower developmental rate into MK cells compared to cells cultured with thrombopoietin alone. Cells cultured with thrombopoietin and Flt3/Flk-2-ligand expressed less CD41, the ploidy level was lower, and they appeared less mature. On the other hand, the cells showed up to 10-fold increase in cell numbers compared to five-fold increase when cultured with thrombopoietin alone. These results suggest that Flt3/Flk-2-ligand in synergy with thrombopoietin may slow down megakaryocyte development by causing increased proliferation of megakaryocyte progenitor cells. PMID:11983110

  9. CD163 and c-Met expression in the lymph node and the correlations between elevated levels of serum free light chain and the different clinicopathological parameters of advanced classical Hodgkin's lymphoma

    PubMed Central

    Bedewy, Magdy; El-Maghraby, Shereen

    2013-01-01

    Background Advances in the understanding of Hodgkin's lymphoma (HL) show various functions of infiltrating immune cells and cytokines in relation to clinical outcomes. The expression of CD163 and c-Met has been suggested to have a role in lymphoid malignancy. Thus, we evaluated the expressions of CD163, c-Met, and serum free light chain (sFLC) in relation to the clinicopathological features of patients with advanced classical HL (cHL). Methods We assessed the expression of CD163 and c-Met in 34 patients with cHL through immunohistochemistry on the lymph node biopsy sections and the levels of pretreatment sFLC were estimated using ELISA. Results High CD163 expression correlated with increased age, B symptoms, International Prognostic Score (IPS) ≥3, mixed cellularity subtype, and low response to treatment. Further, high c-Met expression correlated with increased age at diagnosis, leukocytosis, B symptoms, and lower chance to achieve complete remission. The sFLC levels correlated with increased age at diagnosis, lymphopenia, IPS ≥3, B symptoms, and lower complete remission rates. Conclusion In advanced cHL, increased expression of CD163 and c-Met showed a significant association with adverse prognostic parameters and poor response to treatment. Pretreatment high sFLC level also correlated with poor risk factors, suggesting its use as a candidate prognostic marker. A comprehensive approach for prognostic markers might represent a step towards developing a tailored therapeutic approach for HL. PMID:23826581

  10. Solution-phase parallel synthesis of a pharmacophore library of HUN-7293 analogues: a general chemical mutagenesis approach to defining structure-function properties of naturally occurring cyclic (depsi)peptides.

    PubMed

    Chen, Yan; Bilban, Melitta; Foster, Carolyn A; Boger, Dale L

    2002-05-15

    HUN-7293 (1), a naturally occurring cyclic heptadepsipeptide, is a potent inhibitor of cell adhesion molecule expression (VCAM-1, ICAM-1, E-selectin), the overexpression of which is characteristic of chronic inflammatory diseases. Representative of a general approach to defining structure-function relationships of such cyclic (depsi)peptides, the parallel synthesis and evaluation of a complete library of key HUN-7293 analogues are detailed enlisting solution-phase techniques and simple acid-base liquid-liquid extractions for isolation and purification of intermediates and final products. Significant to the design of the studies and unique to solution-phase techniques, the library was assembled superimposing a divergent synthetic strategy onto a convergent total synthesis. An alanine scan and N-methyl deletion of each residue of the cyclic heptadepsipeptide identified key sites responsible for or contributing to the biological properties. The simultaneous preparation of a complete set of individual residue analogues further simplifying the structure allowed an assessment of each structural feature of 1, providing a detailed account of the structure-function relationships in a single study. Within this pharmacophore library prepared by systematic chemical mutagenesis of the natural product structure, simplified analogues possessing comparable potency and, in some instances, improved selectivity were identified. One potent member of this library proved to be an additional natural product in its own right, which we have come to refer to as HUN-7293B (8), being isolated from the microbial strain F/94-499709. PMID:11996584

  11. Three years of antibacterial consumption in Indonesian Community Health Centers: The application of anatomical therapeutic chemical/defined daily doses and drug utilization 90% method to monitor antibacterial use

