The influence of serum-free culture conditions on skeletal muscle differentiation in a tissue-engineered model

NARCIS (Netherlands)

Gawlitta, D.; Boonen, K.J.M.; Oomens, C.W.J.; Baaijens, F.P.T.; Bouten, C.V.C.

2008-01-01

The influence of differentiation medium (DM) components on C2C12 murine myoblast differentiation has only been studied in monolayer cultures. Serum-free formulations have been applied that omit the use of sera with unknown composition. The goal of the present study was to compare the influence of

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

International Nuclear Information System (INIS)

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

1987-01-01

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. 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 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

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

International Nuclear Information System (INIS)

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

2013-01-01

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:N 2 films showed good attachment of hBM-MSCs at 24 h. However, in the case of silica surface (a-C:SiO x 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 -NH 2 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).

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

Science.gov (United States)

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).

Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21)

Science.gov (United States)

Nollevaux, Géraldine; Devillé, Christelle; El Moualij, Benaïssa; Zorzi, Willy; Deloyer, Patricia; Schneider, Yves-Jacques; Peulen, Olivier; Dandrifosse, Guy

2006-01-01

Background The absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. The cell lineage Caco-2 is a model commonly used to reproduce the features of the bowel epithelium. However, there is a strong debate regarding the value of Caco-2 cell culture to mimick in vivo situation. Indeed, some authors report in Caco-2 a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. The HT29-5M21 intestinal cell lineage is a mucin-secreting cellular population. A co-culture system carried out in a serum-free medium and comprising both Caco-2 and HT29-5M21 cells was developed. The systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure. Results In this study, we investigated the activity and localization of the alkaline phosphatase and the expression of IAP and MUC5AC genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of Caco-2 and HT29-5M21 cells. We observed that the culture conditions used (serum-free medium) did not change the phenotype of each cell type, and produced a reproducible model. The alkaline phosphatase expression characterizing Caco-2 cells was influenced by the presence of HT29-5M21 cells. Conclusion The culture formed by 75% Caco-2 and 25% HT29-5M21 produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules. PMID:16670004

A novel serum-free monolayer culture for orderly hematopoietic differentiation of human pluripotent cells via mesodermal progenitors.

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Akira Niwa

Full Text Available Elucidating the in vitro differentiation of human embryonic stem (ES and induced pluripotent stem (iPS cells is important for understanding both normal and pathological hematopoietic development in vivo. For this purpose, a robust and simple hematopoietic differentiation system that can faithfully trace in vivo hematopoiesis is necessary. In this study, we established a novel serum-free monolayer culture that can trace the in vivo hematopoietic pathway from ES/iPS cells to functional definitive blood cells via mesodermal progenitors. Stepwise tuning of exogenous cytokine cocktails induced the hematopoietic mesodermal progenitors via primitive streak cells. These progenitors were then differentiated into various cell lineages depending on the hematopoietic cytokines present. Moreover, single cell deposition assay revealed that common bipotential hemoangiogenic progenitors were induced in our culture. Our system provides a new, robust, and simple method for investigating the mechanisms of mesodermal and hematopoietic differentiation.

Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21

Directory of Open Access Journals (Sweden)

Schneider Yves-Jacques

2006-05-01

Full Text Available Abstract Background The absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. The cell lineage Caco-2 is a model commonly used to reproduce the features of the bowel epithelium. However, there is a strong debate regarding the value of Caco-2 cell culture to mimick in vivo situation. Indeed, some authors report in Caco-2 a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. The HT29-5M21 intestinal cell lineage is a mucin-secreting cellular population. A co-culture system carried out in a serum-free medium and comprising both Caco-2 and HT29-5M21 cells was developed. The systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure. Results In this study, we investigated the activity and localization of the alkaline phosphatase and the expression of IAP and MUC5AC genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of Caco-2 and HT29-5M21 cells. We observed that the culture conditions used (serum-free medium did not change the phenotype of each cell type, and produced a reproducible model. The alkaline phosphatase expression characterizing Caco-2 cells was influenced by the presence of HT29-5M21 cells. Conclusion The culture formed by 75% Caco-2 and 25% HT29-5M21 produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules.

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

Energy Technology Data Exchange (ETDEWEB)

Tunma, Somruthai [The Graduate School, Chiang Mai University, 239 Huay Kaew Road, Muang District, Chiang Mai 50200 (Thailand); Thailand Center of Excellence in Physics (ThEP), 239 Huay Kaew Road, Muang District, Chiang Mai 50200 (Thailand); Song, Doo-Hoon [Research Center for Orofacial Hard Tissue Regeneration, College of Dentistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Kim, Si-Eun; Kim, Kyoung-Nam [Research Center for Orofacial Hard Tissue Regeneration, College of Dentistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Department and Research Institute of Dental Biomaterials and Bioengineering, College of Dentistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Han, Jeon-Geon [Center for Advanced Plasma Surface Technology, Sungkyunkwan University, 300 Chunchun-dong, Jangan-gu, Suwon 440-746 (Korea, Republic of); Boonyawan, Dheerawan [Thailand Center of Excellence in Physics (ThEP), 239 Huay Kaew Road, Muang District, Chiang Mai 50200 (Thailand); Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, 239 Huay Kaew Road, Muang District, Chiang Mai 50200 (Thailand)

2013-10-15

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:N{sub 2} films showed good attachment of hBM-MSCs at 24 h. However, in the case of silica surface (a-C:SiO{sub x} 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 -NH{sub 2} 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).

Long-term culture of rat hippocampal neurons at low density in serum-free medium: combination of the sandwich culture technique with the three-dimensional nanofibrous hydrogel PuraMatrix.

Science.gov (United States)

Kaneko, Ai; Sankai, Yoshiyuki

2014-01-01

The primary culture of neuronal cells plays an important role in neuroscience. There has long been a need for methods enabling the long-term culture of primary neurons at low density, in defined serum-free medium. However, the lower the cell density, the more difficult it is to maintain the cells in culture. Therefore, we aimed to develop a method for long-term culture of neurons at low density, in serum-free medium, without the need for a glial feeder layer. Here, we describe the work leading to our determination of a protocol for long-term (>2 months) primary culture of rat hippocampal neurons in serum-free medium at the low density of 3×10(4) cells/mL (8.9×10(3) cells/cm2) without a glial feeder layer. Neurons were cultured on a three-dimensional nanofibrous hydrogel, PuraMatrix, and sandwiched under a coverslip to reproduce the in vivo environment, including the three-dimensional extracellular matrix, low-oxygen conditions, and exposure to concentrated paracrine factors. We examined the effects of varying PuraMatrix concentrations, the timing and presence or absence of a coverslip, the timing of neuronal isolation from embryos, cell density at plating, medium components, and changing the medium or not on parameters such as developmental pattern, cell viability, neuronal ratio, and neurite length. Using our method of combining the sandwich culture technique with PuraMatrix in Neurobasal medium/B27/L-glutamine for primary neuron culture, we achieved longer neurites (≥3,000 µm), greater cell viability (≥30%) for 2 months, and uniform culture across the wells. We also achieved an average neuronal ratio of 97%, showing a nearly pure culture of neurons without astrocytes. Our method is considerably better than techniques for the primary culture of neurons, and eliminates the need for a glial feeder layer. It also exhibits continued support for axonal elongation and synaptic activity for long periods (>6 weeks).

Long-term culture of rat hippocampal neurons at low density in serum-free medium: combination of the sandwich culture technique with the three-dimensional nanofibrous hydrogel PuraMatrix.

Directory of Open Access Journals (Sweden)

Ai Kaneko

Full Text Available The primary culture of neuronal cells plays an important role in neuroscience. There has long been a need for methods enabling the long-term culture of primary neurons at low density, in defined serum-free medium. However, the lower the cell density, the more difficult it is to maintain the cells in culture. Therefore, we aimed to develop a method for long-term culture of neurons at low density, in serum-free medium, without the need for a glial feeder layer. Here, we describe the work leading to our determination of a protocol for long-term (>2 months primary culture of rat hippocampal neurons in serum-free medium at the low density of 3×10(4 cells/mL (8.9×10(3 cells/cm2 without a glial feeder layer. Neurons were cultured on a three-dimensional nanofibrous hydrogel, PuraMatrix, and sandwiched under a coverslip to reproduce the in vivo environment, including the three-dimensional extracellular matrix, low-oxygen conditions, and exposure to concentrated paracrine factors. We examined the effects of varying PuraMatrix concentrations, the timing and presence or absence of a coverslip, the timing of neuronal isolation from embryos, cell density at plating, medium components, and changing the medium or not on parameters such as developmental pattern, cell viability, neuronal ratio, and neurite length. Using our method of combining the sandwich culture technique with PuraMatrix in Neurobasal medium/B27/L-glutamine for primary neuron culture, we achieved longer neurites (≥3,000 µm, greater cell viability (≥30% for 2 months, and uniform culture across the wells. We also achieved an average neuronal ratio of 97%, showing a nearly pure culture of neurons without astrocytes. Our method is considerably better than techniques for the primary culture of neurons, and eliminates the need for a glial feeder layer. It also exhibits continued support for axonal elongation and synaptic activity for long periods (>6 weeks.

Effect of IL-6 on proliferation and IG production of human EBV-transformed cell lines in serum free culture media

NARCIS (Netherlands)

Jochems, G. J.; Jordens, R.; van Lier, R. A.; Zeijlemaker, W. P.

1990-01-01

To optimalize growth and Ig production of EBV transformed B cells for large scale tissue culture, we analyzed five stable monoclonal EBV-B cell lines for their responsiveness to interleukin (IL)-6 in standard medium with 5% FCS and in several serum-free media. As we previously demonstrated these

Bovine babesiosis: Cattle protected in the field with a frozen vaccine containing Babesia bovis and Babesia bigemina cultured in vitro with a serum-free medium.

Science.gov (United States)

Rojas-Martínez, Carmen; Rodríguez-Vivas, Roger Iván; Millán, Julio Vicente Figueroa; Bautista-Garfias, Carlos Ramón; Castañeda-Arriola, Roberto Omar; Lira-Amaya, José Juan; Urióstegui, Patricia Vargas; Carrasco, Juan José Ojeda; Martínez, Jesús Antonio Álvarez

2018-04-01

An attenuated live vaccine containing Babesia bovis and B. bigemina cultured in vitro with a serum-free medium was assessed for its clinical protection conferred of naïve cattle, under natural tick-challenge in a high endemicity zone to Babesia spp. Three groups of six animals were treated as follows: group I (GI) received a vaccine derived from parasites cultured with a free-serum medium; group II (GII) were immunized with the standard vaccine, with parasites cultured in a medium supplemented with 40% (v/v) bovine serum; and a control group (GIII) inoculated with non-infected bovine erythrocytes. Inocula were administered by IM route. Experimental animals were kept during 23days after vaccination in a cattle farm free of ticks and Babesia spp. Thereafter, cattle were moved to a high endemicity farm for natural exposure to Babesia spp. transmitted by Rhipicephalus microplus ticks. Protection against clinical babesiosis was observed in bovines belonging to GI (100%) and GII (83.33%), while the control animals (GIII) were not protected, and showed severe clinical signs, closely related to babesiosis, were observed for at least three consecutive days during the challenge. These were fever, anemia, which were measured simultaneously, and circulating parasites were detected by optic light microscopy. All cattle showed B. bovis and B. bigemina in stained blood films during the challenge; B. bovis antibody titers were higher than those to B. bigemina in GI and GII, and lower titers were determined in GIII. The protective capacity of the vaccine derived from B. bovis and B. bigemina cultured in vitro in a serum-free medium was demonstrated. Copyright © 2017 Elsevier B.V. All rights reserved.

Serum-converted platelet lysate can substitute for fetal bovine serum in human mesenchymal stromal cell cultures.

Science.gov (United States)

Mojica-Henshaw, Mariluz P; Jacobson, Pam; Morris, Julie; Kelley, Linda; Pierce, Jan; Boyer, Michael; Reems, Jo-Anna

2013-12-01

Fetal bovine serum (FBS) is commonly used as a serum supplement for culturing human mesenchymal stromal cells (hMSCs). However, human cells grown in FBS, especially for extended periods, risk potential exposure to bovine immunogenic proteins and infectious agents. To address this issue, we investigated the ability of a novel human platelet serum supplement to substitute for FBS in hMSC cultures. Platelet lysate-serum (PL-serum) was converted from platelet lysate-plasma (PL-plasma) that was manufactured from pooled platelet-rich plasma (PRP) apheresis units. Growth factor levels and the number of residual intact platelets in PL-serum and PL-plasma were compared with enzyme-linked immunosorbent assays and flow cytometry, respectively. Proliferation responses of hMSCs cultured in PL-serum, PL-plasma, or FBS were assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the immunophenotype of harvested hMSCs was evaluated by flow cytometry and tri-lineage differentiation potential was evaluated by assessing adipogenic, osteogenic and chondrogenic development. Selected growth factor levels in PL-serum were not significantly different from PL-plasma (P > 0.05). hMSC cultures supplemented with PL-serum had comparable growth kinetics to PL-plasma, and hMSC yields were consistently greater than with FBS. hMSCs harvested from cultures supplemented with PL-serum, PL-plasma or FBS had similar cell surface phenotypes and maintained tri-lineage differentiation potential. PL-serum, similar to PL-plasma, can substitute for FBS in hMSC cultures. Use of PL-serum, in contrast to PL-plasma, has an added advantage of not requiring addition of a xenogeneic source of heparin, providing a completely xeno-free culture medium. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Evolution of triiodothyronine nuclear binding sites in hypothalamic serum-free cultures: evidence for their presence in neurons and astrocytes

International Nuclear Information System (INIS)

Puymirat, J.; Faivre-Bauman, A.

1986-01-01

( 125 I)Triiodothyronine (T 3 ) nuclear binding was studied in hypothalamic cultures from fetal mouse grown in serum-free medium. In enriched neuronal cultures, the apparent dissociation constant of the binding does not change with time in vitro (7 x 10 -11 M), but the maximum binding capacity (MBC) doubles between day 7 and day 14 in vitro. We show here for the first time that homologous astrocyte cell cultures, devoid of neurons as checked by tetanus toxin binding, also display T 3 nuclear binding, with the same affinity as neuronal cultures. However, their MBC is 3 times lower than that of neurons after a week in vitro, and increases more quickly thereafter (Author)

The replacement of serum by hormones in cell culture media.

Science.gov (United States)

Sato, G; Hayashi, I

1976-12-01

The replacement of serum by hormones in cell culture media. (Reemplazo del suero por hormonas en el medio de cultivo de células). Arch. Biol. Med. Exper. 10: 120-121, 1976. The serum used in cell culture media can be replaced by a mixture of hormones and some accesory blood factors. The pituitary cell line GH3 can be grown in a medium in which serum is replaced by triiodothyronine, transferrin, parathormone, tyrotrophin releasing hormone and somatomedins. Hela and BHK cell strains can also be grown in serum free medium supplemented with hormones. Each cell type appears to have different hormonal requirements yet it may found that some hormones are required for most cell types.

Immunohistochemical expression of stem cell, endothelial cell, and chemosensitivity markers in primary glioma spheroids cultured in serum-containing and serum-free medium

DEFF Research Database (Denmark)

Christensen, Karina; Aaberg-Jessen, Charlotte; Andersen, Claus

2010-01-01

To investigate the influence of serum-free medium (SFM) supplemented with epidermal growth factor and basic fibroblast growth factor compared with conventional serum-containing medium (SCM) on the phenotype of organotypic primary spheroids from seven gliomas.......To investigate the influence of serum-free medium (SFM) supplemented with epidermal growth factor and basic fibroblast growth factor compared with conventional serum-containing medium (SCM) on the phenotype of organotypic primary spheroids from seven gliomas....

Influence of serum extraction from the culture medium and of sublethal X-ray irradiation upon microvilli and invaginations of the membrane of Ehrlich ascites tumor cells in monolayer culture

International Nuclear Information System (INIS)

Laudenbach, G.; Pfab, R.; Hess, F.; Schachtschabel, D.O.

1984-01-01

In order to find out modifications of microvilli and invaginations, the cellular surfaces of Ehrlich ascites tumor cells in monolayer culture (basal medium of Eagle + 10% fetal calf serum) were investigated with the aid of electron-microscopic cross-sections. The tumor cells had been cultured without serum 24 hours prior to investigation or irradiated with 2 Gy. Morphometric evaluation after cell culture in a serum-free medium showed a reduced number of microvilli and a diminution of sections of microvilli. As already described before, a reduction of cell proliferation, of the microtubule-microfilament system, and of the endocytosis activity occurs under these serum-free conditions. The number of invaginations (related to a constant membrane part) was reduced by nearly 50% after serum extraction. Similarly to serum extraction, sublethal X-ray irradiation reduced the sections of microvilli, whereas the number of microvilli increased slightly. Contrary to the effect of serum extraction, the irradiated cells showed twice as many invaginations as the non-irradiated control cells. These differences in the surface structures are interpreted as a result of modified growth stimulations (+- serum) and radiogenic reparation processes. (orig.) [de

In vitro culture of human osteosarcoma cell lines: a comparison of functional characteristics for cell lines cultured in medium without and with fetal calf serum.

Science.gov (United States)

Bruserud, Oystein; Tronstad, Karl Johan; Berge, Rolf

2005-06-01

Experimental in vitro models including well-characterised cell lines can be used to identify possible new therapeutic targets for the treatment of osteosarcoma. Culture media including inactivated serum is often recommended for in vitro culture of osteosarcoma cells, but the serum component then represents a nonstandardised parameter including a wide range of unidentified mediators. To improve the standardisation we have investigated whether serum-free culture media can be used in experimental in vitro studies of osteosarcoma cell lines. The seven osteosarcoma cell lines Cal72, SJSA-1, Saos-2, SK-ES-1, U2OS, 143.98.2, and KHOS-32IH were cultured in vitro in various serum-free media and media supplemented with 10% heat-inactivated fetal calf serum (FCS). Although proliferation often was relatively low in serum-free media (X-vivo 10, X-vivo 15, X-vivo 20, Stem Span SFEM), some cell lines (Cal72, KHOS-32IH, Saos-2) showed proliferation comparable with the recommended FCS-containing media even when using serum-free conditions. The optimal serum-free medium then varied between cell lines. We also compared 6 different FCS-containing media (including Stem Span with 10% FCS) and the optimal FCS-containing medium varied between cell lines. However, all cell lines proliferated well in Stem Span with FCS, and this medium was regarded as optimal for four of the lines. FCS could not be replaced by fatty acids or low density lipoprotein when testing the Stem Span medium. The release of a wide range of soluble mediators showed only minor differences when using serum-free and FCS-containing media (including Stem Span with and without FCS), and serum-free Stem Span could also be used for in vitro studies of mitogen-stimulated T cell activation in the presence of accessory osteosarcoma cells. The use of Stem Span with 10% FCS allowed the release of a wide range of chemokines by osteosarcoma cell lines (Cal72, SJSA-1), and the chemokine release profile was very similar to the

Serum-Free Media and the Immunoregulatory Properties of Mesenchymal Stem Cells In Vivo and In Vitro

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Mei Wu

2014-02-01

Full Text Available Background: Mesenchymal stem cells are capable of self-renewal and multi-lineage differentiation. They are used extensively to treat several diseases. Traditionally, mesenchymal stem cells are cultured in serum-containing media, typically supplemented with fetal bovine serum (FBS. However, the variability of FBS is likely to skew experimental results. Although serum-free media used to expand mesenchymal stem cells has facilitated remarkable achievements, immunomodulation of these cells in under serum-free conditions is poorly understood. We hypothesized that mesenchymal stem cells expanded in serum-free media will retain powerful immunoregulatory functions in vitro and in vivo. Design and Methods: Immunosuppressive activity and the immunomodulatory cytokines produced by mesenchymal stem cells in serum-free media were characterized in vitro. Immunomodulation by serum-free mesenchymal stem cell expansion in monocrotaline-induced pulmonary hypertension was explored in vivo. Results: Similar to cells in serum-containing media, mesenchymal stem cells expanded in serum-free media inhibited proliferation and apoptosis of CD4+T cells. They also exhibited strong immunosuppressive activities and secreted high levels of immunomodulatory cytokines such as PGE2, IDO1, COX2, IL-6, and IL-1β, but not HGF. On the other hand, growth of mesenchymal stem cells in serum-free media attenuated pulmonary vascular remodeling and inhibited mRNA expression of proinflammatory cytokines TNF-α, IFN-γ, IL-6, IL-1β, and IL-18. Conclusions: Mesenchymal stem cells in serum-free media maintained powerful immunomodulatory function in vitro and in vivo; serum-free media may replace serum-containing media for basic research and clinical applications.