    PubMed Central

    Pradipta, Ivan S.; Ronasih, Elis; Kartikawati, Arrum D.; Hartanto, Hartanto; Amelia, Rizki; Febrina, Ellin; Abdulah, Rizky

    2015-01-01

    Context: Irrational use of antibacterial drugs in Community Health-Care Centers (CHCs) may lead to increased resistance, morbidity, and mortality. Aims: The aim of this study was to determine patterns of antibacterial use at CHCs in a district of Indonesia and use this as data for an antibiotic policy. Settings and Design: The observational-descriptive study was conducted in a district of Indonesia to obtain antibacterial use from 2008 to 2010. Subjects and Methods: The data obtained from the report on the use of medicines were classified and processed using the anatomical therapeutic chemical (ATC) and defined daily doses (DDD) method, with DDD/1000 patients as a unit measurement. The number of patients was obtained from attending patients in that research period. The most abundant antibacterial drugs use segment was identified by the drug utilization 90% (DU90%) method. Statistical Analysis Used: Descriptive analysis were performed in this study. Results: Fourteen kinds of antibacterial drugs were used in 61 CHCs. The total of antibacterial drug use during the period 2008–2010 was 871.36 DDD/1000 patients/day. Declining antibacterial use was observed between 2008 and 2010. Six kinds of antibacterial drugs were the most commonly used. The data show that the average use per visit was as high as 24.41 DDD. Conclusions: Amoxicillin, sulfamethoxazole and trimethoprim are antibacterials that have to be reconsidered by physicians for use in the Bandung CHC. The high use of antibacterial drugs, as described in the study, can be used as reference to develop an antimicrobial stewardship program and increase awareness of resistance, adverse drug reaction and drug interaction of antibacterial drugs. PMID:25983606

  12. PER.C6(®) cells as a serum-free suspension cell platform for the production of high titer poliovirus: a potential low cost of goods option for world supply of inactivated poliovirus vaccine.

    PubMed

    Sanders, Barbara P; Edo-Matas, Diana; Custers, Jerome H H V; Koldijk, Martin H; Klaren, Vincent; Turk, Marije; Luitjens, Alfred; Bakker, Wilfried A M; Uytdehaag, Fons; Goudsmit, Jaap; Lewis, John A; Schuitemaker, Hanneke

    2013-01-21

    There are two highly efficacious poliovirus vaccines: Sabin's live-attenuated oral polio vaccine (OPV) and Salk's inactivated polio vaccine (IPV). OPV can be made at low costs per dose and is easily administrated. However, the major drawback is the frequent reversion of the OPV vaccine strains to virulent poliovirus strains which can result in Vaccine Associated Paralytic Poliomyelitis (VAPP) in vaccinees. Furthermore, some OPV revertants with high transmissibility can circulate in the population as circulating Vaccine Derived Polioviruses (cVDPVs). IPV does not convey VAPP and cVDPVs but the high costs per dose and insufficient supply have rendered IPV an unfavorable option for low and middle-income countries. Here, we explored whether the human PER.C6(®) cell-line, which has the unique capability to grow at high density in suspension, under serum-free conditions, could be used as a platform for high yield production of poliovirus. PER.C6(®) cells supported replication of all three poliovirus serotypes with virus titers ranging from 9.4 log(10) to 11.1 log(10)TCID(50)/ml irrespective of the volume scale (10 ml in shaker flasks to 2 L in bioreactors). This production yield was 10-30 fold higher than in Vero cell cultures performed here, and even 100-fold higher than what has been reported for Vero cell cultures in literature [38]. In agreement, the D-antigen content per volume PER.C6(®)-derived poliovirus was on average 30-fold higher than Vero-derived poliovirus. Interestingly, PER.C6(®) cells produced on average 2.5-fold more D-antigen units per cell than Vero cells. Based on our findings, we are exploring PER.C6(®) as an interesting platform for large-scale production of poliovirus at low costs, potentially providing the basis for global supply of an affordable IPV. PMID:23123018

  13. Directed Endothelial Progenitor Differentiation from Human Pluripotent Stem Cells Via Wnt Activation Under Defined Conditions.

    PubMed

    Bao, Xiaoping; Lian, Xiaojun; Palecek, Sean P

    2016-01-01

    Efficient derivation of endothelial cells and their progenitors from human pluripotent stem cells (hPSCs) can facilitate studies of human vascular development, disease modeling, drug discovery, and cell-based therapy. Here we provide a detailed protocol for directing hPSCs to functional endothelial cells and their progenitors in a completely defined, growth factor- and serum-free system by temporal modulation of Wnt/β-catenin signaling via small molecules. We demonstrate a 10-day, two-stage process that recapitulates endothelial cell development, in which hPSCs first differentiate to endothelial progenitors that then generate functional endothelial cells and smooth muscle cells. Methods to characterize endothelial cell identity and function are also described. PMID:27590162

  14. Interactions between oocytes and cumulus cells during in vitro maturation of porcine cumulus-oocyte complexes in a chemically defined medium: effect of denuded oocytes on cumulus expansion and oocyte maturation.

    PubMed

    Appeltant, R; Somfai, T; Nakai, M; Bodó, S; Maes, D; Kikuchi, K; Van Soom, A

    2015-03-01

    The aim of the present study was to clarify interactions between oocytes and cumulus cells (CCs) on the level of cumulus expansion and oocyte maturation during IVM of cumulus-oocyte complexes (COCs) in a chemically defined medium using a system that allows individual tracking of oocytes. Especially, the influence of oocyte-secreted factors was investigated by the aid of addition of denuded oocytes (DOs) as a possible approach to improve the IVM system. The basic maturation medium was porcine oocyte medium with addition of gonadotropins only during the first 20 hours of IVM. During IVM, COCs were kept fixed to the bottom of culture dish by adhesive Cell-Tak coating, which enabled individual tracking of COCs during IVM. Size changes in COCs during IVM were measured by digital image analysis. Cumulus expansion in a porcine oocyte medium of intact COCs increased in a typical manner until 20 hours and decreased in size subsequently until 48 hours of IVM (P < 0.05). Removal of oocytes from COCs by oocytectomy allowed the expansion of CCs to some extent, although their expansion ability was lower than that of COCs (P < 0.05). Addition of DOs (COCs to DOs ratio of 9:16) did not improve cumulus expansion and oocyte maturation rates of intact COCs (P > 0.05) but did enhance cumulus expansion of oocytectomized complexes (P < 0.05). Furthermore, removal of CCs before IVM increased oocyte maturation rates compared with COCs (52.3% and 32.9%, respectively) (P < 0.05) and a similar effect was observed in COCs when the gap junction inhibitor carbenoxolone was added to the IVM medium: carbenoxolone repressed the expansion of COCs at 20 hours of IVM. In conclusion, the porcine oocyte enhances cumulus expansion both by gap junctional communications and presumably by oocyte-secreted factor production. Nevertheless, the presence of oocytes is not a prerequisite for this process. In return, CCs maintain meiotic arrest in cumulus-enclosed oocytes during the initial culture

  15. Defining Overweight and Obesity

    MedlinePlus

    ... Physical Activity Overweight & Obesity Healthy Weight Breastfeeding Micronutrient Malnutrition State and Local Programs Defining Adult Overweight and ... Physical Activity Overweight & Obesity Healthy Weight Breastfeeding Micronutrient Malnutrition State and Local Programs File Formats Help: How ...

  16. Interpolation and Definability

    NASA Astrophysics Data System (ADS)

    Gabbay, Dov M.; Maksimova, Larisa L.

    This chapter is on interpolation and definability. This notion is not only central in pure logic, but has significant meaning and applicability in all areas where logic itself is applied, especially in computer science, artificial intelligence, logic programming, philosophy of science and natural language. The notion may sometimes appear to the reader as too technical/mathematical but it does also have a general meaning in terms of expressibility and definability.

  17. Comparison between the cultures of human induced pluripotent stem cells (hiPSCs) on feeder-and serum-free system (Matrigel matrix), MEF and HDF feeder cell lines.