Synthetic surface for expansion of human mesenchymal stem cells in xeno-free, chemically defined culture conditions.

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Paula J Dolley-Sonneville

Full Text Available Human mesenchymal stem cells (HMSCS possess three properties of great interest for the development of cell therapies and tissue engineering: multilineage differentiation, immunomodulation, and production of trophic factors. Efficient ex vivo expansion of hMSCs is a challenging requirement for large scale production of clinical grade cells. Low-cost, robust, scalable culture methods using chemically defined materials need to be developed to address this need. This study describes the use of a xeno-free synthetic peptide acrylate surface, the Corning® Synthemax® Surface, for culture of hMSCs in serum-free, defined medium. Cell performance on the Corning Synthemax Surface was compared to cells cultured on biological extracellular matrix (ECM coatings in xeno-free defined medium and in traditional conditions on tissue culture treated (TCT plastic in fetal bovine serum (FBS supplemented medium. Our results show successful maintenance of hMSCs on Corning Synthemax Surface for eight passages, with cell expansion rate comparable to cells cultured on ECM and significantly higher than for cells in TCT/FBS condition. Importantly, on the Corning Synthemax Surface, cells maintained elongated, spindle-like morphology, typical hMSC marker profile and in vitro multilineage differentiation potential. We believe the Corning Synthemax Surface, in combination with defined media, provides a complete synthetic, xeno-free, cell culture system for scalable production of hMSCs.

Optimization of a serum-free culture medium for mouse embryonic stem cells using design of experiments (DoE) methodology.

Science.gov (United States)

Knöspel, Fanny; Schindler, Rudolf K; Lübberstedt, Marc; Petzolt, Stephanie; Gerlach, Jörg C; Zeilinger, Katrin

2010-12-01

The in vitro culture behaviour of embryonic stem cells (ESC) is strongly influenced by the culture conditions. Current culture media for expansion of ESC contain some undefined substances. Considering potential clinical translation work with such cells, the use of defined media is desirable. We have used Design of Experiments (DoE) methods to investigate the composition of a serum-free chemically defined culture medium for expansion of mouse embryonic stem cells (mESC). Factor screening analysis according to Plackett-Burman revealed that insulin and leukaemia inhibitory factor (LIF) had a significant positive influence on the proliferation activity of the cells, while zinc and L: -cysteine reduced the cell growth. Further analysis using minimum run resolution IV (MinRes IV) design indicates that following factor adjustment LIF becomes the main factor for the survival and proliferation of mESC. In conclusion, DoE screening assays are applicable to develop and to refine culture media for stem cells and could also be employed to optimize culture media for human embryonic stem cells (hESC).

Serum-Free Cryopreservation of Five Mammalian Cell Lines in Either a Pelleted or Suspended State

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Corsini Joe

2004-01-01

Full Text Available Herein we have explored two practical aspects of cryopreserving cultured mammalian cells during routine laboratory maintenance. First, we have examined the possibility of using a serum-free, hence more affordable, cryopreservative. Using five mammalian lines (Crandell Feline Kidney, MCF7, A72, WI 38 and NB324K, we found that the serum-free alternative preserves nearly as efficiently as the serum-containing preservatives. Second, we compared cryostorage of those cells in suspended versus a pellet form using both aforementioned cryopreservatives. Under our conditions, cells were in general recovered equally well in a suspended versus a pellet form.

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

Science.gov (United States)

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

Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells.

Science.gov (United States)

Hemeda, Hatim; Giebel, Bernd; Wagner, Wolfgang

2014-02-01

Culture media for therapeutic cell preparations-such as mesenchymal stromal cells (MSCs)-usually comprise serum additives. Traditionally, fetal bovine serum is supplemented in basic research and in most clinical trials. Within the past years, many laboratories adapted their culture conditions to human platelet lysate (hPL), which further stimulates proliferation and expansion of MSCs. Particularly with regard to clinical application, human alternatives for fetal bovine serum are clearly to be preferred. hPL is generated from human platelet units by disruption of the platelet membrane, which is commonly performed by repeated freeze and thaw cycles. Such culture supplements are notoriously ill-defined, and many parameters contribute to batch-to-batch variation in hPL such as different amounts of plasma, a broad range of growth factors and donor-specific effects. The plasma components of hPL necessitate addition of anticoagulants such as heparins to prevent gelatinization of hPL medium, and their concentration must be standardized. Labels for description of hPL-such as "xenogen-free," "animal-free" and "serum free"-are not used consistently in the literature and may be misleading if not critically assessed. Further analysis of the precise composition of relevant growth factors, attachment factors, microRNAs and exosomes will pave the way for optimized and defined culture conditions. The use of hPL has several advantages and disadvantages: they must be taken into account because the choice of cell culture additive has major impact on cell preparations. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Serum-Free Media and the Immunoregulatory Properties of Mesenchymal Stem Cells In Vivo and In Vitro

OpenAIRE

Mei Wu; Zhi-Bo Han; Jun Feng Liu; You Wei Wang; Jian Zhong Zhang; Chun Tuan Li; Peng Liang Xin; Zhong Chao Han; Xiong Peng Zhu

2014-01-01

Background: Mesenchymal stem cells are capable of self-renewal and multi-lineage differentiation. They are used extensively to treat several diseases. Traditionally, mesenchymal stem cells are cultured in serum-containing media, typically supplemented with fetal bovine serum (FBS). However, the variability of FBS is likely to skew experimental results. Although serum-free media used to expand mesenchymal stem cells has facilitated remarkable achievements, immunomodulation of these cells in un...

Shift of microRNA profile upon glioma cell migration using patient-derived spheroids and serum-free conditions

DEFF Research Database (Denmark)

Munthe, Sune; Halle, Bo; Boldt, Henning B

2017-01-01

Glioblastoma multiforme (GBM) is the most frequent malignant primary brain tumor. A major reason for the overall median survival being only 14.6 months is migrating tumor cells left behind after surgery. Another major reason is tumor cells having a so-called cancer stem cell phenotype being...... therefore resistant towards traditional chemo- and radiotherapy. A group of novel molecular targets are microRNAs (miRNAs). MiRNAs are small non-coding RNAs exerting post-transcriptional regulation of gene expression. The aim of this study was to identify differentially expressed miRNAs in migrating GBM...... cells using serum-free stem cell conditions. We used patient-derived GBM spheroid cultures for a novel serum-free migration assay. MiRNA expression of migrating tumor cells isolated at maximum migration speed was compared with corresponding spheroids using an OpenArray Real-Time PCR System. The mi...

Serum-free Erythroid Differentiation for Efficient Genetic Modification and High-Level Adult Hemoglobin Production.

Science.gov (United States)

Uchida, Naoya; Demirci, Selami; Haro-Mora, Juan J; Fujita, Atsushi; Raines, Lydia N; Hsieh, Matthew M; Tisdale, John F

2018-06-15

In vitro erythroid differentiation from primary human cells is valuable to develop genetic strategies for hemoglobin disorders. However, current erythroid differentiation methods are encumbered by modest transduction rates and high baseline fetal hemoglobin production. In this study, we sought to improve both genetic modification and hemoglobin production among human erythroid cells in vitro . To model therapeutic strategies, we transduced human CD34 + cells and peripheral blood mononuclear cells (PBMCs) with lentiviral vectors and compared erythropoietin-based erythroid differentiation using fetal-bovine-serum-containing media and serum-free media. We observed more efficient transduction (85%-93%) in serum-free media than serum-containing media (20%-69%), whereas the addition of knockout serum replacement (KSR) was required for serum-free media to promote efficient erythroid differentiation (96%). High-level adult hemoglobin production detectable by electrophoresis was achieved using serum-free media similar to serum-containing media. Importantly, low fetal hemoglobin production was observed in the optimized serum-free media. Using KSR-containing, serum-free erythroid differentiation media, therapeutic adult hemoglobin production was detected at protein levels with β-globin lentiviral transduction in both CD34 + cells and PBMCs from sickle cell disease subjects. Our in vitro erythroid differentiation system provides a practical evaluation platform for adult hemoglobin production among human erythroid cells following genetic manipulation.

Organotypic hippocampal slice culture from the adult mouse brain: a versatile tool for translational neuropsychopharmacology.

Science.gov (United States)

Kim, Hyunjeong; Kim, Eosu; Park, Minsun; Lee, Eun; Namkoong, Kee

2013-03-05

One of the most significant barriers towards translational neuropsychiatry would be an unavailability of living brain tissues. Although organotypic brain tissue culture could be a useful alternative enabling observation of temporal changes induced by various drugs in living brain tissues, a proper method to establish a stable organotypic brain slice culture system using adult (rather than neonatal) hippocampus has been still elusive. In this study, we evaluated our simple method using the serum-free culture medium for successful adult organotypic hippocampal slice culture. Several tens of hippocampal slices from a single adult mouse (3-5 months old) were cultured in serum-free versus serum-containing conventional culture medium for 30 days and underwent various experiments to validate the effects of the existence of serum in the culture medium. Neither the excessive regression of neuronal viability nor metabolic deficiency was observed in the serum-free medium culture in contrast to the serum-containing medium culture. Despite such viability, newly generated immature neurons were scarcely detected in the serum-free culture, suggesting that the original neurons in the brain slice persist rather than being replaced by neurogenesis. Key structural features of in vivo neural tissue constituting astrocytes, neural processes, and pre- and post-synapses were also well preserved in the serum-free culture. In conclusion, using the serum-free culture medium, the adult hippocampal slice culture system will serve as a promising ex vivo tool for various fields of neuroscience, especially for studies on aging-related neuropsychiatric disorders or for high throughput screening of potential agents working against such disorders. Copyright © 2012 Elsevier Inc. All rights reserved.

Specific estrogen-induced cell proliferation of cultured Syrian hamster renal proximal tubular cells in serum-free chemically defined media

International Nuclear Information System (INIS)

Oberley, T.D.; Lauchner, L.J.; Pugh, T.D.; Gonzalez, A.; Goldfarb, S.; Li, S.A.; Li, J.J.

1989-01-01

It has long been recognized that the renal proximal tubular epithelium of the hamster is a bona fide estrogen target tissue. The effect of estrogens on the growth of proximal tubule cell explants and dissociated single cells derived from these explant outgrowths has been studied in culture. Renal tubular cells were grown on a PF-HR-9 basement membrane under serum-free chemically defined culture conditions. At 7-14 days in culture, cell number was enhanced 3-fold in the presence of either 17β-estradiol or diethylstilbestrol. A similar 3-fold increase in cell number was also seen at 1 nM 17β-estradiol in subcultured dissociated single tubular cells derived from hamster renal tubular explant outgrowths at 21 days in culture. Concomitant exposure of tamoxifen at 3-fold molar excess in culture completely abolished the increase in cell number seen with 17β-estradiol. The proliferation effect of estrogens on proximal tubular cell growth appears to be species specific since 17β-estradiol did not alter the growth of either rat or guinea pig proximal tubules in culture. In addition, at 7-10 days in culture in the presence of 17β-estradiol, [ 3 H]thymidine labeling of hamster tubular cells was enhanced 3-fold. These results clearly indicate that estrogens can directly induce primary epithelial cell proliferation at physiologic concentrations and provide strong additional evidence for an important hormonal role in the neoplastic transformation of the hamster kidney

Serum Immunoglobulin Free Light Chain Assessment in IgG4-Related Disease

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Aurélie Grados

2013-01-01

Full Text Available Immunoglobulin free light chains are produced in excess during normal antibody synthesis. Their evaluation is commonly used in case of a monoclonal gammopathy. In polyclonal hypergammaglobulinemia related to the Sjögren syndrome or systemic lupus, erythematosus serum free light chain levels are increased and could correlate with disease activity. We show here that the κ ( and λ ( free light chains and the κ : λ ratio ( are increased in sixteen patients with IgG4-related disease when compared to healthy controls. The increase of κ and λ free light chains probably reflects the marked polyclonal B cell activation of the disease. We could not assess in this small cohort of patients a significative correlation of serum free light chain levels and disease activity or extension.

Establishment of feeder-free culture system for human induced pluripotent stem cell on DAS nanocrystalline graphene

Science.gov (United States)

Lee, Hyunah; Nam, Donggyu; Choi, Jae-Kyung; Araúzo-Bravo, Marcos J.; Kwon, Soon-Yong; Zaehres, Holm; Lee, Taehee; Park, Chan Young; Kang, Hyun-Wook; Schöler, Hans R.; Kim, Jeong Beom

2016-02-01

The maintenance of undifferentiated human pluripotent stem cells (hPSC) under xeno-free condition requires the use of human feeder cells or extracellular matrix (ECM) coating. However, human-derived sources may cause human pathogen contamination by viral or non-viral agents to the patients. Here we demonstrate feeder-free and xeno-free culture system for hPSC expansion using diffusion assisted synthesis-grown nanocrystalline graphene (DAS-NG), a synthetic non-biological nanomaterial which completely rule out the concern of human pathogen contamination. DAS-NG exhibited advanced biocompatibilities including surface nanoroughness, oxygen containing functional groups and hydrophilicity. hPSC cultured on DAS-NG could maintain pluripotency in vitro and in vivo, and especially cell adhesion-related gene expression profile was comparable to those of cultured on feeders, while hPSC cultured without DAS-NG differentiated spontaneously with high expression of somatic cell-enriched adhesion genes. This feeder-free and xeno-free culture method using DAS-NG will facilitate the generation of clinical-grade hPSC.

Determination of thymidine in serum used for cell culture media

International Nuclear Information System (INIS)

Schaer, J.C.; Maurer, U.; Schindler, R.

1978-01-01

Thymidine concentrations in serum used for cell culture media were determined with an assay based on isotope dilution. In this assay, incorporation of (3H)-thymidine into DNA of cultured cells was measured in the presence of 5 and 20% serum as a function of the concentration of unlabeled thymidine added to the medium. Thymidine concentrations were measured using horse serum as well as fetal calf serum in the culture media. Dialysis of serum resulted in a reduction of thymidine levels by factors of at least 10

Tissue culture media supplemented with 10% fetal calf serum contains a castrate level of testosterone.

NARCIS (Netherlands)

Sedelaar, J.P.M.; Isaacs, J.T.

2009-01-01

BACKGROUND: Human prostate cancer cells are routinely maintained in media supplemented with 10% Fetal Calf Serum (FCS) to provide androgen. In the present study, total and free testosterone levels in 10%FCS supplemented tissue culture media were determined and compared to levels in intact and

Isolation and characterization of Wharton’s jelly-derived multipotent mesenchymal stromal cells obtained from bovine umbilical cord and maintained in a defined serum-free three-dimensional system

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Cardoso Tereza C

2012-05-01

Full Text Available Abstract Background The possibility for isolating bovine mesenchymal multipotent cells (MSCs from fetal adnexa is an interesting prospect because of the potential for these cells to be used for biotechnological applications. Bone marrow and adipose tissue are the most common sources of MSCs derived from adult animals. However, little knowledge exists about the characteristics of these progenitors cells in the bovine species. Traditionally most cell cultures are developed in two dimensional (2D environments. In mammalian tissue, cells connect not only to each other, but also support structures called the extracellular matrix (ECM. The three-dimensional (3D cultures may play a potential role in cell biotechnology, especially in tissue therapy. In this study, bovine-derived umbilical cord Wharton’s jelly (UC-WJ cells were isolated, characterized and maintained under 3D-free serum condition as an alternative of stem cell source for future cell banking. Results Bovine-derived UC-WJ cells, collected individually from 5 different umbilical cords sources, were successfully cultured under serum-free conditions and were capable to support 60 consecutive passages using commercial Stemline® mesenchymal stem cells expansion medium. Moreover, the UC-WJ cells were differentiated into osteocytes, chondrocytes, adipocytes and neural-like cells and cultured separately. Additionally, the genes that are considered important embryonic, POU5F1 and ITSN1, and mesenchymal cell markers, CD105+, CD29+, CD73+ and CD90+ in MSCs were also expressed in five bovine-derived UC-WJ cultures. Morphology of proliferating cells typically appeared fibroblast-like spindle shape presenting the same viability and number. These characteristics were not affected during passages. There were 60 chromosomes at the metaphase, with acrocentric morphology and intense telomerase activity. Moreover, the proliferative capacity of T cells in response to a mitogen stimulus was suppressed when

Serum triiodothyronine and free triiodothyronine index in normal pregnancy

International Nuclear Information System (INIS)

Karunanidhi, A.; Charles, S.X.; Kanagasabapathy, A.S.

1981-01-01

The thyroid function at various stages of pregnancy and after delivery was assessed by measuring serum total triiodothyronine (T 3 ), T 3 resin uptake (T 3 RU) and free triiodothyronine index (FT 3 I). In first, second and third trimesters of pregnancy, the mean values of serum total T 3 were significantly elevated (P 3 I remained unaltered. During labour, both serum total T 3 and FT 3 I observed during labour and after delivery may be due to the changes in blood oestrogen levels. This study indicates that serum free T 3 concentrations in pregnancy remained unaltered in the presence of elevated serum total T 3 levels. FT 3 I determination would thus appear to be a reliable in vitro thyroid function test for the assessment of thyroid function throughout pregnancy. Serum total T 3 was measured by radioimmunoassay using radioiodinated T 3 ( 125 I-T 3 ). (author)

Short-term, serum-free, static culture of cord blood-derived CD34+ cells: effects of FLT3-L and MIP-1alpha on in vitro expansion of hematopoietic progenitor cells.

Science.gov (United States)

Capmany, G; Querol, S; Cancelas, J A; García, J

1999-08-01

The use of ex vivo expanded cells has been suggested as a possible means to accelerate the speed of engraftment in cord blood (CB) transplantation. The aim of this study was to fix the optimal condition for the generation of committed progenitors without affecting the stem cell compartment. Analysis of the effects of FLT3-L and MIP-1alpha when combined with SCF, IL-3 and IL-6, in short-term (6 days), serum-free expansion cultures of CB-selected CD34+ cells. An important expansion was obtained that ranged between 8-15 times for CFU-GM, 21-51 times for the BFU-E/CFU-Mix population and 11 to 30 times for CD34+ cells assessed by flow cytometry. From the combinations tested, those in which FLT3-L was present had a significant increase in the expansion of committed progenitors, while the presence of MIP-1alpha had a detrimental effect on the generation of more differentiated cells. However, stem cell candidates assessed by week 5 CAFC assay could be maintained in culture when both MIP-1a and FLT3-L were present (up to 91% recovery). This culture system was also able to expand megakaryocytic precursors as determined by the co-expression of CD34 and CD61 antigens (45-70 times), in spite of the use of cytokines non-specific for the megakaryocytic lineage. The results obtained point to the combination of SCF, IL-3, IL-6, FLT3-L and MIP-1alpha as the best suited for a pre-clinical short-term serum-free static ex vivo expansion protocol of CB CD34+ cells, since it can generate large numbers of committed progenitor cells as well as maintaining week 5 CAFC.