    PubMed

    Ghasemi-Dehkordi, Payam; Allahbakhshian-Farsani, Mehdi; Abdian, Narges; Mirzaeian, Amin; Saffari-Chaleshtori, Javad; Heybati, Fatemeh; Mardani, Gashtasb; Karimi-Taghanaki, Alireza; Doosti, Abbas; Jami, Mohammad-Saeid; Abolhasani, Marziyeh; Hashemzadeh-Chaleshtori, Morteza

    2015-09-01

    Human induced pluripotent stem cells (hiPSCs) are a type of pluripotent stem cells artificially derived from an adult somatic cell (typically human fibroblast) by forced expression of specific genes. In recent years, different feeders like inactivated mouse embryonic fibroblasts (MEFs), human dermal fibroblasts (HDFs), and feeder free system have commonly been used for supporting the culture of stem cells in undifferentiated state. In the present work, the culture of hiPSCs and their characterizations on BD Matrigel (feeder-and serum-free system), MEF and HDF feeders using cell culture methods and molecular techniques were evaluated and compared. The isolated HDFs from foreskin samples were reprogrammed to hiPSCs using gene delivery system. Then, the pluripotency ability of hiPSCs cultured on each layer was determined by teratoma formation and immunohistochemical staining. After EBs generation the expression level of three germ layers genes were evaluated by Q-real-time PCR. Also, the cytogenetic stability of hiPSCs cultured on each condition was analyzed by karyotyping and comet assay. Then, the presence of pluripotency antigens were confirmed by Immunocytochemistry (ICC) test and alkaline phosphatase staining. This study were showed culturing of hiPSCs on BD Matrigel, MEF and HDF feeders had normal morphology and could maintain in undifferentiated state for prolonged expansion. The hiPSCs cultured in each system had normal karyotype without any chromosomal abnormalities and the DNA lesions were not observed by comet assay. Moreover, up-regulation in three germ layers genes in cultured hiPSCs on each layer (same to ESCs) compare to normal HDFs were observed (p < 0.05). The findings of the present work were showed in stem cells culturing especially hiPSCs both MEF and HDF feeders as well as feeder free system like Matrigel are proper despite benefits and disadvantages. Although, MEFs is suitable for supporting of stem cell culturing but it can animal pathogens

  18. Defining Airflow Obstruction

    PubMed Central

    Eschenbacher, William L.

    2016-01-01

    Airflow obstruction has been defined using spirometric test results when the forced expiratory volume in 1 second (FEV1) to forced vital capacity (FVC) ratio is below a fixed cutoff (<70%) or lower limits of normal (LLN) from reference equations that are based on values from a normal population. However, similar to other positive or abnormal diagnostic test results that are used to identify the presence of disease, perhaps airflow obstruction should be defined based on the values of FEV1/FVC for a population of individuals with known disease such as chronic obstructive pulmonary disease (COPD). Unfortunately, we do not know such a distribution of values of FEV1/FVC for patients with COPD since there is no gold standard for this syndrome or condition. Yet, we have used this physiologic definition of airflow obstruction based on a normal population to identify patients with COPD. In addition, we have defined airflow obstruction as either being present or absent. Instead, we should use a different approach to define airflow obstruction based on the probability or likelihood that the airflow obstruction is present which in turn would give us the probability or likelihood of a disease state such as COPD. PMID:27239557

  19. Defining Equality in Education

    ERIC Educational Resources Information Center

    Benson, Ronald E.

    1977-01-01

    Defines equality of education in three areas: 1) by the degree of integration of school systems; 2) by a comparison of material resources and assets in education; and 3) by the effects of schooling as measured by the mean scores of groups on standardized tests. Available from: College of Education, 107 Quadrangle, Iowa State University, Ames, Iowa…

  20. Defining Supports Geometry

    ERIC Educational Resources Information Center

    Stephan, Michelle L.; McManus, George E.; Dickey, Ashley L.; Arb, Maxwell S.