Human autologous serum as a substitute for fetal bovine serum in human Schwann cell culture.

Directory of Open Access Journals (Sweden)

Parisa Goodarzi

2014-04-01

Full Text Available Nowadays, cell -based and tissue engineered products have opened new horizons in treatment of incurable nervous system disorders. The number of studies on the role of Schwann cells (SC in treating nervous disorders is higher than other cell types. Different protocols have been suggested for isolation and expansion of SC which most of them have used multiple growth factors, mitogens and fetal bovine sera (FBS in culture medium. Because of potential hazards of animal-derived reagents, this study was designed to evaluate the effect of replacing FBS with human autologous serum (HAS on SC's yield and culture parameters. Samples from 10 peripheral nerve biopsies were retrieved and processed under aseptic condition. The isolated cells cultured in FBS (1st group or autologous serum (2nd group. After primary culture the cells were seeded at 10000 cell/cm2 in a 12 wells cell culture plate for each group. At 100% confluency, the cell culture parameters (count, viability, purity and culture duration of 2 groups were compared using paired t-test. The average donors' age was 35.80 (SD=13.35 and except for 1 sample the others cultured successfully. In first group, the averages of cell purity, viability and culture duration were 97% (SD=1.32, 97/33% (SD=1.22 and 11.77 (SD=2.58 days respectively. This parameters were 97.33% (SD=1.00, 97.55% (SD=1.33 and 10.33 days (SD=1.65 in second group. The difference of cell count, purity and viability were not significant between 2 groups (P>0.05. The cells of second group reached to 100% confluency in shorter period of time (P=0.03. The results of this study showed that autologous serum can be a good substitute for FBS in human SC culture. This can reduce the costs and improve the safety of cell product for clinical application.

Optimized human platelet lysate as novel basis for a serum-, xeno-, and additive-free corneal endothelial cell and tissue culture.

Science.gov (United States)

Thieme, Daniel; Reuland, Lynn; Lindl, Toni; Kruse, Friedrich; Fuchsluger, Thomas

2018-02-01

The expansion of donor-derived corneal endothelial cells (ECs) is a promising approach for regenerative therapies in corneal diseases. To achieve the best Good Manufacturing Practice standard the entire cultivation process should be devoid of nonhuman components. However, so far, there is no suitable xeno-free protocol for clinical applications. We therefore introduce a processed variant of a platelet lysate for the use in corneal cell and tissue culture based on a Good Manufacturing Practice-grade thrombocyte concentrate. This processed human platelet lysate (phPL), free of any animal components and of anticoagulants such as heparin with a physiological ionic composition, was used to cultivate corneal ECs in vitro and ex vivo in comparison to standard cultivation with fetal calf serum (FCS). Human donor corneas were cut in quarters while 2 quarters of each cornea were incubated with the respective medium supplement. Three fields of view per quarter were taken into account for the analysis. Evaluation of phPL as a medium supplement in cell culture of immortalized EC showed a superior viability compared with FCS control with reduced cell proliferation. Furthermore, the viability during the expansion of primary cells is significantly (3-fold ±0.5) increased with phPL compared with FCS standard medium. Quartering donor corneas was traumatic for the endothelium and therefore resulted in increased EC loss. Interestingly, however, cultivation of the quartered pieces for 2 weeks in 0.1-mg/ml pHPL in Biochrome I showed a 21 (±10) % EC loss compared with 67 (±12) % EC loss when cultivated in 2% FCS in Biochrome I. The cell culture protocol with pHPL as FCS replacement seems to be superior to the standard FCS protocols with respect to EC survival. It offers a xeno-free and physiological environment for corneal endothelial cells. This alternative cultivation protocol could facilitate the use of EC for human corneal cell therapy. Copyright © 2017 John Wiley & Sons, Ltd.

Free serum thyroxine

International Nuclear Information System (INIS)

Degrossi, O.J.; Altschuler, Noe; Cabrejas, M.L. de; Pinkas, Mirta; Garcia del Rio, Hernan

1982-01-01

The use of radiommunoassay (RIA) tehcniques has increased the diagnosis of thyroid functional alterations. A solid phase RIA method for free thyroxine (FT4) measurement was tested. Serum FT4, Total T4, T3 and TSH were determined by radioimmunoassay in 179 subjects. One hundred twenty two patients were normal (8 to 75 years old); FT4 was 1.42 +- 0.03 ng/100 ml (avg. value and std. error). In 27 cases of thyrotoxicosis the values were 4.66 +- 0.48 ng/100 ml and in 15 cases of hypothyroidism 0.50 +- 0.06 ng/100 ml (statistics probability [es

Normal human serum (HS) prevents oxidant-induced lysis of cultured endothelial cells (ECs)

International Nuclear Information System (INIS)

Callahan, K.S.; Harlan, J.M.

1986-01-01

Most studies demonstrating oxidant lysis of cultured ECs are performed in serum-free media or media containing low concentrations of bovine serum. The authors found that HS protects human and bovine ECs from lysis caused by reagent H 2 O 2 or glucose/glucose oxidase (GO)-generated H 2 O 2 . EC injury was assessed by 51 Cr release, cell detachment, or trypan blue dye exclusion. Protective HS activity was dose-dependent with concentrations greater than or equal to 25% preventing lethal injury. Cytotoxicity at 24 hrs, induced by 20 mU/ml GO, was 90.1 +/- 5.2% without HS vs 1.7 +/- 4.6% with 25% HS present (20 exp). Similar protection was observed with heparinized plasma. Of note, comparable concentrations of bovine serum were devoid of protective activity. Addition of fatty acid-free albumin to the media was also without protective effect. Preliminary characterization showed HS activity was stable to 60 0 C for 30 min, non-dialyzable at 25,000 MW cutoff, and retained in delipidated serum. The HS protection was not merely due to scavenging of exogenous H 2 O 2 as A23187-induced EC lysis was also prevented by HS. Protective activity was not reproduced by purified cerruloplasmin or transferrin. In conclusion, unidentified factor(s) present in HS protect cultured ECs from oxidant-induced lysis. Since endothelium is normally exposed to 100% plasma, the authors suggest that in vitro studies of oxidant-mediated injury be performed in the presence of HS. Factor(s) in HS may play an important role in modulating oxidant-induced vascular injury in vivo

21 CFR 862.1730 - Free tyrosine test system.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Free tyrosine test system. 862.1730 Section 862....1730 Free tyrosine test system. (a) Identification. A free tyrosine test system is a device intended to measure free tyrosine (an amono acid) in serum and urine. Measurements obtained by this device are used in...

An improved ultrafiltration method for determining free testosterone in serum

International Nuclear Information System (INIS)

Vlahos, I.; MacMahon, W.; Sgoutas, D.; Bowers, W.; Thompson, J.; Trawick, W.

1982-01-01

In this method, we use the Amicon MPS-1 centrifugal ultrafiltration device and the YMB membrane in measuring free testosterone in serum. Two independent assays are combined: total testosterone and the ultrafiltrable fraction of added [ 3 H]testosterone. The unbound fraction is determined in 0.15-0.5 mL ultrafiltrates of 0.6 to 1 mL of variably diluted serum that has been equilibrated with [ 3 H]testosterone at 37 degrees C. The assay is rapid (less than 1 h), practicable (requires 0.6 mL of serum), and reproducible (CV 3.2% within assay, 3.9% between assays). Accuracy was evaluated as the fraction of free testosterone in the ultrafiltrate of dialyzed serum vs that in a prior dialysate; they were the same confirming the validity of the free testosterone measurement. Samples from ostensibly healthy men and women and from hirsute and pregnant women gave results that agreed with those obtained by equilibrium dialysis. Total testosterone concentrations for normal and hirsute women showed considerable overlap, but data on free testosterone concentrations in these populations were better resolved

Generation of megakaryocytic progenitors from human embryonic stem cells in a feeder- and serum-free medium.

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Marjorie Pick

Full Text Available BACKGROUND: The production of human platelets from embryonic stem cells in a defined culture system is a prerequisite for the generation of platelets for therapeutic use. As an important step towards this goal, we report the differentiation of human embryonic stem cells (hESCs towards the megakaryocyte (Mk lineage using a 'spin embryoid body' method in serum-free differentiation medium. METHODOLOGY AND PRINCIPAL FINDINGS: Immunophenotypic analyses of differentiating hESC identified a subpopulation of cells expressing high levels of CD41a that expressed other markers associated with the Mk lineage, including CD110, CD42b and CD61. Differentiated cells were sorted on the basis of their expression of CD41a, CD34 and CD45 and assessed for Mk colony formation, expression of myeloid and Mk genes and ability to endoreplicate DNA. In a collagen-based colony assay, the CD41a⁺ cells sorted from these differentiation cultures produced 100-800 Mk progenitors at day 13 and 25-160 Mk progenitors at day 20 of differentiation per 100,000 cells assayed. Differentiated Mk cells produced platelet-like particles which expressed CD42b and were activated by ADP, similar to platelets generated from precursors in cord blood. These studies were complemented by real time PCR analyses showing that subsets of cells enriched for CD41a⁺ Mk precursors expressed high levels of Mk associated genes such as PF4 and MPL. Conversely, high levels of myeloid and erythroid related transcripts, such as GATA1, TAL1/SCL and PU.1, were detected in sorted fractions containing CD34⁺ and CD45⁺ cells. CONCLUSIONS: We describe a serum- and feeder-free culture system that enabled the generation of Mk progenitors from human embryonic stem cells. These cells formed colonies that included differentiated Mks that fragmented to form platelet-like particles. This protocol represents an important step towards the generation of human platelets for therapeutic use.

Glucose and phosphate modulation of intracellular 45Ca incorporated into pancreatic islets during culture in the absence and presence of serum

International Nuclear Information System (INIS)

Bergsten, P.

1985-01-01

The effects of glucose and phosphate on the intracellular 45 Ca content were measured in β cell-rich pancreatic islets cultured in media containing or lacking serum. Irrespective of the glucose and serum concentrations there were no or very small increments of 45 Ca contents when phosphate was raised from 0.8 to 5.8 mM during culture for 1 day. However, after 7 days of culture in serum-free medium there was a massive accumulation of 45 Ca in the islets in response to the higher phosphate concentration. Glucose markedly reduced, and serum eliminated, the extensive accumulation probably due to increased cell viability. In the cells cultured in the presence of serum, raising the glucose concentration from 1.0 to 5.5 mM resulted in an increased incorporation of 45 Ca. This effect was particularly pronounced after culture for 7 days in 5.8 mM phosphate. A further increase of glucose to 20 mM reduced the 45 Ca content. The results are consistent with the concept that glucose both stimulates 45 Ca uptake into different β-cell pools and degranulates the cell with associated loss of intracellular calcium from the granular calcium pool. (author)

Optimization of chemically defined cell culture media--replacing fetal bovine serum in mammalian in vitro methods

DEFF Research Database (Denmark)

van der Valk, J; Brunner, D; De Smet, K

2010-01-01

with an undefined and variable composition. Defined media supplements are commercially available for some cell types. However, information on the formulation by the companies is often limited and such supplements can therefore not be regarded as completely defined. The development of defined media is difficult......, reproducible and reduce the use of experimental animals. Good cell culture practice (GCCP) is an attempt to develop a common standard for in vitro methods. The implementation of the use of chemically defined media is part of the GCCP. This will decrease the dependence on animal serum, a supplement...... and often takes place in isolation. A workshop was organised in 2009 in Copenhagen to discuss strategies to improve the development and use of serum-free defined media. In this report, the results from the meeting are discussed and the formulation of a basic serum-free medium is suggested. Furthermore...

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

NARCIS (Netherlands)

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.

2014-01-01

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

Multiple free-radical scavenging (MULTIS) capacity in cattle serum.

Science.gov (United States)

Sueishi, Yoshimi; Kamogawa, Erisa; Kimura, Anna; Kitahara, Go; Satoh, Hiroyuki; Asanuma, Taketoshi; Oowada, Shigeru

2017-01-01

Multiple free-radical scavenging (MULTIS) activity in cattle and human sera was evaluated with electron spin resonance spectroscopy. Scavenging rates against six active species, namely hydroxyl radical, superoxide anion, alkoxyl radical, alkylperoxyl radical, methyl radical, and singlet oxygen were quantified. The difference in the electron spin resonance signal intensity in the presence and absence of the serum was converted into the scavenging rates. Comparative MULTIS measurements were made in sera from eight beef cattle, three fetal calves and fifteen healthy human volunteers. Further, we determined the MULTIS value of albumin, the most abundant component in serum. MULTIS values in cattle sera indicated higher scavenging activity against most free radical species tested than human sera. In particular, cattle serum scavenging activities against superoxide and methyl radical were higher than human serum by 2.6 and 3.7 fold, respectively. In cattle serum, albumin appears to play a dominant role in MULTIS activity, but in human serum that is not the case. Previous data indicated that the abundance of uric acid in bovine blood is nearly 80% less than humans; however, this difference does not explain the deviation in MULTIS profile.

Supplements in human islet culture: human serum albumin is inferior to fetal bovine serum.

Science.gov (United States)

Avgoustiniatos, Efstathios S; Scott, William E; Suszynski, Thomas M; Schuurman, Henk-Jan; Nelson, Rebecca A; Rozak, Phillip R; Mueller, Kate R; Balamurugan, A N; Ansite, Jeffrey D; Fraga, Daniel W; Friberg, Andrew S; Wildey, Gina M; Tanaka, Tomohiro; Lyons, Connor A; Sutherland, David E R; Hering, Bernhard J; Papas, Klearchos K

2012-01-01

Culture of human islets before clinical transplantation or distribution for research purposes is standard practice. At the time the Edmonton protocol was introduced, clinical islet manufacturing did not include culture, and human serum albumin (HSA), instead of fetal bovine serum (FBS), was used during other steps of the process to avoid the introduction of xenogeneic material. When culture was subsequently introduced, HSA was also used for medium supplementation instead of FBS, which was typically used for research islet culture. The use of HSA as culture supplement was not evaluated before this implementation. We performed a retrospective analysis of 103 high-purity islet preparations (76 research preparations, all with FBS culture supplementation, and 27 clinical preparations, all with HSA supplementation) for oxygen consumption rate per DNA content (OCR/DNA; a measure of viability) and diabetes reversal rate in diabetic nude mice (a measure of potency). After 2-day culture, research preparations exhibited an average OCR/DNA 51% higher (p < 0.001) and an average diabetes reversal rate 54% higher (p < 0.05) than clinical preparations, despite 87% of the research islet preparations having been derived from research-grade pancreata that are considered of lower quality. In a prospective paired study on islets from eight research preparations, OCR/DNA was, on average, 27% higher with FBS supplementation than that with HSA supplementation (p < 0.05). We conclude that the quality of clinical islet preparations can be improved when culture is performed in media supplemented with serum instead of albumin.

Defined culture medium for stem cell differentiation: applicability of serum-free conditions in the mouse embryonic stem cell test.

Science.gov (United States)

Riebeling, Christian; Schlechter, Katharina; Buesen, Roland; Spielmann, Horst; Luch, Andreas; Seiler, Andrea

2011-06-01

The embryonic stem cell test (EST) is a validated method to assess the developmental toxicity potency of chemicals. It was developed to reduce animal use and allow faster testing for hazard assessment. The cells used in this method are maintained and differentiated in media containing foetal calf serum. This animal product is of considerable variation in quality, and individual batches require extensive testing for their applicability in the EST. Moreover, its production involves a large number of foetuses and possible animal suffering. We demonstrate the serum-free medium and feeder cell-free maintenance of the mouse embryonic stem cell line D3 and investigate the use of specific growth factors for induction of cardiac differentiation. Using a combination of bone morphogenetic protein-2, bone morphogenetic protein-4, activin A and ascorbic acid, embryoid bodies efficiently differentiated into contracting myocardium. Additionally, examining levels of intracellular marker proteins by flow cytometry not only confirmed differentiation into cardiomyocytes, but demonstrated significant differentiation into neuronal cells in the same time frame. Thus, this approach might allow for simultaneous detection of developmental effects on both early mesodermal and neuroectodermal differentiation. The serum-free conditions for maintenance and differentiation of D3 cells described here enhance the transferability and standardisation and hence the performance of the EST. Copyright © 2011 Elsevier Ltd. All rights reserved.

Salivary Cortisol Can Replace Free Serum Cortisol Measurements in Patients With Septic Shock

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Orlander, Philip R.

2011-01-01

Background: There is a renewed interest in adrenal function during severe sepsis. Most studies have used total serum cortisol levels; however, only free serum cortisol is biologically active. The aim of this study was to determine the validity of salivary cortisol levels as a surrogate for free serum cortisol levels during septic shock. Methods: Fifty-seven patients with septic shock were studied to determine the correlation between total serum cortisol and salivary cortisol to free serum cortisol levels. Thirty-eight patients were included in the salivary to free serum cortisol correlation. Salivary cortisol level was tested by enzyme immunoassay. Serum total cortisol, free cortisol, and cortisol-binding globulin (CBG) levels were determined by liquid chromatography-mass spectrometry, equilibrium analysis, and radioimmunoassay, respectively. Results: The mean ± SD age was 56.6 ± 18.5 years. Fifty-seven percent were women. APACHE (Acute Physiology and Chronic Health Evaluation) II score median was 26, Simplified Acute Physiology Score II median was 61, and Sequential Organ Failure Assessment median was 13. The correlation between salivary and free serum cortisol levels was 0.79 (95% CI, 0.63-0.89; P cortisol and total serum cortisol levels was 0.86 (95% CI, 0.78-0.92; P cortisol level was 2.27 ± 1.64 μg/dL. The mean ± SD salivary cortisol level was 2.60 ± 2.69 μg/dL. The mean ± SD total serum cortisol level was 21.56 ± 8.71 μg/dL. The mean ± SD CBG level was 23.54 ± 8.33 mg/dL. Conclusions: Salivary cortisol level can be used as a surrogate of free serum cortisol level in patients with septic shock with very good correlation. Salivary cortisol testing is noninvasive, easy to perform, and can be conducted daily. Trial registry: ClinicalTrials.gov; No.: NCT00523198; URL: www.clinicaltrials.gov PMID:21816912

Ultrasensitive radioimmunoassay for direct determination of free triiodothyronine concentration in serum

International Nuclear Information System (INIS)

Weeke, J.; Oerskov, H.

1975-01-01

Free triiodothyronine (T 3 ) in serum has been measured directly in dialysates of serum, using a wick chromatographic radioimmunoassay. Adequate sensitivity was attained by the use of [ 125 I]T 3 with a very high specific activity (2,000 to 3,000μCi/μg). Sera were dialysed against a Krebs-Ringer bicarbonate buffer modified so as to be similar to plasma water. Dialysis took place under carefully controlled circumstances. The influence on the equilibrium of total to free T 3 of temperature, serum dilution and dialysis time was studied. By the present method, free T 3 in serum from groups of subjects including 20 men, 10 women taking oral contraceptives and 20 women with normal menstrual cycles were identical, averaging 5.2 pg/ml. A chromatographic radioimmunoassay of total T 3 using high specific activity [ 125 I]T 3 and a very small test sample is also described. (auth.)