    2012-01-01

    The process of developing definitions is underemphasized in most mathematics instruction. Investing time in constructing meaning is well worth the return in terms of the knowledge it imparts. In this article, the authors present a third approach to "defining," called "constructive." It involves modifying students' previous understanding of a term…

  1. On Defining Mass

    ERIC Educational Resources Information Center

    Hecht, Eugene

    2011-01-01

    Though central to any pedagogical development of physics, the concept of mass is still not well understood. Properly defining mass has proven to be far more daunting than contemporary textbooks would have us believe. And yet today the origin of mass is one of the most aggressively pursued areas of research in all of physics. Much of the excitement…

  2. Defining Effective Teaching

    ERIC Educational Resources Information Center

    Layne, L.

    2012-01-01

    The author looks at the meaning of specific terminology commonly used in student surveys: "effective teaching." The research seeks to determine if there is a difference in how "effective teaching" is defined by those taking student surveys and those interpreting the results. To investigate this difference, a sample group of professors and students…

  3. Defining structural limit zones

    NASA Technical Reports Server (NTRS)

    Merchant, D. H.

    1978-01-01

    Method for defining limit loads uses probability distribution of largest load occurring during given time intervals. Method is compatible with both deterministic and probabilistic structural design criteria. It also rationally accounts for fact that longer structure is exposed to random loading environment, greater is possibility that it will experience extreme load.

  4. Defining Faculty Work.

    ERIC Educational Resources Information Center

    Gray, Peter J.; Diamond, Robert M.

    1994-01-01

    A process of planned change is proposed for redefining college faculty work. Legitimate faculty work is defined in broad terms, and information sources and methods for collecting information to support redefinition are identified. The final step in the redefinition process is the development of new mission statements for the institution and its…

  5. Defined by Limitations

    ERIC Educational Resources Information Center

    Arriola, Sonya; Murphy, Katy

    2010-01-01

    Undocumented students are a population defined by limitations. Their lack of legal residency and any supporting paperwork (e.g., Social Security number, government issued identification) renders them essentially invisible to the American and state governments. They cannot legally work. In many states, they cannot legally drive. After the age of…

  6. Defining Dynamic Route Structure

    NASA Technical Reports Server (NTRS)

    Zelinski, Shannon; Jastrzebski, Michael

    2011-01-01

    This poster describes a method for defining route structure from flight tracks. Dynamically generated route structures could be useful in guiding dynamic airspace configuration and helping controllers retain situational awareness under dynamically changing traffic conditions. Individual merge and diverge intersections between pairs of flights are identified, clustered, and grouped into nodes of a route structure network. Links are placed between nodes to represent major traffic flows. A parametric analysis determined the algorithm input parameters producing route structures of current day flight plans that are closest to todays airway structure. These parameters are then used to define and analyze the dynamic route structure over the course of a day for current day flight paths. Route structures are also compared between current day flight paths and more user preferred paths such as great circle and weather avoidance routing.

  7. Defining the paramedic process.

    PubMed

    Carter, Holly; Thompson, James

    2015-01-01

    The use of a 'process of care' is well established in several health professions, most evidently within the field of nursing. Now ingrained within methods of care delivery, it offers a logical approach to problem solving and ensures an appropriate delivery of interventions that are specifically suited to the individual patient. Paramedicine is a rapidly advancing profession despite a wide acknowledgement of limited research provisions. This frequently results in the borrowing of evidence from other disciplines. While this has often been useful, there are many concerns relating to the acceptable limit of evidence transcription between professions. To date, there is no formally recognised 'process of care'-defining activity within the pre-hospital arena. With much current focus on the professional classification of paramedic work, it is considered timely to formally define a formula that underpins other professional roles such as nursing. It is hypothesised that defined processes of care, particularly the nursing process, may have features that would readily translate to pre-hospital practice. The literature analysed was obtained through systematic searches of a range of databases, including Ovid MEDLINE, Cumulative Index to Nursing and Allied Health. The results demonstrated that the defined process of care provides nursing with more than just a structure for practice, but also has implications for education, clinical governance and professional standing. The current nursing process does not directly articulate to the complex and often unstructured role of the paramedic; however, it has many principles that offer value to the paramedic in their practice. Expanding the nursing process model to include the stages of Dispatch Considerations, Scene Assessment, First Impressions, Patient History, Physical Examination, Clinical Decision-Making, Interventions, Re-evaluation, Transport Decisions, Handover and Reflection would provide an appropriate model for pre

  8. Defining functional dyspepsia.

    PubMed

    Mearin, Fermín; Calleja, José Luis

    2011-12-01

    Dyspepsia and functional dyspepsia represent a highly significant public health issue. A good definition of dyspepsia is key for helping us to better approach symptoms, decision making, and therapy indications.During the last few years many attempts were made at establishing a definition of dyspepsia. Results were little successful on most occasions, and clear discrepancies arose on whether symptoms should be associated with digestion, which types of symptoms were to be included, which anatomic location should symptoms have, etc.The Rome III Committee defined dyspepsia as "a symptom or set of symptoms that most physicians consider to originate from the gastroduodenal area", including the following: postprandial heaviness, early satiety, and epigastric pain or burning. Two new entities were defined: a) food-induced dyspeptic symptoms (postprandial distress syndrome); and b) epigastric pain (epigastric pain syndrome). These and other definitions have shown both strengths and weaknesses. At times they have been much too complex, at times much too simple; furthermore, they have commonly erred on the side of being inaccurate and impractical. On the other hand, some (the most recent ones) are difficult to translate into the Spanish language. In a meeting of gastroenterologists with a special interest in digestive functional disorders, the various aspects of dyspepsia definition were discussed and put to the vote, and the following conclusions were arrived at: dyspepsia is defined as a set of symptoms, either related or unrelated to food ingestion, localized on the upper half of the abdomen. They include: a) epigastric discomfort (as a category of severity) or pain; b) postprandial heaviness; and c) early satiety. Associated complaints include: nausea, belching, bloating, and epigastric burn (heartburn). All these must be scored according to severity and frequency. Furthermore, psychological factors may be involved in the origin of functional dyspepsia. On the other hand

  9. Defining periodontal health

    PubMed Central

    2015-01-01

    Assessment of the periodontium has relied exclusively on a variety of physical measurements (e.g., attachment level, probing depth, bone loss, mobility, recession, degree of inflammation, etc.) in relation to various case definitions of periodontal disease. Periodontal health was often an afterthought and was simply defined as the absence of the signs and symptoms of a periodontal disease. Accordingly, these strict and sometimes disparate definitions of periodontal disease have resulted in an idealistic requirement of a pristine periodontium for periodontal health, which makes us all diseased in one way or another. Furthermore, the consequence of not having a realistic definition of health has resulted in potentially questionable recommendations. The aim of this manuscript was to assess the biological, environmental, sociological, economic, educational and psychological relationships that are germane to constructing a paradigm that defines periodontal health using a modified wellness model. The paradigm includes four cardinal characteristics, i.e., 1) a functional dentition, 2) the painless function of a dentition, 3) the stability of the periodontal attachment apparatus, and 4) the psychological and social well-being of the individual. Finally, strategies and policies that advocate periodontal health were appraised. I'm not sick but I'm not well, and it's a sin to live so well. Flagpole Sitta, Harvey Danger PMID:26390888

  10. TAPERED DEFINING SLOT

    DOEpatents

    Pressey, F.W.

    1959-09-01

    An improvement is reported in the shape and formation of the slot or opening in the collimating slot member which forms part of an ion source of the type wherein a vapor of the material to be ionized is bombarded by electrons in a magnetic field to strike an arc-producing ionization. The defining slot is formed so as to have a substantial taper away from the cathode, causing the electron bombardment from the cathode to be dispersed over a greater area reducing its temperature and at the same time bringing the principal concentration of heat from the electron bombardment nearer the anode side of the slot, thus reducing deterioration and prolonging the life of the slot member during operation.