Platelet lysate as replacement for fetal bovine serum in mesenchymal stromal cell cultures.

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Bieback, Karen

2013-10-01

Mesenchymal stromal cells (MSC) emerged as highly attractive in cell-based regenerative medicine. Initially thought to provide cells capable of differentiation towards mesenchymal cell types (osteoblasts, chondrocytes, adipocytes etc.), by and by potent immunoregulatory and pro-regenerative activities have been discovered, broadening the field of potential applications from bone and cartilage regeneration to wound healing and treatment of autoimmune diseases. Due to the limited frequency in most tissue sources, ex vivo expansion of MSC is required compliant with good manufacturing practice (GMP) guidelines to yield clinically relevant cell doses. Though, still most manufacturing protocols use fetal bovine serum (FBS) as cell culture supplement to isolate and to expand MSC. However, the high lot-to-lot variability as well as risk of contamination and immunization call for xenogenic-free culture conditions. In terms of standardization, chemically defined media appear as the ultimate achievement. Since these media need to maintain all key cellular and therapy-relevant features of MSC, the development of chemically defined media is still - albeit highly investigated - only in its beginning. The current alternatives to FBS rely on human blood-derived components: plasma, serum, umbilical cord blood serum, and platelet derivatives like platelet lysate. Focusing on quality aspects, the latter will be addressed within this review.

Differentiation of Dental Pulp Stem Cells into Neuron-Like Cells in Serum-Free Medium

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Shahrul Hisham Zainal Ariffin

2013-01-01

Full Text Available Dental pulp tissue contains dental pulp stem cells (DPSCs. Dental pulp cells (also known as dental pulp-derived mesenchymal stem cells are capable of differentiating into multilineage cells including neuron-like cells. The aim of this study was to examine the capability of DPSCs to differentiate into neuron-like cells without using any reagents or growth factors. DPSCs were isolated from teeth extracted from 6- to 8-week-old mice and maintained in complete medium. The cells from the fourth passage were induced to differentiate by culturing in medium without serum or growth factors. RT-PCR molecular analysis showed characteristics of Cd146+, Cd166+, and Cd31− in DPSCs, indicating that these cells are mesenchymal stem cells rather than hematopoietic stem cells. After 5 days of neuronal differentiation, the cells showed neuron-like morphological changes and expressed MAP2 protein. The activation of Nestin was observed at low level prior to differentiation and increased after 5 days of culture in differentiation medium, whereas Tub3 was activated only after 5 days of neuronal differentiation. The proliferation of the differentiated cells decreased in comparison to that of the control cells. Dental pulp stem cells are induced to differentiate into neuron-like cells when cultured in serum- and growth factor-free medium.

Attachment and growth of human keratinocytes in a serum-free environment.

Science.gov (United States)

Gilchrest, B A; Calhoun, J K; Maciag, T

1982-08-01

Using a serum-free system, we have investigated the influence of human fibronectin (HFN) and selected growth factors (GF) on the attachment and growth of normal human keratinocytes in vitro. Single-cell suspensions of keratinocytes from near-confluent primary plates, plated on 5-10 microgram/cm2 HFN, showed approximately 30-40% attachment after 2-24 hours of incubation at 37 degrees C, compared with 4-6% attachment on uncoated platic plates. Percentage of attached cells was independent of seed density, tissue donor age, in vitro culture age, or medium composition, while subsequent cellular proliferation was strongly dependent on these factors. Keratinocytes grown on an adequate HFN matrix in a previously described hormone-supplemented medium (Maciag et al., 1981a) achieved four to eight population doubling over 7-12 days at densities greater than or equal to 104 cell/cm2. Removal of most GF individually from the medium had little or no effect on growth, while removal of epidermal growth factor (EGF) alone reduced growth by 30-35% and removal of bovine brain extract (BE) alone reduced growth by approximately 90%. Conversely, EGF alone in basal medium supported approximately 10% control growth, BE alone supported 30-40% control growth, and the combination of EGF and BE approximately 70%. In addition to its major effect on proliferation in this system, BE was necessary to preserve normal keratinocyte morphology and protein production. These findings expand earlier observations that HFN facilitates keratinocyte attachment in vitro and that a brain-derived extract can exert a major positive influence on cultured keratinocytes.

A Continuous-Exchange Cell-Free Protein Synthesis System Based on Extracts from Cultured Insect Cells

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Stech, Marlitt; Quast, Robert B.; Sachse, Rita; Schulze, Corina; Wüstenhagen, Doreen A.; Kubick, Stefan

2014-01-01

In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds. PMID:24804975

Human serum-derived protein removes the need for coating in defined human pluripotent stem cell culture

Science.gov (United States)

Pijuan-Galitó, Sara; Tamm, Christoffer; Schuster, Jens; Sobol, Maria; Forsberg, Lars; Merry, Catherine L. R.; Annerén, Cecilia

2016-01-01

Reliable, scalable and time-efficient culture methods are required to fully realize the clinical and industrial applications of human pluripotent stem (hPS) cells. Here we present a completely defined, xeno-free medium that supports long-term propagation of hPS cells on uncoated tissue culture plastic. The medium consists of the Essential 8 (E8) formulation supplemented with inter-α-inhibitor (IαI), a human serum-derived protein, recently demonstrated to activate key pluripotency pathways in mouse PS cells. IαI efficiently induces attachment and long-term growth of both embryonic and induced hPS cell lines when added as a soluble protein to the medium at seeding. IαI supplementation efficiently supports adaptation of feeder-dependent hPS cells to xeno-free conditions, clonal growth as well as single-cell survival in the absence of Rho-associated kinase inhibitor (ROCKi). This time-efficient and simplified culture method paves the way for large-scale, high-throughput hPS cell culture, and will be valuable for both basic research and commercial applications. PMID:27405751

Serum total and free carnitine levels in children with asthma.

Science.gov (United States)

Asilsoy, Suna; Bekem, Ozlem; Karaman, Ozkan; Uzuner, Nevin; Kavukçu, Salih

2009-02-01

Serum carnitine is decreased in recurrent pulmonary infections. We aimed to evaluate serum carnitine levels in asthmatic children. Study group consisted of children with stable asthma and those with acute asthma attacks, while control group included healthy children. Attack severity was determined by the pulmonary score system. Total and free carnitine levels were studied in one blood sample from the control group and stable asthmatics and in two samples from children with acute asthma exacerbation during and after the attack. All the 40 patients in the study group had moderate asthma including 30 with acute attack (13 mild and 17 moderate) and 10 with stable asthma. Carnitine levels were significantly lower in acute attack asthmatics than in the stable asthmatics and controls, while there was no significant difference between the latter two groups. Carnitine levels were not different between asthmatics with mild and moderate attack, and were similar during and after an acute attack. Serum carnitine levels decrease in children with moderate asthma during exacerbation of asthma and shortly thereafter. Further studies are needed to evaluate the effect of carnitine treatment on serum carnitine level.

Metaphase II oocytes from human unilaminar follicles grown in a multi-step culture system.

Science.gov (United States)

McLaughlin, M; Albertini, D F; Wallace, W H B; Anderson, R A; Telfer, E E

2018-03-01

Can complete oocyte development be achieved from human ovarian tissue containing primordial/unilaminar follicles and grown in vitro in a multi-step culture to meiotic maturation demonstrated by the formation of polar bodies and a Metaphase II spindle? Development of human oocytes from primordial/unilaminar stages to resumption of meiosis (Metaphase II) and emission of a polar body was achieved within a serum free multi-step culture system. Complete development of oocytes in vitro has been achieved in mouse, where in vitro grown (IVG) oocytes from primordial follicles have resulted in the production of live offspring. Human oocytes have been grown in vitro from the secondary/multi-laminar stage to obtain fully grown oocytes capable of meiotic maturation. However, there are no reports of a culture system supporting complete growth from the earliest stages of human follicle development through to Metaphase II. Ovarian cortical biopsies were obtained with informed consent from women undergoing elective caesarean section (mean age: 30.7 ± 1.7; range: 25-39 years, n = 10). Laboratory setting. Ovarian biopsies were dissected into thin strips, and after removal of growing follicles were cultured in serum free medium for 8 days (Step 1). At the end of this period secondary/multi-laminar follicles were dissected from the strips and intact follicles 100-150 μm in diameter were selected for further culture. Isolated follicles were cultured individually in serum free medium in the presence of 100 ng/ml of human recombinant Activin A (Step 2). Individual follicles were monitored and after 8 days, cumulus oocyte complexes (COCs) were retrieved by gentle pressure on the cultured follicles. Complexes with complete cumulus and adherent mural granulosa cells were selected and cultured in the presence of Activin A and FSH on membranes for a further 4 days (Step 3). At the end of Step 3, complexes containing oocytes >100 μm diameter were selected for IVM in SAGE medium (Step 4) then

Development of a new mouse palate organ culture system and effect of X-irradiation on palatogenesis

International Nuclear Information System (INIS)

Hiranuma, Hiroko; Jikko, Akitoshi; Maeda, Takashi; Furukawa, Souhei

1999-01-01

On the basis of an already established suspension system of organ culture for mouse palate explants, we developed a new culture system, which has several advantages over the previous methods. We used a 48-well culture plate in which the explants can be cultured individually, and only 300 μl of medium is needed for each well. In order to optimize the culture results, we systematically studied the influence of main ''culture conditions'' such as tilt degree of the culturing palate, rotation speed, and addition of ascorbic acid to the medium. This system allows culturing of palates from day 13.5 of gestation to day 16.5 under serum-free conditions using a chemically defined medium, which resulted in 78% of the palates growing fused. Utilizing this culture system, the direct effect of X-irradiation on palataogesis was analyzed. A 4 Gy dose of X-irradiation was administrated at the beginning of culture period. The incidence of palatal fusion was not significantly different from that of the non-irradiated group. (author)

Multiple free-radical scavenging capacity in serum

Science.gov (United States)

Oowada, Shigeru; Endo, Nobuyuki; Kameya, Hiromi; Shimmei, Masashi; Kotake, Yashige

2012-01-01

We have developed a method to determine serum scavenging-capacity profile against multiple free radical species, namely hydroxyl radical, superoxide radical, alkoxyl radical, alkylperoxyl radical, alkyl radical, and singlet oxygen. This method was applied to a cohort of chronic kidney disease patients. Each free radical species was produced with a common experimental procedure; i.e., uv/visible-light photolysis of free-radical precursor/sensitizer. The decrease in free-radical concentration by the presence of serum was quantified with electron spin resonance spin trapping method, from which the scavenging capacity was calculated. There was a significant capacity change in the disease group (n = 45) as compared with the healthy control group (n = 30). The percent values of disease’s scavenging capacity with respect to control group indicated statistically significant differences in all free-radical species except alkylperoxyl radical, i.e., hydroxyl radical, 73 ± 12% (p = 0.001); superoxide radical, 158 ± 50% (p = 0.001); alkoxyl radical, 121 ± 30% (p = 0.005); alkylperoxyl radical, 123 ± 32% (p>0.1); alkyl radical, 26 ± 14% (p = 0.001); and singlet oxygen, 57 ± 18% (p = 0.001). The scavenging capacity profile was illustrated using a radar chart, clearly demonstrating the characteristic change in the disease group. Although the cause of the scavenging capacity change by the disease state is not completely understood, the profile of multiple radical scavenging capacities may become a useful diagnostic tool. PMID:22962529

The B-domain of factor VIII reduces cell membrane attachement to host cells in serum free conditions

DEFF Research Database (Denmark)

Kolind, Mille Petersen; Nørby, Peder Lisby; Flintegaard, Thomas Veje

2010-01-01

engineered extensively throughout the years to increase the low production yields that initially were obtained from mammalian cell cultures. The scope of this work was to investigate the interaction of rFVIII with the cell membrane surface of the producing cells in serum free medium. We wondered whether...... binding of rFVIII to the cell membrane could be a factor diminishing the production yield. We studied the contribution of the rFVIII B-domain to membrane attachment by transfecting several constructs containing increasing lengths of the B-domain into cells under serum free conditions. We found that 90......% of rFVIII is attached to the cell membrane of the producing cell when the rFVIII variant contains a short B-domain (21 aa). By increasing the length of the B-domain the membrane attached fraction can be reduced to 50% of the total expressed rFVIII. Further, our studies show that the N...

Interaction of estradiol and high density lipoproteins on proliferation of the human breast cancer cell line MCF-7 adapted to grow in serum free conditions

International Nuclear Information System (INIS)

Jozan, S.; Faye, J.C.; Tournier, J.F.; Tauber, J.P.; David, J.F.; Bayard, F.

1985-01-01

The responsiveness of the human mammary carcinoma cell line MCF-7 to estradiol and tamoxifen treatment has been studied in different culture conditions. Cells from exponentially growing cultures were compared with cells in their initial cycles after replating from confluent cultures (''confluent-log'' cells). It has been observed that estradiol stimulation of tritiated thymidine incorporation decreases with cell density and that ''confluent-log'' cells are estrogen unresponsive for a period of four cell cycles in serum-free medium conditions. On the other hand, growth of cells replated from exponentially growing, as well as from confluent cultures, can be inhibited by tamoxifen or a combined treatment with tamoxifen and the progestin levonorgestrel. This growth inhibitory effect can be rescued by estradiol when cells are replated from exponentially growing cultures. The growth inhibitory effect cannot be rescued by estradiol alone (10(-10) to 10(-8) M) when cells are replated from confluent cultures. In this condition, the addition of steroid depleted serum is necessary to reverse the state of estradiol unresponsiveness. Serum can be replaced by high density lipoproteins but not by low density lipoproteins or lipoprotein deficient serum. The present data show that estradiol and HDL interact in the control of MCF-7 cell proliferation

Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression studies.

Science.gov (United States)

Yang, R; Elankumaran, Y; Hijjawi, N; Ryan, U

2015-06-01

A cell-free culture system for Cryptosporidium parvum was analysed using scanning electron microscopy (SEM) to characterise life cycle stages and compare gene expression in cell-free culture and cell culture using HCT-8 cells. Cryptosporidium parvum samples were harvested at 2 h, 8 h, 14 h, 26 h, 50 h, 74 h, 98 h, 122 h and 170 h, chemically fixed and specimens were observed using a Zeiss 1555 scanning electron microscope. The presence of sporozoites, trophozoites and type I merozoites were identified by SEM. Gene expression in cell culture and cell-free culture was studied using reverse transcriptase quantitative PCR (RT-qPCR) of the sporozoite surface antigen protein (cp15), the glycoprotein 900 (gp900), the Cryptosporidium oocyst wall protein (COWP) and 18S ribosomal RNA (rRNA) genes in both cell free and conventional cell culture. In cell culture, cp15 expression peaked at 74 h, gp900 expression peaked at 74 h and 98 h and COWP expression peaked at 50 h. In cell-free culture, CP15 expression peaked at 98 h, gp900 expression peaked at 74 h and COWP expression peaked at 122 h. The present study is the first to compare gene expression of C. parvum in cell culture and cell-free culture and to characterise life cycle stages of C. parvum in cell-free culture using SEM. Findings from this study showed that gene expression patterns in cell culture and cell-free culture were similar but in cell-free culture, gene expression was delayed for CP15 and COWP in cell free culture compared with the cell culture system and was lower. Although three life cycle stageswere conclusively identified, improvements in SEM methodology should lead to the detection of more life cycle stages. Copyright © 2015 Elsevier Inc. All rights reserved.

An improved method for the radioimmunoassay of free-thyroxine in serum dialysates

International Nuclear Information System (INIS)

Giles, A.F.

1982-01-01

A convenient, sensitive radioimmunoassay (using 125 I) of free thyroxine in serum dialysates is described. The method utilizes a solid phase separation system (pre-formed double antibody) and a relatively short incubation period (220 min) with a staggered addition of tracer. Blanks were low and consistent. The normal range for non-pregnant euthyroid samples (n = 59) was 11-23 pmol/l. Third trimester pregnancy samples were mostly within the normal range but at the lower end. Patients on T4 replacement showed a much wider variation in free T4 levels with many samples in the hyperthyroid region. Some hypothyroid samples had undetectable free T4 levels and hyperthyroid samples were frequently greater than 80 pmol/l. (author)

Verification of serum reference intervals for free light chains in a local South African population.

Science.gov (United States)

Zemlin, Annalise E; Rensburg, Megan A; Ipp, Hayley; Germishuys, Jurie J; Erasmus, Rajiv T

2013-11-01

Monoclonal serum free light chain measurements are used to follow up and manage patients with monoclonal gammopathies, and abnormal serum free light chain ratios are associated with risk of progression in certain diseases. We aimed to validate the reference intervals in our population. Reference intervals for κ and λ free light chains were established on 120 healthy adults. Creatinine levels were measured to exclude renal dysfunction and serum protein electrophoresis was performed. All creatinine values were within normal limits. After exclusion of subjects with abnormal serum protein electrophoreses, 113 subjects were available for analysis. The 95% reference interval was 6.3-20.6 mg/L for κ free light chains, 8.7-25.9 mg/L for λ free light chains and 0.46-1.23 for free light chain ratio. Most of the values fell within the manufacturer's recommended limits and therefore could be used for our population.

Comparison between human cord blood serum and platelet-rich plasma supplementation for Human Wharton's Jelly Stem Cells and dermal fibroblasts culture

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Hashemi SS

2016-08-01

Full Text Available We carried out a side-by-side comparison of the effects of Human cord blood serum (HcbS versus embryonic PRP on Human Wharton's Jelly Stem Cells(hWMSCand dermal fibroblasts proliferation. Human umbilical cord blood was collected to prepare activated serum (HCS and platelet-rich plasma (CPRP.Wharton's Jelly Stem Cells and dermal fibroblasts were cultured in complete medium with10% CPRP, 10%HCSor 10% fetal bovine serumand control (serum-free media.The efficiency of the protocols was evaluated in terms of the number of adherent cells and their expansion and Cell proliferation. We showed that proliferation of fibroblasts and mesenchymal stem cells in the presence of cord blood serum and platelet-rich plasma significantly more than the control group (p≤0/05. As an alternative to FBS, cord blood serum has been proved as an effective component in cell tissue culture applications and embraced a vast future in clinical applications of regenerative medicine. However, there is still a need to explore the potential of HCS and its safe applications in humanized cell therapy or tissue engineering.

Production of inactivated influenza H5N1 vaccines from MDCK cells in serum-free medium.

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Alan Yung-Chih Hu

Full Text Available 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 × 10(6 cells/mL after 5 days. The maximum virus titers of 1024 Hemagglutinin (HA units/50 µL and 7.1 ± 0.3 × 10(8 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.

Testing of serum atherogenicity in cell cultures: questionable data published

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Sergei V. Jargin

2012-01-01

Full Text Available In a large series of studies was reported that culturing of smooth muscle cells with serum from atherosclerosis patients caused intracellular lipid accumulation, while serum from healthy controls had no such effect. Cultures were used for evaluation of antiatherogenic drugs. Numerous substances were reported to lower serum atherogenicity: statins, trapidil, calcium antagonists, garlic derivatives etc. On the contrary, beta-blockers, phenothiazines and oral hypoglycemics were reported to be pro-atherogenic. Known antiatherogenic agents can influence lipid metabolism and cholesterol synthesis, intestinal absorption or endothelium-related mechanisms. All these targets are absent in cell monocultures. Inflammatory factors, addressed by some antiatherogenic drugs, are also not reproduced. In vivo, relationship between cholesterol uptake by cells and atherogenesis must be inverse rather than direct: in familial hypercholesterolemia, inefficient clearance of LDL-cholesterol by cells predisposes to atherosclerosis. Accordingly, if a pharmacological agent reduces cholesterol uptake by cells in vitro, it should be expected to elevate cholesterol in vivo. Validity of clinical recommendations, based on serum atherogenicity testing in cell monocultures, is therefore questionable. These considerations pertain also to the drugs developed on the basis of the cell culture experiments.

Derivation of transgene-free human induced pluripotent stem cells from human peripheral T cells in defined culture conditions.

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Yoshikazu Kishino

Full Text Available Recently, induced pluripotent stem cells (iPSCs were established as promising cell sources for revolutionary regenerative therapies. The initial culture system used for iPSC generation needed fetal calf serum in the culture medium and mouse embryonic fibroblast as a feeder layer, both of which could possibly transfer unknown exogenous antigens and pathogens into the iPSC population. Therefore, the development of culture systems designed to minimize such potential risks has become increasingly vital for future applications of iPSCs for clinical use. On another front, although donor cell types for generating iPSCs are wide-ranging, T cells have attracted attention as unique cell sources for iPSCs generation because T cell-derived iPSCs (TiPSCs have a unique monoclonal T cell receptor genomic rearrangement that enables their differentiation into antigen-specific T cells, which can be applied to novel immunotherapies. In the present study, we generated transgene-free human TiPSCs using a combination of activated human T cells and Sendai virus under defined culture conditions. These TiPSCs expressed pluripotent markers by quantitative PCR and immunostaining, had a normal karyotype, and were capable of differentiating into cells from all three germ layers. This method of TiPSCs generation is more suitable for the therapeutic application of iPSC technology because it lowers the risks associated with the presence of undefined, animal-derived feeder cells and serum. Therefore this work will lead to establishment of safer iPSCs and extended clinical application.

Free Publishing Culture. Sustainable Models?

Directory of Open Access Journals (Sweden)

Silvia Nanclares Escudero

2013-03-01

Full Text Available As a result of the collective research on the possibilities for publishing production and distribution offered nowadays by the Free Culture scenario, we present here a mapping of symptoms in order to propose a transitory diagnostic of the question: Is it possible to generate an economically sustainable publishing model based on the uses and customs generated and provided by Free Culture? Data, intuitions, experiences and ideas attempt to back up our affirmative answer.

Increasing synthetic serum substitute (SSS) concentrations in P1 glucose/phosphate-free medium improves implantation rate: a comparative study.

Science.gov (United States)

Ben-Yosef, D; Yovel, I; Schwartz, T; Azem, F; Lessing, J B; Amit, A

2001-11-01

To assess the comparative efficacy of IVF medium (MediCult, with 5.2 mM glucose) and a glucose/phosphate-free medium, P1 (Irvine Scientific), and to investigate the influence of increasing the serum supplementation (synthetic serum substitute; SSS; Irvine Scientific) to P1 on embryo development and implantation. Patients were randomly assigned to IVF medium (Group 1, cycles n = 172) or P1 supplemented with 10% SSS (Group 2, cycles n = 229) according to the medium scheduled for use on the day of oocyte retrieval. Another 555 IVF consequent cycles (Group 3) were performed using increased SSS concentrations (20%) in P1 medium. In this large series of IVF cycles, we herein demonstrate that significantly higher pregnancy and implantation rates were found when embryos were cultured in glucose/phosphate-free medium P1 supplemented with 20% SSS compared to supplementation with the lower SSS concentration and with IVF medium.

A simplified ultrafiltration method for determination of serum free cortisol

International Nuclear Information System (INIS)

MacMahon, W.; Sgoutas, D.

1983-01-01

The authors describe the suitability of the Amicon MPS-1 centrifugal ultrafiltration device and the YMB membrane for measuring free cortisol in serum. The method combines two independent assays: total cortisol and the ultrafiltrate fraction of added [ 3 H]cortisol. The unbound fraction is determined in 0.25-0.30 ml of ultrafiltrate collected from 0.6 to 1 ml of serum that has been equilibrated with [ 3 H]cortisol at 37 0 C for 20 min. The assay is rapid (less than 1 h), practical (no more than 0.6 ml of serum is necessary) and repeatable (CV: 3.8% within-assay and 12.2% in different assays). Error introduced in free cortisol measurement due to dilution effects in dialysis is systematically defined, and the effect of tracer purity on the ultrafiltration method is examined. Dialyzed sera from normal men and women, from patients with Cushing's disease and adrenal insufficiency, and from pregnant women gave ultrafiltration results that accurately duplicated those obtained by previous dialysis. (Auth.)

A new method for culture of zona-included or zona-free embryos: the Well of the Well (WOW) system

DEFF Research Database (Denmark)

Vajta, Gabor; Peura, T T; Holm, Peter

2000-01-01

(WOW) system. Small wells (WOWs) were formed in four-well dishes by melting the bottom with heated steel rods. The WOWs were then rinsed, the wells were filled with medium, and the embryos were placed into the WOWs. To test the value of the WOW system a 3 x 3 factorial experiment was performed. Bovine......Culture of mammalian zygotes individually and in small groups results in lower developmental rates than culture of large groups. Zona-free zygotes also have impaired developmental potential in current culture systems. This paper describes a new approach to resolve the problems, the Well of the Well...... embryos cultured in 400 microl medium. The WOW system resulted in higher blastocyst/oocyte rates for all three modules (single: 59 group of five: 61 single zona-digested: 53 than the culture in drops or in wells (P well...

Active Stat3 is required for survival of human squamous cell carcinoma cells in serum-free conditions

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DiGiovanni John

2006-04-01

Full Text Available Abstract Background Squamous cell carcinoma (SCC of the skin is the most aggressive form of non-melanoma skin cancer (NMSC, and is the single most commonly diagnosed cancer in the U.S., with over one million new cases reported each year. Recent studies have revealed an oncogenic role of activated signal transducer and activator of transcription 3 (Stat3 in many human tumors, especially in those of epithelial origin, including skin SCC. Stat3 is a mediator of numerous growth factor and cytokine signaling pathways, all of which activate it through phosphorylation of tyrosine 705. Results To further address the role of Stat3 in skin SCC tumorigenesis, we have analyzed a panel of human skin-derived cell lines ranging from normal human epidermal keratinocytes (NHEK, to non-tumorigenic transformed skin cells (HaCaT, to highly tumorigenic cells (SRB1-m7 and SRB12-p9 and observed a positive correlation between Stat3 phosphorylation and SCC malignancy. We next determined the role of Stat3 activity in cell proliferation and viability under serum-free culture conditions. This was accomplished by suppressing Stat3 activity in the SRB12-p9 cells through stable expression of a dominant negative acting form of Stat3β, which contains a tyrosine 705 to phenylalanine mutation (S3DN. The S3DN cells behaved similar to parental SRB12-p9 cells when cultured in optimal growth conditions, in the presence of 10% fetal calf serum. However, unlike the SRB12-p9 cells, S3DN cells underwent apoptotic cell death when cultured in serum-free medium (SFM. This was evidenced by multiple criteria, including accumulation of sub-G1 particles, induced PARP cleavage, and acquisition of the characteristic morphological changes associated with apoptosis. Conclusion This study provides direct evidence for a role for Stat3 in maintaining cell survival in the conditions of exogenous growth factor deprivation produced by culture in SFM. We also propose that delivery of the S3DN gene or

Serum-free microcarrier based production of replication deficient Influenza vaccine candidate virus lacking NS1 using Vero cells

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Yan Mylene L

2011-08-01

Full Text Available Abstract Background Influenza virus is a major health concern that has huge impacts on the human society, and vaccination remains as one of the most effective ways to mitigate this disease. Comparing the two types of commercially available Influenza vaccine, the live attenuated virus vaccine is more cross-reactive and easier to administer than the traditional inactivated vaccines. One promising live attenuated Influenza vaccine that has completed Phase I clinical trial is deltaFLU, a deletion mutant lacking the viral Nonstructural Protein 1 (NS1 gene. As a consequence of this gene deletion, this mutant virus can only propagate effectively in cells with a deficient interferon-mediated antiviral response. To demonstrate the manufacturability of this vaccine candidate, a batch bioreactor production process using adherent Vero cells on microcarriers in commercially available animal-component free, serum-free media is described. Results Five commercially available animal-component free, serum-free media (SFM were evaluated for growth of Vero cells in agitated Cytodex 1 spinner flask microcarrier cultures. EX-CELL Vero SFM achieved the highest cell concentration of 2.6 × 10^6 cells/ml, whereas other SFM achieved about 1.2 × 10^6 cells/ml. Time points for infection between the late exponential and stationary phases of cell growth had no significant effect in the final virus titres. A virus yield of 7.6 Log10 TCID50/ml was achieved using trypsin concentration of 10 μg/ml and MOI of 0.001. The Influenza vaccine production process was scaled up to a 3 liter controlled stirred tank bioreactor to achieve a cell density of 2.7 × 10^6 cells/ml and virus titre of 8.3 Log10 TCID50/ml. Finally, the bioreactor system was tested for the production of the corresponding wild type H1N1 Influenza virus, which is conventionally used in the production of inactivated vaccine. High virus titres of up to 10 Log10 TCID50/ml were achieved. Conclusions We describe for the

Embryogenesis-promoting factors in rat serum.

Science.gov (United States)

Katoh, M; Kimura, R; Shoji, R

1998-06-15

Regarding whole rat embryo cultures in vitro, rat serum as a culture medium is known to support the normal growth of rat embryos in the organogenesis phase. The purpose of the present study was to isolate the embryogenesis-promoting factors from rat serum as a first step in the development of a defined serum-free medium for a whole embryo culture system. Pooled rat serum after heat inactivation was fractionated into three major peaks (frA, containing a region of void volume, frB, and frC) by gel filtration. The 9.5-day rat embryos that were cultivated for 48 hr in essential salt medium containing frB (with a molecular size range of 100-500 kDa) revealed normal growth. Three proteins (27 kDa, 76 kDa, and 190 kDa) that had the embryogenesis-promoting effects were isolated from 3-hr delayed centrifuged rat serum by the ion exchange chromatography. The 76-kDa protein was found to be rat transferrin by immunoblotting. The 27-kDa protein was identified as apo-AI (the major apoprotein of high-density lipoprotein) by immunoblotting. High-density lipoprotein obtained from pooled rat serum by a NaBr density gradient ultracentrifugation was found to have a positive effect on embryogenesis. The 10-kDa protein was also identified as alpha 1-inhibitor 3 by immunoblotting. In addition, the embryogenesis-promoting effect of the fraction containing 27-kDa and 190-kDa proteins declined within a short period of storage at -20 degrees C. This decrease was countered by supplementing its fraction (D-2) with albumin isolated from rat serum. These results in the present study suggest that transferrin, high-density lipoprotein, and alpha 1-inhibitor 3 in rat serum may be embryogenesis-promoting factors, and that albumin appeared to play a role in the embryogenesis of rat embryos in whole embryo cultures.

Optimization of Serum Immunoglobulin Free Light Chain Analysis for Subclassification of Cardiac Amyloidosis.

Science.gov (United States)

Halushka, Marc K; Eng, George; Collins, A Bernard; Judge, Daniel P; Semigran, Marc J; Stone, James R

2015-06-01

Accurate and rapid classification of cardiac amyloidosis is important for patient management. We have optimized the use of serum free light chain kappa and lambda values to differentiate immunoglobulin light chain amyloid (AL) amyloidosis from transthyretin amyloid and amyloid A using 85 cases of tissue-proven cardiac amyloidosis, in which there was direct classification of amyloidosis by mass spectrometry or immunofluorescence. The serum free light chain kappa/lambda ratios were non-overlapping for the three major groups: AL-lambda (0.01-0.41, n = 30), non-AL (0.52-2.7, n = 43), and AL-kappa (6.7-967, n = 12). A kappa/lambda ratio value between 0.5 and 5.0 had 100 % sensitivity and 100 % specificity for distinguishing AL amyloidosis from non-AL amyloidosis. This optimized range for serum light chain kappa/lambda ratio provides extremely robust classification of cardiac amyloidosis. Cases of cardiac amyloidosis in which the serum kappa/lambda free light chain ratio falls close to these new cutoff values may benefit most from direct amyloid subtyping.

Clinical significance of the measurements of serum free thyroxine and free triiodothyronine concentrations

International Nuclear Information System (INIS)

Kubota, Ken; Sasaki, Norio; Takaku, Fumimaro; Uchimura, Hidemasa

1988-01-01

A commercially available ''DPC'' radioimmunoassay kit was used to study the serum concentrations of free triiodothyronine (FT3) and free thyroxine (FT4) in a series of 189 patients with various thyroid diseases and 120 healthy controls. The basal serum concentrations of FT3 and FT4 in normal controls ranged from 0.98 to 2.04 ng/dl and from 1.43 to 3.66 pg/ml, respectively. All untreated patients with Graves' disease had abnormally high FT3 and FT4 values, indicating the discrimination between hyperthyroid and normal subjects. A decreased ratio of FT4 to FT3 was observed in patients managed with antithyroid drugs. In diagnosing hypothyroidism, ''DPC'' FT4 kits were more sensitive than ''DPC'' TT4 and ''Amerlex'' FT4 kits. In the case of non-thyroid diseases, FT4 and FT3 values, as well as total T4 and total T3, were normal or decreased, with the exception of occasionally unknown high values. The interference of thyroxine binding globulin abnormablity was successfully eliminated by using new versions of ''DPC'' kits. (Namekawa, K.)

Platelet lysate as a novel serum-free media supplement for the culture of equine bone marrow-derived mesenchymal stem cells.

Science.gov (United States)

Naskou, Maria C; Sumner, Scarlett M; Chocallo, Anna; Kemelmakher, Hannah; Thoresen, Merrilee; Copland, Ian; Galipeau, Jacques; Peroni, John F

2018-03-22

Mesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) pooled from donor horses in place of FBS to culture equine MSCs. We hypothesized that ePL, produced following apheresis, will function as the sole media supplement to accelerate the expansion of equine bone marrow-derived MSCs without altering their phenotype and their immunomodulatory capacity. Platelet concentrate was obtained via plateletpheresis and ePL were produced via freeze-thaw and centrifugation cycles. Population doublings (PD) and doubling time (DT) of bone marrow-derived MSCs (n = 3) cultured with FBS or ePL media were calculated. Cell viability, immunophenotypic analysis, and trilineage differentiation capacity of MSCs were assessed accordingly. To assess the ability of MSCs to modulate inflammatory responses, E. coli lipopolysaccharide (LPS)-stimulated monocytes were cocultured with MSCs cultured in the two different media formulations, and cell culture supernatants were assayed for the production of tumor necrosis factor (TNF)-α. Our results showed that MSCs cultured in ePL media exhibited similar proliferation rates (PD and DT) compared with those cultured in FBS at individual time points. MSCs cultured in ePL showed a statistically significant increased viability following a single washing step, expressed similar levels of MSC markers compared to FBS, and were able to differentiate towards the three lineages. Finally, MSCs cultured in ePL efficiently suppressed

Optimization of the culturing conditions of human umbilical cord blood-derived endothelial colony-forming cells under xeno-free conditions applying a transcriptomic approach

NARCIS (Netherlands)

Zeisberger, Steffen M.; Zoller, Stefan; Riegel, Mariluce; Chen, Shuhua; Krenning, Guido; Harmsen, Martin C.; Sachinidis, Agapios; Zisch, Andreas H.

Establishment of fetal bovine serum (FBS)-free cell culture conditions is essential for transplantation therapies. Blood-derived endothelial colony-forming cells (ECFCs) are potential candidates for regenerative medicine applications. ECFCs were isolated from term umbilical cord blood units and

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

Science.gov (United States)

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. Copyright © 2013 Elsevier Ltd. All rights reserved.

Treatment of serum with supernatants from cultures of Candida albicans reduces its serum-dependent phagocytosis Tratamento de soro com sobrenadante de cultura de Candida albicans reduz a fagocitose soro-dependente

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Aderbal Antonio dos Santos

2002-01-01

Full Text Available Candida albicans is a potent activator of the complement system, and heat labile opsonins produced by activation of C3 (C3b and iC3b enhance phagocytosis of C. albicans mediated by complement receptors. In this study we treated mouse serum with supernatants from cultures of a protease producer strain of C. albicans and evaluated the ability of this serum to enhance phagocytosis of C. albicans. Cell-free supernatants from cultures of C. albicans were concentrated 5 fold and added to mouse serum for 30 min at 37ºC, before using this serum for opsonization of glutaraldehyde-fixed yeast cells. We observed that normal mouse serum increased about 3 fold the phagocytosis of C. albicans by mice peritoneal macrophages, whereas supernatant-treated serum did not increase phagocytosis. This effect of supernatants on serum was prevented by addition of pepstatin (5 µg/ ml; an inhibitor of C. albicans acid proteases to the medium. Serum treated with supernatants from cultures of a protease-deficient mutant of C. albicans also increased about 3 fold phagocytosis of the yeast. These results suggest that a protease produced by C. albicans causes proteolysis of serum opsonins, thereby reducing the phagocytosis of the yeast.Candida albicans é um potente ativador do sistema complemento, e opsoninas lábeis ao calor produzidas por ativação de C3 (C3b e iC3b aumentam a fagocitose de C. albicans mediada por receptores de complemento. Neste estudo, tratamos o soro de camundongo com sobrenadante de culturas de uma cepa de C. albicans produtora de proteases e avaliamos a capacidade deste soro reduzir a fagocitose de C. albicans. Sobrenadantes livres de células obtidos de cultura de C. albicans foram concentrados 5 vezes e adicionados ao soro de camundongo por 30 minutos a 37ºC, antes deste soro ser usado para opsonização de C. albicans na forma de levedura e fixadas em glutaraldeido. Nós observamos que soro normal aumentou 3 vezes a fagocitose de C. albicans por

New method for culture of zona-included or zona-free embryos: the Well of the Well (WOW) system.

Science.gov (United States)

Vajta, G; Peura, T T; Holm, P; Páldi, A; Greve, T; Trounson, A O; Callesen, H

2000-03-01

Culture of mammalian zygotes individually and in small groups results in lower developmental rates than culture of large groups. Zona-free zygotes also have impaired developmental potential in current culture systems. This paper describes a new approach to resolve the problems, the Well of the Well (WOW) system. Small wells (WOWs) were formed in four-well dishes by melting the bottom with heated steel rods. The WOWs were then rinsed, the wells were filled with medium, and the embryos were placed into the WOWs. To test the value of the WOW system a 3 x 3 factorial experiment was performed. Bovine presumptive zygotes were cultured from day 1 to day 7 (day 0: day of insemination) using three modules (single embryos, embryo groups of five, or single zona-digested embryos) and three different culture systems (400 microl medium, 200 microl drops, or WOWs). An additional control group consisted of 40 to 50 embryos cultured in 400 microl medium. The WOW system resulted in higher blastocyst/oocyte rates for all three modules (single: 59%; group of five: 61%; single zona-digested: 53%) than the culture in drops or in wells (P WOWs per well. The cell number of blastocysts cultured in the WOW system did not differ from that of the controls. Apart from its theoretical value in revealing the role of different factors influencing embryo development in vitro, the WOW system may have immediate practical consequences in certain areas of mammalian embryo production. Copyright 2000 Wiley-Liss, Inc.

Elevated serum free thyroxine by thyroxine analog radioimmunoassays in euthyroid patients with familial dysalbuminemic hyperthyroxinemia

International Nuclear Information System (INIS)

Rajatanavin, R.; Fournier, L.; DeCosimo, D.; Abreau, C.; Braverman, L.E.

1982-01-01

A study was done to ascertain whether the serum free T4 measured by free T4 radioimmunoassay kits would, like equilibrium dialysis, be normal in patients with familial dysalbuminemic hyperthyroxinemia. Five free T4 radioimmunoassay kits were used to measure free T4 in serum samples from 19 patients with familial dysalbuminemic hyperthyroxinemia and 20 healthy volunteers. Values (mean +/- SE) for T4, free T4 index, and free T4 (equilibrium dialysis) in these normal subjects and patients with familial dysalbuminemic hyperthyroxinemia, respectively, were as follows: T4, 8.1 +/- 0.2 and 18.3 +/- 0.7 μg/dL; free T4 index, 3.1 +/- 0.1 and 7.3 +/- 0.3 μg/dL; free T4, 1.4 +/- 0.1 and 1.2 +/- 0.1 ng/dL. The following free T4 radioimmunoassay methods were used: antibody coated microfine silica, microencapsulated antibody, two-step antibody-coated tube, and one-step 125 I-T4 analog (2 kits). The present findings in patients with familial dysalbuminemic hyperthyroxinemia and previous observations in ill euthyroid patients suggest that serum free T4 measured by some radioimmunoassay methods must be interpreted with caution in these two clinical situations

Free Speech as a Cultural Value in the United States

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Mauricio J. Alvarez

2018-02-01

Full Text Available Political orientation influences support for free speech, with liberals often reporting greater support for free speech than conservatives. We hypothesized that this effect should be moderated by cultural context: individualist cultures value individual self-expression and self-determination, and collectivist cultures value group harmony and conformity. These different foci should differently influence liberals and conservatives’ support for free speech within these cultures. Two studies evaluated the joint influence of political orientation and cultural context on support for free speech. Study 1, using a multilevel analysis of data from 37 U.S. states (n = 1,001, showed that conservatives report stronger support for free speech in collectivist states, whereas there were no differences between conservatives and liberals in support for free speech in individualist states. Study 2 (n = 90 confirmed this pattern by priming independent and interdependent self-construals in liberals and conservatives. Results demonstrate the importance of cultural context for free speech. Findings suggest that in the U.S. support for free speech might be embraced for different reasons: conservatives’ support for free speech appears to be motivated by a focus on collectively held values favoring free speech, while liberals’ support for free speech might be motivated by a focus on individualist self-expression.

Higher Serum Levels of Free ĸ plus λ Immunoglobulin Light Chains Ameliorate Survival of Hemodialysis Patients

DEFF Research Database (Denmark)

Thilo, Florian; Caspari, Christina; Scholze, Alexandra

2011-01-01

Background/Aims: Impaired immune function is common in patients with chronic renal failure. Now, we determined whether serum levels of free immunoglobulin light chains predict mortality in patients with chronic kidney disease stage 5 on hemodialysis. Methods: We performed a prospective cohort study...... of 160 hemodialysis patients with a median follow-up of 15 months (interquartile range, 3-44 months). Serum levels of free κ and λ immunoglobulin light chains were measured at the start of the study. The primary end point was mortality from any cause. Results: In survivors, median serum levels of free κ...... plus λ immunoglobulin light chains were significantly higher compared with nonsurvivors (p light chains above the median compared with patients with serum levels below the median of 210 mg...

Comparison of serum fractionation methods by data independent label-free proteomics

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D. Baiwir

2015-12-01

Full Text Available Off-line sample prefractionations applied prior to biomarker discovery proteomics are options to enable more protein identifications and detect low-abundance proteins. This work compared five commercial methods efficiency to raw serum analysis using label-free proteomics. The variability of the protein quantities determined for each process was similar to the unprefractionated serum. A 49% increase in protein identifications and 12.2% of reliable quantification were obtained. A 61 times lower limit of protein quantitation was reached compared to protein concentrations observed in raw serum. The concentrations of detected proteins were confronted to estimated reference values.

Can serum free fatty acids assessment predict severe preeclampsia?

African Journals Online (AJOL)

Nermeen Saad El Beltagy

2011-10-20

Oct 20, 2011 ... Methods: Twenty cases with severe preeclampsia (blood pressure P 160/110 after 20th week of ges- tation and ... ing factor with preeclampsia in non-obese pregnant women. ... Preeclampsia (PE) is a common pregnancy disorder that is ... centration of free fatty acids in the serum was measured by an.

Defined xenogeneic-free and hypoxic environment provides superior conditions for long-term expansion of human adipose-derived stem cells.

Science.gov (United States)

Yang, Sufang; Pilgaard, Linda; Chase, Lucas G; Boucher, Shayne; Vemuri, Mohan C; Fink, Trine; Zachar, Vladimir

2012-08-01

Development and implementation of therapeutic protocols based on stem cells or tissue-engineered products relies on methods that enable the production of substantial numbers of cells while complying with stringent quality and safety demands. In the current study, we aimed to assess the benefits of maintaining cultures of adipose-derived stem cells (ASCs) in a defined culture system devoid of xenogeneic components (xeno-free) and hypoxia over a 49-day growth period. Our data provide evidence that conditions involving StemPro mesenchymal stem cells serum-free medium (SFM) Xeno-Free and hypoxia (5% oxygen concentration) in the culture atmosphere provide a superior proliferation rate compared to a standard growth environment comprised of alpha-modified Eagle medium (A-MEM) supplemented with fetal calf serum (FCS) and ambient air (20% oxygen concentration) or that of A-MEM supplemented with FCS and hypoxia. Furthermore, a flow cytometric analysis and in vitro differentiation assays confirmed the immunophenotype stability and maintained multipotency of ASCs when expanded under xeno-free conditions and hypoxia. In conclusion, our data demonstrate that growth conditions utilizing a xeno-free and hypoxic environment not only provide an improved environment for the expansion of ASCs, but also set the stage as a culture system with the potential broad spectrum utility for regenerative medicine and tissue engineering applications.

Neuroprotection against oxidative stress by serum from heat acclimated rats.

Science.gov (United States)

Beit-Yannai, E; Trembovler, V; Horowitz, M; Lazarovici, P; Kohen, R; Shohami, E

1998-09-25

Exposure of PC12 cells, to 1% serum derived from normothermic (CON) rats resulted in 79% cell death. Sister cultures treated with 1% serum derived from heat acclimated (ACC) rats, were neuroprotected and expressed a significant reduction in cell death. In PC12 cells exposed to a free radical generator causing an oxidative stress, 90% cell death was measured in CON serum treated cultures, while ACC serum treated cultures were neuroprotected. Xanthine oxidase activity and uric acid (UA) levels were lower in ACC serum compared to CON. Addition of UA to both sera abolished the difference in cell viability, and toxicity of ACC serum reached that of CON. These findings suggest a causal relationship between the lower levels of UA in ACC and the neuroprotective effect observed. The present study proposes heat acclimation as an experimental and/or clinical tool for the achievement of neuroprotection.

Stepwise Differentiation of Pluripotent Stem Cells into Osteoblasts Using Four Small Molecules under Serum-free and Feeder-free Conditions

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Kosuke Kanke

2014-06-01

Full Text Available 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-methoxyphenylpyrido[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.

Comparison of serum hormone levels of captive and free-living maned wolves Chrysocyon brachyurus

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O.B. Maia

2008-02-01

Full Text Available Serum hormone levels were compared between captive and free-living maned wolves and seasonal variations of sex hormones were studied. Blood samples were collected from 16 male and 26 female adult animals from Brazilian zoos, and from 30 male and 24 female free-living adults to determine serum progesterone and testosterone by radioimmunoassay. Serum testosterone concentrations varied (P 0.05. Sixteen captive males showed higher testosterone concentration during winter and spring compared with 30 free-living animals (P < 0.05. Progesterone concentration varied among seasons in 26 captive females (P < 0.05, being higher in autumn (15.3 ± 3.1 ng/mL than in summer (6.6 ± 1.5 ng/mL, winter (5.3 ± 3.1 ng/mL and spring (4.3 ± 0.7 ng/mL. Progesterone concentration of 24 free-living females varied between autumn (17.1 ± 6.0 ng/mL and winter (1.7 ± 0.3 ng/mL (P < 0.05, but we could not obtain data for spring or summer. No difference in progesterone levels was observed between captive and free-living females in autumn and winter.

Free Speech as a Cultural Value in the United States

OpenAIRE

Alvarez, Mauricio J.; Kemmelmeier, Markus

2017-01-01

Political orientation influences support for free speech, with liberals often reporting greater support for free speech than conservatives. We hypothesized that this effect should be moderated by cultural context: individualist cultures value individual self-expression and self-determination, and collectivist cultures value group harmony and conformity. These different foci should differently influence liberals and conservatives’ support for free speech within these cultures. Two studies eval...

Opium consumption is negatively associated with serum prostate-specific antigen (PSA), free PSA, and percentage of free PSA levels.

Science.gov (United States)

Safarinejad, Mohammad Reza; Asgari, Seyyed Alaeddin; Farshi, Alireza; Iravani, Shahrokh; Khoshdel, Alireza; Shekarchi, Babak

2013-01-01

Addiction to opium continues to be a major worldwide medical and social problem. The study addressing the association between opium consumption and serum prostate-specific antigen (PSA) level is lacking. We determined the effects of opium consumption on serum PSA levels in opium-addict men. Our study subjects comprised 438 opium-addict men with a mean age of 52.2 ± 6.4 years (group 1). We compared these men with 446 men who did not indicate current or past opium use (group 2). Serum total PSA (tPSA), free PSA (fPSA), % fPSA, and sex hormones were compared between the 2 groups. The mean serum tPSA level was significantly lower in group 1 (1.05 ng/mL) than in controls (1.45 ng/mL) (P = 0.001). Opium consumption was also associated with lower fPSA (P = 0.001) and % fPSA (P = 0.001). Serum free testosterone level in opium-addict patients (132.5 ± 42 pg/mL) was significantly lower than that in controls (156.2 ± 43 pg/mL) (P = 0.03). However, no significant correlation existed between tPSA and free testosterone levels (r = 0.28, 95% CI, -0.036 to 0.51, P = 0.34). Among the patients with cancer in group 1, 35% were found to have high-grade tumor (Gleason score ≥ 7) compared with 26.7% in group 2 (P = 0.02). Total PSA and fPSA were strongly correlated with duration of opium use (r = -0.06, 95% CI, -0.04 to -0.08, P = 0.0001; and r = -0.05, 95% CI, -0.03 to -0.07, P = 0.0001, respectively). Opium consumption is independently and negatively associated with serum tPSA, fPSA, and % fPSA levels.

Changes in Serum Free Amino Acids and Muscle Fatigue Experienced during a Half-Ironman Triathlon.

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Francisco Areces

Full Text Available The aim of this study was to investigate the relationship between changes in serum free amino acids, muscle fatigue and exercise-induced muscle damage during a half-ironman triathlon. Twenty-six experienced triathletes (age = 37.0 ± 6.8 yr; experience = 7.4 ± 3.0 yr competed in a real half-ironman triathlon in which sector times and total race time were measured by means of chip timing. Before and after the race, a countermovement jump and a maximal isometric force test were performed, and blood samples were withdrawn to measure serum free amino acids concentrations, and serum creatine kinase levels as a blood marker of muscle damage. Total race time was 320 ± 37 min and jump height (-16.3 ± 15.2%, P 20%. However, neither the changes in serum free amino acids nor the tryptophan/BCAA ratio were related muscle fatigue or muscle damage during the race.

Prognostic value of the serum free light chain ratio in newly diagnosed myeloma: proposed incorporation into the international staging system.

Science.gov (United States)

Snozek, C L H; Katzmann, J A; Kyle, R A; Dispenzieri, A; Larson, D R; Therneau, T M; Melton, L J; Kumar, S; Greipp, P R; Clark, R J; Rajkumar, S V

2008-10-01

To determine if the serum free light chain (FLC) ratio has prognostic value in patients with symptomatic multiple myeloma (MM), baseline serum samples from a well-characterized cohort of 790 newly diagnosed MM patients were tested with the FLC assay. FLC ratio was calculated as kappa/lambda (reference range 0.26-1.65). On the basis of the distribution of values, a cutpoint kappa/lambda FLC ratio of 32 was chosen for further analysis. Overall survival was significantly inferior in patients with an abnormal FLC ratio of 32 (n=479) compared with those with an FLC ratio between 0.03 and 32 (n=311), with median survival of 30 versus 39 months, respectively. We incorporated abnormal FLC ratio with the International Staging System (ISS) risk factors (that is, albumin or=3.5 g/l), to create a risk stratification model with improved prognostic capabilities. Patients with 0, 1, 2 or 3 adverse risk factors had significantly different overall survival, with median survival times of 51, 39, 30 and 22 months, respectively (P<0.001). These findings suggest that the serum FLC ratio at initial diagnosis is an important predictor of prognosis in myeloma, and can be incorporated into the ISS for improved risk stratification.

Utility of serum periostin and free IgE levels in evaluating responsiveness to omalizumab in patients with severe asthma.

Science.gov (United States)

Tajiri, T; Matsumoto, H; Gon, Y; Ito, R; Hashimoto, S; Izuhara, K; Suzukawa, M; Ohta, K; Ono, J; Ohta, S; Ito, I; Oguma, T; Inoue, H; Iwata, T; Kanemitsu, Y; Nagasaki, T; Niimi, A; Mishima, M

2016-10-01

Omalizumab, a humanized anti-IgE monoclonal antibody, has demonstrated efficacy in patients with severe allergic asthma. However, treatment responses vary widely among individuals. Despite a lack of data, free serum IgE levels following omalizumab treatment have been proposed as a marker of treatment responsiveness. In this prospective, observational study, we assessed the utility of biomarkers of type 2 inflammation in predicting omalizumab treatment responses, as determined by the absence of asthma exacerbation during the first year of treatment. Free serum IgE levels were monitored for 2 years to examine their association with baseline biomarker levels and the number of exacerbations. We enrolled thirty patients who had been treated with omalizumab for at least 1 year, of whom 27 were treated for 2 years. Baseline serum periostin levels and blood eosinophil counts were significantly higher in patients without exacerbations during the first year of treatment than in patients with exacerbations. Baseline serum periostin levels, but not eosinophil counts, were negatively associated with free serum IgE levels after 16 or 32 weeks of treatment. Reduced free serum IgE levels during treatment from those at baseline were associated with reduced exacerbation numbers at 2 years. In 14 patients who continued to have exacerbations during the first year of treatment, exacerbation numbers gradually and significantly decreased over the 2-year study period, with concurrent significant reductions in free serum IgE levels. Baseline serum periostin levels and serum free IgE levels during treatment follow-up may be useful in evaluating responses to omalizumab treatment. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids.

Science.gov (United States)

Bonazza, Camila; Andrade, Sheila Siqueira; Sumikawa, Joana Tomomi; Batista, Fabrício Pereira; Paredes-Gamero, Edgar J; Girão, Manoel J B C; Oliva, Maria Luiza V; Castro, Rodrigo Aquino

2016-01-01

Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 β-estradiol (E2) and progesterone (P4) effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation). These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity.

Elevated Concentrations of Serum Immunoglobulin Free Light Chains in Systemic Lupus Erythematosus Patients in Relation to Disease Activity, Inflammatory Status, B Cell Activity and Epstein-Barr Virus Antibodies

DEFF Research Database (Denmark)

Draborg, Anette H; Lydolph, Magnus; Westergaard, Marie

2015-01-01

, FLCs' association with Epstein-Barr virus (EBV) antibodies was examined. METHODS: Using a nephelometric assay, κFLC and λFLC concentrations were quantified in sera from 45 SLE patients and 40 healthy controls. SLE patients with renal insufficiency were excluded in order to preclude high concentrations......OBJECTIVE: In this study, we examined the concentration of serum immunoglobulin free light chains (FLCs) in systemic lupus erythematosus (SLE) patients and investigated its association with various disease parameters in order to evaluate the role of FLCs as a potential biomarker in SLE. Furthermore...... of serum FLCs due to decreased clearance. RESULTS: Serum FLC concentrations were significantly elevated in SLE patients compared to healthy controls (pdisease activity (SLE disease activity...

Influence of cell type and cell culture media on the propagation of foot-and-mouth disease virus with regard to vaccine quality.

Science.gov (United States)

Dill, Veronika; Hoffmann, Bernd; Zimmer, Aline; Beer, Martin; Eschbaumer, Michael

2018-03-16

Suspension culture of BHK cells allows large-scale virus propagation and cost-efficient vaccine production, while the shift to animal-component-free cell culture media without serum is beneficial for the quality and downstream processing of the product. Foot-and-mouth disease virus is still endemic in many parts of the world and high-quality vaccines are essential for the eradication of this highly contagious and economically devastating disease. Changes to the viral genome sequence during passaging in an adherent and a suspension cell culture system were compared and the impact of amino acid substitutions on receptor tropism, antigenicity and particle stability was examined. Virus production in suspension cells in animal-component-free media and in serum-containing media as well as in adherent cells in serum-containing media was compared. Infection kinetics were determined and the yield of intact viral particles was estimated in all systems using sucrose density gradient centrifugation. Capsid protein sequence alterations were serotype-specific, but varied between cell lines. But The A 24 -2P virus variant had expanded its receptor tropism, but virus neutralization tests found no changes in the antigenic profile in comparison to the original viruses. There were no differences in viral titer between a suspension and an adherent cell culture system, independent of the type of media used. Also, the usage of a serum-free suspension culture system promoted viral growth and allowed an earlier harvest. For serotype O isolates, no differences were seen in the yield of 146S particles. Serotype A preparations revealed a decreased yield of 146S particles in suspension cells independent of the culture media. The selective pressure of the available surface receptors in different cell culture systems may be responsible for alterations in the capsid coding sequence of culture-grown virus. Important vaccine potency characteristics such as viral titer and the neutralization

Serum and plasma for total and free anticonvulsant drug analyses: effects on EMIT assays and ultrafiltration devices.

Science.gov (United States)

Godolphin, W; Trepanier, J; Farrell, K

1983-01-01

The suitability of serum and plasma anticoagulated with heparin, EDTA, citrate, or oxalate was assessed for analysis of free and total phenytoin, carbamazepine, and valproic acid. The free fraction was isolated by ultrafiltration through FreeLevel devices (Syva, Palo Alto, CA). Serum, heparin, and EDTA plasma were satisfactory for both free and total phenytoin and carbamazepine. EDTA could not be used for EMIT (Syva) analysis of valproate. Citrate and, to a lesser degree, oxalate cause a significant negative interference in the concentration of these three drugs as measured both by EMIT and gas-liquid chromatography.

Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids.

Directory of Open Access Journals (Sweden)

Camila Bonazza

Full Text Available Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 β-estradiol (E2 and progesterone (P4 effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation. These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity.

Determination of free amino acids of porcine serum responsible for ...

African Journals Online (AJOL)

The 1H NMR spectra of serum metabolites at 600 MHz showed that free amino acids such as alanine, leucine, phenylalanine, and valine were qualitatively higher in the HpHG than in the LpHG. The relative abundance of three amino acids was quantitatively verified by HPLC: Phenylalanine and valine (P<0.01) and leucine ...

Development of a serum-free medium for in vitro expansion of human cytotoxic T lymphocytes using a statistical design

Directory of Open Access Journals (Sweden)

Lee Gyun

2010-09-01

Full Text Available Abstract Background Serum-containing medium (SCM, which has a number of poorly defined components with varying concentrations, hampers standardization of lymphocyte cultures. In order to develop a serum-free medium (SFM for the expansion of human lymphocytes from peripheral blood mononuclear cells (PBMCs, a statistical optimization approach based on a fractional factorial method and a response surface method was adopted. A basal medium was prepared by supplementing RPMI1640 medium with insulin, albumin, ferric citrate, ethanolamine, fatty acids, glutamine, sodium pyruvate, 2-mercaptoethanol, 1-thioglycerol, nonessential amino acids, and vitamins. We identified additional positive determinants and their optimal concentrations for cell growth through a statistical analysis. Results From a statistical analysis using the fractional factorial method, cholesterol and polyamine supplement were identified as positive determinants for cell growth. Their optimal concentrations were determined by the response surface method. The maximum viable cell concentration in the developed SFM was enhanced by more than 1.5-fold when compared to that in RPMI1640 supplemented with 10% fetal bovine serum (FBS. Furthermore, a cytotoxicity assay and an enzyme-linked immunospot assay revealed that the effector function of cytotoxic T lymphocytes generated from PBMCs grown in SFM, by stimulation of peptide-presenting dendritic cells, was retained or even better than that in SCM. Conclusions The use of a developed SFM with cholesterol and polyamine supplement for human lymphocyte culture resulted in better growth without loss of cellular function when compared to SCM.

Development of a serum-free medium for in vitro expansion of human cytotoxic T lymphocytes using a statistical design.

Science.gov (United States)

Jeon, Min Kyoung; Lim, Jong-Baeck; Lee, Gyun Min

2010-09-21

Serum-containing medium (SCM), which has a number of poorly defined components with varying concentrations, hampers standardization of lymphocyte cultures. In order to develop a serum-free medium (SFM) for the expansion of human lymphocytes from peripheral blood mononuclear cells (PBMCs), a statistical optimization approach based on a fractional factorial method and a response surface method was adopted. A basal medium was prepared by supplementing RPMI1640 medium with insulin, albumin, ferric citrate, ethanolamine, fatty acids, glutamine, sodium pyruvate, 2-mercaptoethanol, 1-thioglycerol, nonessential amino acids, and vitamins. We identified additional positive determinants and their optimal concentrations for cell growth through a statistical analysis. From a statistical analysis using the fractional factorial method, cholesterol and polyamine supplement were identified as positive determinants for cell growth. Their optimal concentrations were determined by the response surface method. The maximum viable cell concentration in the developed SFM was enhanced by more than 1.5-fold when compared to that in RPMI1640 supplemented with 10% fetal bovine serum (FBS). Furthermore, a cytotoxicity assay and an enzyme-linked immunospot assay revealed that the effector function of cytotoxic T lymphocytes generated from PBMCs grown in SFM, by stimulation of peptide-presenting dendritic cells, was retained or even better than that in SCM. The use of a developed SFM with cholesterol and polyamine supplement for human lymphocyte culture resulted in better growth without loss of cellular function when compared to SCM.

Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems

Science.gov (United States)

Badenes, Sara M.; Fernandes, Tiago G.; Cordeiro, Cláudia S. M.; Boucher, Shayne; Kuninger, David; Vemuri, Mohan C.; Diogo, Maria Margarida; Cabral, Joaquim M. S.

2016-01-01

Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in spinner flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm2 of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x106 cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells. PMID:26999816

Serum Levels of Follistatin Are Positively Associated With Serum-Free Thyroxine Levels in Patients With Hyperthyroidism or Euthyroidism

Science.gov (United States)

Tseng, Fen-Yu; Chen, Yen-Ting; Chi, Yu-Chao; Chen, Pei-Lung; Yang, Wei-Shiung

2016-01-01

Abstract Follistatin is a glycoprotein with various biologic functions that plays a role in adipocyte differentiation, muscle stimulation, anti-inflammation, and energy homeostasis. Thyroid hormones influence energy expenditure, glucose, and lipid metabolism. The association between serum follistatin level and thyroid function statuses has seldom been evaluated. The objectives of this study were to compare serum follistatin concentrations in different thyroid function statuses and to evaluate the associations between serum follistatin and free thyroxine (fT4) levels. In this study, 30 patients with hyperthyroidism (HY group) and 30 euthyroid individuals (EU group) were recruited. The patients of HY group were treated with antithyroid regimens as clinically indicated, whereas no medication was given to EU group. The demographic and anthropometric characteristics, biochemical data, serum levels of follistatin, and thyroid function of both groups at baseline and at the 6th month were compared. Data of all patients were pooled for the analysis of the associations between the levels of follistatin and fT4. At baseline, the HY group had significantly higher serum follistatin levels than the EU group (median [Q1, Q3]: 1.81 [1.33, 2.78] vs 1.13 [0.39, 1.45] ng/mL, P hyperthyroidism had higher serum follistatin levels, which decreased after receiving antithyroid treatment. In addition, the serum follistatin concentrations were positively associated with serum fT4 levels in patients with hyperthyroidism or euthyroidism. PMID:26844494

Hematopoietic stem cell cytokines and fibroblast growth factor-2 stimulate human endothelial cell-pericyte tube co-assembly in 3D fibrin matrices under serum-free defined conditions.

Directory of Open Access Journals (Sweden)

Annie O Smith

Full Text Available We describe a novel 3D fibrin matrix model using recombinant hematopoietic stem cell cytokines under serum-free defined conditions which promotes the assembly of human endothelial cell (EC tubes with co-associated pericytes. Individual ECs and pericytes are randomly mixed together and EC tubes form that is accompanied by pericyte recruitment to the EC tube abluminal surface over a 3-5 day period. These morphogenic processes are stimulated by a combination of the hematopoietic stem cell cytokines, stem cell factor, interleukin-3, stromal derived factor-1α, and Flt-3 ligand which are added in conjunction with fibroblast growth factor (FGF-2 into the fibrin matrix. In contrast, this tube morphogenic response does not occur under serum-free defined conditions when VEGF and FGF-2 are added together in the fibrin matrices. We recently demonstrated that VEGF and FGF-2 are able to prime EC tube morphogenic responses (i.e. added overnight prior to the morphogenic assay to hematopoietic stem cell cytokines in collagen matrices and, interestingly, they also prime EC tube morphogenesis in 3D fibrin matrices. EC-pericyte interactions in 3D fibrin matrices leads to marked vascular basement membrane assembly as demonstrated using immunofluorescence and transmission electron microscopy. Furthermore, we show that hematopoietic stem cell cytokines and pericytes stimulate EC sprouting in fibrin matrices in a manner dependent on the α5β1 integrin. This novel co-culture system, under serum-free defined conditions, allows for a molecular analysis of EC tube assembly, pericyte recruitment and maturation events in a critical ECM environment (i.e. fibrin matrices that regulates angiogenic events in postnatal life.

A defined and xeno-free culture method enabling the establishment of clinical-grade human embryonic, induced pluripotent and adipose stem cells.

Directory of Open Access Journals (Sweden)

Kristiina Rajala

2010-04-01

Full Text Available The growth of stem cells in in vitro conditions requires optimal balance between signals mediating cell survival, proliferation, and self-renewal. For clinical application of stem cells, the use of completely defined conditions and elimination of all animal-derived materials from the establishment, culture, and differentiation processes is desirable.Here, we report the development of a fully defined xeno-free medium (RegES, capable of supporting the expansion of human embryonic stem cells (hESC, induced pluripotent stem cells (iPSC and adipose stem cells (ASC. We describe the use of the xeno-free medium in the derivation and long-term (>80 passages culture of three pluripotent karyotypically normal hESC lines: Regea 06/015, Regea 07/046, and Regea 08/013. Cardiomyocytes and neural cells differentiated from these cells exhibit features characteristic to these cell types. The same formulation of the xeno-free medium is capable of supporting the undifferentiated growth of iPSCs on human feeder cells. The characteristics of the pluripotent hESC and iPSC lines are comparable to lines derived and cultured in standard undefined culture conditions. In the culture of ASCs, the xeno-free medium provided significantly higher proliferation rates than ASCs cultured in medium containing allogeneic human serum (HS, while maintaining the differentiation potential and characteristic surface marker expression profile of ASCs, although significant differences in the surface marker expression of ASCs cultured in HS and RegES media were revealed.Our results demonstrate that human ESCs, iPSCs and ASCs can be maintained in the same defined xeno-free medium formulation for a prolonged period of time while maintaining their characteristics, demonstrating the applicability of the simplified xeno-free medium formulation for the production of clinical-grade stem cells. The basic xeno-free formulation described herein has the potential to be further optimized for specific

Differentiation of oligodendrocyte progenitor cells from dissociated monolayer and feeder-free cultured pluripotent stem cells.

Science.gov (United States)

Yamashita, Tomoko; Miyamoto, Yuki; Bando, Yoshio; Ono, Takashi; Kobayashi, Sakurako; Doi, Ayano; Araki, Toshihiro; Kato, Yosuke; Shirakawa, Takayuki; Suzuki, Yutaka; Yamauchi, Junji; Yoshida, Shigetaka; Sato, Naoya

2017-01-01

Oligodendrocytes myelinate axons and form myelin sheaths in the central nervous system. The development of therapies for demyelinating diseases, including multiple sclerosis and leukodystrophies, is a challenge because the pathogenic mechanisms of disease remain poorly understood. Primate pluripotent stem cell-derived oligodendrocytes are expected to help elucidate the molecular pathogenesis of these diseases. Oligodendrocytes have been successfully differentiated from human pluripotent stem cells. However, it is challenging to prepare large amounts of oligodendrocytes over a short amount of time because of manipulation difficulties under conventional primate pluripotent stem cell culture methods. We developed a proprietary dissociated monolayer and feeder-free culture system to handle pluripotent stem cell cultures. Because the dissociated monolayer and feeder-free culture system improves the quality and growth of primate pluripotent stem cells, these cells could potentially be differentiated into any desired functional cells and consistently cultured in large-scale conditions. In the current study, oligodendrocyte progenitor cells and mature oligodendrocytes were generated within three months from monkey embryonic stem cells. The embryonic stem cell-derived oligodendrocytes exhibited in vitro myelinogenic potency with rat dorsal root ganglion neurons. Additionally, the transplanted oligodendrocyte progenitor cells differentiated into myelin basic protein-positive mature oligodendrocytes in the mouse corpus callosum. This preparative method was used for human induced pluripotent stem cells, which were also successfully differentiated into oligodendrocyte progenitor cells and mature oligodendrocytes that were capable of myelinating rat dorsal root ganglion neurons. Moreover, it was possible to freeze, thaw, and successfully re-culture the differentiating cells. These results showed that embryonic stem cells and human induced pluripotent stem cells maintained in a

Establishing elements of a synthetic biology platform for Vaccinia virus production: BioBrick™ design, serum-free virus production and microcarrier-based cultivation of CV-1 cells.

Science.gov (United States)

Liu, Shuchang; Ruban, Ludmila; Wang, Yaohe; Zhou, Yuhong; Nesbeth, Darren N

2017-02-01

Vaccinia virus (VACV) is an established vector for vaccination and is beginning to prove effective as an oncolytic agent. Industrial production of VACV stands to benefit in future from advances made by synthetic biology in genome engineering and standardisation. The CV-1 cell line can be used for VACV propagation and has been used extensively with the CRISPR/Cas9 system for making precise edits of the VACV genome. Here we take first steps toward establishing a scalable synthetic biology platform for VACV production with CV-1 cells featuring standardised biological tools and serum free cell cultivation. We propose a new BioBrick™ plasmid backbone format for inserting transgenes into VACV. We then test the performance of CV-1 cells in propagation of a conventional recombinant Lister strain VACV, VACVL-15 RFP, in a serum-free process. CV-1 cells grown in 5% foetal bovine serum (FBS) Dulbecco's Modified Eagle Medium (DMEM) were adapted to growth in OptiPRO and VP-SFM brands of serum-free media. Specific growth rates of 0.047 h -1 and 0.044 h -1 were observed for cells adapted to OptiPRO and VP-SFM respectively, compared to 0.035 h -1 in 5% FBS DMEM. Cells adapted to OptiPRO and to 5% FBS DMEM achieved recovery ratios of over 96%, an indication of their robustness to cryopreservation. Cells adapted to VP-SFM showed a recovery ratio of 82%. Virus productivity in static culture, measured as plaque forming units (PFU) per propagator cell, was 75 PFU/cell for cells in 5% FBS DMEM. VP-SFM and OptiPRO adaptation increased VACV production to 150 PFU/cell and 350 PFU/cell respectively. Boosted PFU/cell from OptiPRO-adapted cells persisted when 5% FBS DMEM or OptiPRO medium was observed during the infection step and when titre was measured using cells adapted to 5% FBS DMEM or OptiPRO medium. Finally, OptiPRO-adapted CV-1 cells were successfully cultivated using Cytodex-1 microcarriers to inform future scale up studies.

Establishing elements of a synthetic biology platform for Vaccinia virus production: BioBrick™ design, serum-free virus production and microcarrier-based cultivation of CV-1 cells

Directory of Open Access Journals (Sweden)

Shuchang Liu

2017-02-01

Full Text Available Vaccinia virus (VACV is an established vector for vaccination and is beginning to prove effective as an oncolytic agent. Industrial production of VACV stands to benefit in future from advances made by synthetic biology in genome engineering and standardisation. The CV-1 cell line can be used for VACV propagation and has been used extensively with the CRISPR/Cas9 system for making precise edits of the VACV genome. Here we take first steps toward establishing a scalable synthetic biology platform for VACV production with CV-1 cells featuring standardised biological tools and serum free cell cultivation. We propose a new BioBrick™ plasmid backbone format for inserting transgenes into VACV. We then test the performance of CV-1 cells in propagation of a conventional recombinant Lister strain VACV, VACVL-15 RFP, in a serum-free process. CV-1 cells grown in 5% foetal bovine serum (FBS Dulbecco’s Modified Eagle Medium (DMEM were adapted to growth in OptiPRO and VP-SFM brands of serum-free media. Specific growth rates of 0.047 h−1 and 0.044 h−1 were observed for cells adapted to OptiPRO and VP-SFM respectively, compared to 0.035 h−1 in 5% FBS DMEM. Cells adapted to OptiPRO and to 5% FBS DMEM achieved recovery ratios of over 96%, an indication of their robustness to cryopreservation. Cells adapted to VP-SFM showed a recovery ratio of 82%. Virus productivity in static culture, measured as plaque forming units (PFU per propagator cell, was 75 PFU/cell for cells in 5% FBS DMEM. VP-SFM and OptiPRO adaptation increased VACV production to 150 PFU/cell and 350 PFU/cell respectively. Boosted PFU/cell from OptiPRO-adapted cells persisted when 5% FBS DMEM or OptiPRO medium was observed during the infection step and when titre was measured using cells adapted to 5% FBS DMEM or OptiPRO medium. Finally, OptiPRO-adapted CV-1 cells were successfully cultivated using Cytodex-1 microcarriers to inform future scale up studies.

Platelet lysate: a replacement for fetal bovine serum in animal cell culture?

OpenAIRE

Johansson, Liselott; Klinth, Jeanna; Holmqvist, Olov; Ohlson, Sten

2003-01-01

A new cell culture supplement, platelet lysate, was evaluated with reference to fetal bovine serum (FBS), an established industrial medium for animal cell culture. Chemical and bacteriological profiles were conducted including the presence of platelet-derived growth factor (PDGF). PDGF was detected in the platelet lysate but it was not present in FBS. The platelet lysate medium demonstrated lack of microorganisms, mycoplasma and endotoxins. The platelet lysate was investigated in culture stud...

Free radical scavenging activity and secondary metabolites from in vitro cultures of Sanicula graveolens.

Science.gov (United States)

Cheel, José; Schmeda-Hirschmann, Guillermo; Jordan, Miguel; Theoduloz, Cristina; Rodríguez, Jaime A; Gerth, André; Wilken, Dirk

2007-01-01

An in vitro propagation system was developed to obtain shoot and root cultures from the Andean spice Sanicula graveolens (Apiaceae). Propagation of shoots, roots and plantlets was achieved by the temporary immersion system. The free radical scavenging effect of the methanol/water (7:3 v/v) extracts was determined by the discoloration of the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH). Total phenolic, flavonoid, chlorogenic acid (CA) and quercetin 3-O-glucoside content in the samples was assessed by spectrophotometry and DAD-HPLC analysis, respectively. On a dry weight basis, the crude extracts showed total phenolic values ranging from 3.57 to 6.93%, with highest content for the root culture sample. Total flavonoid content ranged from 1.23 to 2.23% and was lower for the root culture. Chlorogenic acid and neochlorogenic acid were identified by TLC in all samples. Highest free radical scavenging effect was observed for the root culture which also presented the highest CA content. Two of the shoot culture samples, with similar IC50 values in the DPPH discoloration assay, also presented close quercetin-3-O-glucoside content.

Serum free-thyroxine modifications with age and in normal pregnancy

International Nuclear Information System (INIS)

Degrossi, O.J.; Altschuler, N.; Watanabe, T.; Pinkas, M.; Damilano, S.; Garcia del Rio, H.

1982-01-01

The possibility of using radioimmunoassay techniques (RIA) in the assessment of circulating thyroid hormones, total thyroxine (T4) and triiodothyronine (T3) has particularly increases the diagnosis of thyroid diseases. Thyroidal hormones circulate bound to proteins; therefore, variations in the transport capacity of the latter will produce important modifications in the T4 and T3 figures. Only small fractions, less than 0.05% for T4 and than 0.5% for T3, circulate in the free form and are considered as metabolically active forms of both hormones. In order to attain a correct clinical valuation, the rates of the free fractions as well as the total rates of these hormones must be known. Recently, these studies of free hormones are carried out by means of RIA, with the consequent advantages. The variations in serum free thyroxine (FT4) under certain physiogical conditions, such as for different ages and during pregnancy, were particularly studied

Increased serum free tryptophan in patients with diarrhea-predominant irritable bowel syndrome.

Science.gov (United States)

Christmas, David M; Badawy, Abdulla A-B; Hince, Dana; Davies, Simon J C; Probert, Christopher; Creed, Tom; Smithson, John; Afzal, Muhammad; Nutt, David J; Potokar, John P

2010-10-01

Irregularities of serotonin function in irritable bowel syndrome (IBS) may be due to changes in the metabolism of the serotonin precursor l-tryptophan. Dietary alteration of tryptophan intake may impact upon the mood and bowel symptoms of IBS. We hypothesized that diarrhea-predominant irritable bowel syndrome (d-IBS) patients would exhibit an increase in plasma tryptophan due to alterations in tryptophan metabolism. We also hypothesized that a diet low in tryptophan would reverse this change and reduce symptoms. Thirteen patients with d-IBS had fasting serum free and total tryptophan, large neutral amino acids, and 6 kynurenine metabolites measured before and after 2 weeks of a strict dairy-free diet. Baseline tryptophan parameters were compared with an age- and sex-matched control group. Changes in the specific tryptophan parameters before and after dairy-free diet were correlated with symptoms of IBS and mood. Compared with the control group, d-IBS patients at baseline exhibited significantly higher free serum tryptophan (10.5 ± 4.35 vs 4.75 ± 2.43 μmol/L [means ± standard deviation], P = .006) and significantly lower tryptophan dioxygenase and total tryptophan oxidation as measured by the kynurenine to free tryptophan and total kynurenines to free tryptophan ratios (23.37 ± 10.12 vs 55.33 ± 16.02, P < .001 and 49.34 ± 17.84 vs 258.46 ± 98.67, P < .001, respectively). Dairy-free diet did not modulate metabolites of the kynurenine pathway or symptoms. Tryptophan metabolism along the kynurenine pathway is inhibited in d-IBS, and a dairy-free diet does not alter this. Our findings are consistent with possible enhanced serotonin activity in d-IBS. Copyright © 2010 Elsevier Inc. All rights reserved.

Is the color trails culture free?

Science.gov (United States)

Fasfous, Ahmed F; Puente, Antonio E; Pérez-Marfil, María Nieves; Cruz-Quintana, Francisco; Peralta-Ramirez, Isabel; Pérez-García, Miguel

2013-11-01

Increasingly clinical neuropsychology has been addressing the effects of culture on neuropsychological functioning. However, that focus has been on comparing performance on standardized tests across two or more groups, often Hispanic. In this study, Arabic children were tested in Morocco using a "culture-free test," Children's Color Trails. Children of different ages and living in rural and urban centers were tested. The results suggest that the Color Trails Test scores from Arab children differed from U.S. norms available. Furthermore, the location of testing and the age of the child were of significance. The role of culture-specific tests was considered.

Calorimetric and spectroscopic properties of small globular proteins (bovine serum albumin, hemoglobin) after free radical generation

International Nuclear Information System (INIS)

Farkas, N.; Belagyi, J.; Lorinczy, D.

2003-01-01

Mild oxidation of -SH-containing proteins (serum albumin, hemoglobin) by Ce(IV)-ions in the presence of the spin trap phenyl-tert-butylnitrone (PBN) resulted in the appearance of strongly immobilized nitroxide free radicals which evidences the formation of thiyl radicals on the thiol site of the proteins. In hydroxyl free radical generating system a fraction of strongly immobilized nitroxide radicals was also detected in these proteins, which implies that the oxidation of a fraction of the thiol groups was also involved in the free radical reaction. According to the differential scanning calorimetry (DSC) experiments the melting processes of the proteins were calorimetrically irreversible, therefore the two-state kinetic model was used to evaluate the experiments. The results support the view that site-specific interaction of SH-containing proteins with hydroxyl and thiyl free radicals is able to modify the internal dynamics of proteins and affect the conformation of large molecules

A solid phase radioimmunoassay for free triiodothyronine in serum: assay development and validation

International Nuclear Information System (INIS)

Liu Fangyan; Zhou Meiying; Yin Linxiang; Yang Jianzhong; Hua Chenglin

1999-01-01

A solid phase radioimmunoassay for free triiodothyronine in serum was developed based on double-antibody coated tubes. The method was turned out to be reliable with good reproducibility, higher sensitivity and easy performance. The measurable range of FT 3 in serum was 1.2 to 38 pmol/L. The mean coefficients of variation within and between assays were 1.79%∼3.18% and 4.72%∼9.31%, respectively. The FT 3 concentrations in euthyroid serum as determined by this method were 2.8 to 7.8 pmol/L. The FT 3 values determined by this new method correlated well with those measured by a commercial radioimmunoassay (r = 0.853)

Changes in serum concentrations of total and free testosterone in young and middle-aged men

International Nuclear Information System (INIS)

Wang Guohong; Xu Ruiji; Zhang Zhongshu

2011-01-01

To determine changes in serum concentrations of total and free testosterone in young and middle-aged men.the healthy men (n=126) were divided 20-29 yr, 30-39 yr and 40-49 yr three groups,their serum levels of total testosterone (T), free testosterone (FT), luteinizing hormone (LH), follicle-stimulating hormone (FSH), sex hormone binding globulin (SHBG) and estradiol (E2) were detected. The results were statically analyzed. The results showed that the levels of serum T and FT was found significantly decreased in 30-39 yr group than in 20-29 yr group (15.06±13 nmol/L vs 20.41±86 nmol/L, P<0.01; 9.36±95 pg/L vs 11.48±88 pg/L, P<0.05; respectively). There were young trends that age-related decline in androgen levels. (authors)

Analysis of relationship between tumor markers and quantification of free DNA in serum of lung cancer patients

International Nuclear Information System (INIS)

Yang Shunfang; Zhang Peiling; Cao Jie; Zeng Jun; Dong Qianggang

2006-01-01

To evaluate the diagnostic value and relationship between five tumor markers (CA19- 9,CA125,CYFRA21-1 ,CEA,NSE) and free DNA in serum for lung cancer detection and try to find a new and more efficient tumor marker, the amounts of CA19-9, CA125, CYFRA21-1, CEA, NSE were determined by RIA and free DNA was determined by the use of quantitative real time PCR amplification of the human epidermal growth factor receptor (EGFR) in 52 lung cancer patients and 8 cases of benign pulmonary disease and 10 healthy controls. The resulls showed that average concentration of free DNA in serum of lung cancer patients, benign pulmo- nary disease and healthy controls was 107.6ng/mL, 76.86ng/mL and 18.8ng/mL, respective- ly. The diagnostic sensitivity, specificity and accuracy of free DNA for lung cancer were 71. 2%, 50% and 68.3%, same as the diagnostic value of combined detection of five tumor markers. The sensitivity, specificity and accuracy of the five tumor markers and free DNA combinend detection for lung cancer were 94.2%, 25% and 85%, respectively. The free DNA in the serum of lung cancer patients may be a new and better tumor marker. (authors)

Bone Marrow Mesenchymal Stem Cells Enhance the Differentiation of Human Switched Memory B Lymphocytes into Plasma Cells in Serum-Free Medium

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Guillaume Bonnaure

2016-01-01

Full Text Available The differentiation of human B lymphocytes into plasma cells is one of the most stirring questions with regard to adaptive immunity. However, the terminal differentiation and survival of plasma cells are still topics with much to be discovered, especially when targeting switched memory B lymphocytes. Plasma cells can migrate to the bone marrow in response to a CXCL12 gradient and survive for several years while secreting antibodies. In this study, we aimed to get closer to niches favoring plasma cell survival. We tested low oxygen concentrations and coculture with mesenchymal stem cells (MSC from human bone marrow. Besides, all cultures were performed using an animal protein-free medium. Overall, our model enables the generation of high proportions of CD38+CD138+CD31+ plasma cells (≥50% when CD40-activated switched memory B lymphocytes were cultured in direct contact with mesenchymal stem cells. In these cultures, the secretion of CXCL12 and TGF-β, usually found in the bone marrow, was linked to the presence of MSC. The level of oxygen appeared less impactful than the contact with MSC. This study shows for the first time that expanded switched memory B lymphocytes can be differentiated into plasma cells using exclusively a serum-free medium.

A stromal cell free culture system generates mouse pro-T cells that can reconstitute T-cell compartments in vivo.

Science.gov (United States)

Gehre, Nadine; Nusser, Anja; von Muenchow, Lilly; Tussiwand, Roxane; Engdahl, Corinne; Capoferri, Giuseppina; Bosco, Nabil; Ceredig, Rhodri; Rolink, Antonius G

2015-03-01

T-cell lymphopenia following BM transplantation or diseases such as AIDS result in immunodeficiency. Novel approaches to ameliorate this situation are urgently required. Herein, we describe a novel stromal cell free culture system in which Lineage(-) Sca1(+)c-kit(+) BM hematopoietic progenitors very efficiently differentiate into pro-T cells. This culture system consists of plate-bound Delta-like 4 Notch ligand and the cytokines SCF and IL-7. The pro-T cells developing in these cultures express CD25, CD117, and partially CD44; express cytoplasmic CD3ε; and have their TCRβ locus partially D-J rearranged. They could be expanded for over 3 months and used to reconstitute the T-cell compartments of sublethally irradiated T-cell-deficient CD3ε(-/-) mice or lethally irradiated WT mice. Pro-T cells generated in this system could partially correct the T-cell lymphopenia of pre-Tα(-/-) mice. However, reconstituted CD3ε(-/-) mice suffered from a wasting disease that was prevented by co-injection of purified CD4(+) CD25(high) WT Treg cells. In a T-cell-sufficient or T-lymphopenic setting, the development of disease was not observed. Thus, this in vitro culture system represents a powerful tool to generate large numbers of pro-T cells for transplantation and possibly with clinical applications. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hematologic and serum chemistry reference intervals for free-ranging lions (Panthera leo).

Science.gov (United States)

Maas, Miriam; Keet, Dewald F; Nielen, Mirjam

2013-08-01

Hematologic and serum chemistry values are used by veterinarians and wildlife researchers to assess health status and to identify abnormally high or low levels of a particular blood parameter in a target species. For free-ranging lions (Panthera leo) information about these values is scarce. In this study 7 hematologic and 11 serum biochemistry values were evaluated from 485 lions from the Kruger National Park, South Africa. Significant differences between sexes and sub-adult (≤ 36 months) and adult (>36 months) lions were found for most of the blood parameters and separate reference intervals were made for those values. The obtained reference intervals include the means of the various blood parameter values measured in captive lions, except for alkaline phosphatase in the subadult group. These reference intervals can be utilized for free-ranging lions, and may likely also be used as reference intervals for captive lions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Implications of a Culturally Evolved Self for Notions of Free Will

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Lloyd Hawkeye Robertson

2017-10-01

Full Text Available Most schools in psychology have emphasized individual choice despite evidence of genetic and cultural determinism. It is suggested in this paper that the rejection of classical behaviorism by psychology and other humanities flowed from deeply held cultural assumptions about volition and free will. While compatibilists have suggested that notions of free will and determinism are not mutually exclusive, the psychological mechanisms by which such an accommodation could be explained have been inadequately explored. Drawing on research into classical cultures, this paper builds an argument that the notion of free will was adaptive flowing from culturally evolved changes to the self, and that this “evolved self,” containing assumptions of personal volition, continuity, and reason, became benchmarks of what it means to be human. The paper proposes a model of a culturally evolved self that is compatible with understandings of free will and determinism. Implications for therapeutic practice and future research are discussed.

Effects of cultured shrimp (Litopenaeus vannamei consumption on serum lipoproteins of healthy normolipidemic men

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Farzaneh Yousefi

2012-12-01

Full Text Available Background: It has been suggested that moderate shrimp consumption in normolipidemic subjects will not adversely affect the overall lipoprotein profile. Hence, shrimp consumption can be included in “healthy heart" nutritional guidelines. However, the effects of cultured shrimp on serum lipoproteins of normal subjects have not yet investigated. Material and Methods: Twenty-five healthy normolipidemic men who were workers of a shrimp farm in Bushehr province participated in a quasi-experimental study. In a crossover six weeks trial, the effect of three days per week diet (containing 300 g cultured shrimp Litopenaeus vannamei /day on serum lipid profile was compared with a zero-marine baseline diet. Results: After six weeks trial, serum triglyceride and HDL-cholesterol levels were not significantly changed from the baseline levels (p>0.05. However, total cholesterol and LDL-cholesterol levels, total cholesterol to HDL-cholesterol and LDL-cholesterol to HDL-cholesterol ratios were significantly increased (p<0.0001. Conclusion: Moderate cultured shrimp (Litopenaeus vannamei consumption can increase total cholesterol and LDL-cholesterol levels in normolipidemic men. Although a diet containing native shrimp has many benefits for healthy persons, but we do not recommend cultured shrimp in a healthy heart diet for persons with dyslipidemia or cardiovascular diseases.

Induction of calcification by serum depletion in cell culture: a model for focal calcification in aortas related to atherosclerosis

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Villar Maria T

2008-01-01

Full Text Available Abstract Background Since aortic calcification has been shown to initiate in the lower zone of well-thickened plaques (LZP adjacent to the aortic media of rabbits fed supplemental cholesterol diets, a restricted supply of serum to vascular cells could play a role in vascular calcification. This study was designed to use a cell culture model to support this hypothesis. Results Rabbit aortic smooth muscle cells were grown to confluence in a culture media containing 10 % fetal bovine serum (FBS. The confluent cells were then exposed to the media for 2 hrs with or without serum at a Ca × P ion product range of 4.5–9.4 mM2. In contrast to the cells cultured in the presence of FBS, confluent cells in its absence displayed marked mineral-positive alizarin red staining and infrared absorption of mineral phosphate. A kinetic parameter C1/2 was used to designate the concentration of serum or its protein constituents needed to reduce the deposition of Ca and P by half. The C1/2 for FBS and rabbit serum was 0.04–0.07 % The C1/2 value for rabbit serum proteins was 13.5 μg/ml corresponding to the protein concentration in 0.06 % of serum. This C1/2 was markedly smaller than 86.2 μg/ml for bovine serum albumin present in 0.37 % serum (p Conclusion The aortic smooth muscle cell culture model suggests that serum depletion may play a role in the initiation of aortic calcification. The serum exhibits remarkable ability to inhibit cell-mediated calcification.

A low percentage of autologous serum can replace bovine serum to engineer human nasal cartilage

Directory of Open Access Journals (Sweden)

F Wolf

2008-02-01

Full Text Available For the generation of cell-based therapeutic products, it would be preferable to avoid the use of animal-derived components. Our study thus aimed at investigating the possibility to replace foetal bovine serum (FBS with autologous serum (AS for the engineering of cartilage grafts using expanded human nasal chondrocytes (HNC. HNC isolated from 7 donors were expanded in medium containing 10% FBS or AS at different concentrations (2%, 5% and 10% and cultured in pellets using serum-free medium or in Hyaff®-11 meshes using medium containing FBS or AS. Tissue forming capacity was assessed histologically (Safranin O, immunohistochemically (type II collagen and biochemically (glycosaminoglycans -GAG- and DNA. Differences among experimental groups were assessed by Mann Whitney tests. HNC expanded under the different serum conditions proliferated at comparable rates and generated cartilaginous pellets with similar histological appearance and amounts of GAG. Tissues generated by HNC from different donors cultured in Hyaff®-11 had variable quality, but the accumulated GAG amounts were comparable among the different serum conditions. Staining intensity for collagen type II was consistent with GAG deposition. Among the different serum conditions tested, the use of 2% AS resulted in the lowest variability in the GAG contents of generated tissues. In conclusion, a low percentage of AS can replace FBS both during the expansion and differentiation of HNC and reduce the variability in the quality of the resulting engineered cartilage tissues.

A microculture system for the measurement of antigen-induced murine lymphocyte proliferation: advantages of 5% horse serum and 5 X 10(-5) M mercaptoethanol.

Science.gov (United States)

Brummer, E; Vris, T W; Lawrence, H S

1977-01-01

Short term microculture systems which measure murine lymphocyte proliferative responses to mitogens are well established. We demonstrate here that these microculture methods are not suitable for antigen-induced responses because of the high levels of murine lymphocyte proliferation in control cultures associated with the use of fetal calf serum or human serum. We also show that this problem can be eliminated with the use of a combination of 5% horse serum and 5 X 10(-5) M mercaptoethanol. We describe an antigen-induced murine lymphocyte proliferation microculture system in which good stimulation indices are achieved and the lymphocyte proliferation in control cultures remain at a low level throughout the 7 day culture period.

Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches.

Science.gov (United States)

Saury, Charlotte; Lardenois, Aurélie; Schleder, Cindy; Leroux, Isabelle; Lieubeau, Blandine; David, Laurent; Charrier, Marine; Guével, Laëtitia; Viau, Sabrina; Delorme, Bruno; Rouger, Karl

2018-05-02

Canine MuStem cells have demonstrated regenerative efficacy in a dog model of muscular dystrophy, and the recent characterization of human counterparts (hMuStem) has highlighted the therapeutic potential of this muscle-derived stem cell population. To date, these cells have only been generated in research-grade conditions. However, evaluation of the clinical efficacy of any such therapy will require the production of hMuStem cells in compliance with good manufacturing practices (GMPs). Because the current use of fetal bovine serum (FBS) to isolate and expand hMuStem cells raises several ethical, safety, and supply concerns, we assessed the use of two alternative xeno-free blood derivatives: human serum (HS) and a human platelet lysate (hPL). hMuStem cells were isolated and expanded in vitro in either HS-supplemented or hPL-supplemented media and the proliferation rate, clonogenicity, myogenic commitment potential, and oligopotency compared with that observed in FBS-supplemented medium. Flow cytometry and high-throughput 3'-digital gene expression RNA sequencing were used to characterize the phenotype and global gene expression pattern of hMuStem cells cultured with HS or hPL. HS-supplemented and hPL-supplemented media both supported the isolation and long-term proliferation of hMuStem cells. Compared with FBS-based medium, both supplements enhanced clonogenicity and allowed for a reduction in growth factor supplementation. Neither supplement altered the cell lineage pattern of hMuStem cells. In vitro differentiation assays revealed a decrease in myogenic commitment and in the fusion ability of hMuStem cells when cultured with hPL. In return, this reduction of myogenic potential in hPL-supplemented cultures was rapidly reversed by substitution of hPL with HS or fibrinogen-depleted hPL. Moreover, culture of hMuStem cells in hPL hydrogel and fibrinogen-depleted hPL demonstrated that myogenic differentiation potential is maintained in heparin-free hPL derivatives. Our

Effects of serum on cytotoxicity of nano- and micro-sized ZnO particles

Energy Technology Data Exchange (ETDEWEB)

Hsiao, I-Lun; Huang, Yuh-Jeen, E-mail: yjhuang@mx.nthu.edu.tw [National Tsing Hua University, Department of Biomedical Engineering and Environmental Sciences (China)

2013-09-15

Although an increasing number of in vitro studies are being published regarding the cytotoxicity of nanomaterials, the components of the media for toxicity assays have often varied according to the needs of the scientists. Our aim for this study was to evaluate the influence of serum-in this case, fetal bovine serum-in a cell culture medium on the toxicity of nano-sized (50-70 nm) and micro-sized (<1 {mu}m) ZnO on human lung epithelial cells (A549). The nano- and micro-sized ZnO both exhibited their highest toxicity when exposed to serum-free media, in contrast to exposure in media containing 5 or 10 % serum. This mainly comes not only from the fact that ZnO particles in the serum-free media have a higher dosage-per-cell ratio, which results from large aggregates of particles, rapid sedimentation, absence of protein protection, and lower cell growth rate, but also that extracellular Zn{sup 2+} release contributes to cytotoxicity. Although more extracellular Zn{sup 2+} release was observed in serum-containing media, it did not contribute to nano-ZnO cytotoxicity. Furthermore, non-dissolved particles underwent size-dependent particle agglomeration, resulting in size-dependent toxicity in both serum-containing and serum-free media. A low correlation between cytotoxicity and inflammation endpoints in the serum-free medium suggested that some signaling pathways were changed or induced. Since cell growth, transcription behavior for protein production, and physicochemical properties of ZnO particles all were altered in serum-free media, we recommend the use of a serum-containing medium when evaluating the cytotoxicity of NPs.

The influence of metronidazole on free thymidine content of blood serum of irradiated mice

International Nuclear Information System (INIS)

Konov, A.V.; Ryabchenko, N.I.

1986-01-01

The influence of a radiosensitizer, metronidazole, on the free thymidine content of blood serum of irradiated mice was studied in aerobic and hypoxic conditions. A heated metronidazole solution (1 mg/g) was administered intraperitoneally 30 min before irradiation of animals with a dose of 3 Gy. Thymidine concentration in blood serum was determined by the radioimmunological technique. The influence of metronidazole on the level of thymidinemia was only noted in the animals exposed under hypoxic conditions

Comparative Serum Fatty Acid Profiles of Captive and Free-Ranging Cheetahs (Acinonyx jubatus) in Namibia.

Science.gov (United States)

Tordiffe, Adrian S W; Wachter, Bettina; Heinrich, Sonja K; Reyers, Fred; Mienie, Lodewyk J

2016-01-01

Cheetahs (Acinonyx jubatus) are highly specialised large felids, currently listed as vulnerable on the IUCN red data list. In captivity, they are known to suffer from a range of chronic non-infectious diseases. Although low heterozygosity and the stress of captivity have been suggested as possible causal factors, recent studies have started to focus on the contribution of potential dietary factors in the pathogenesis of these diseases. Fatty acids are an important component of the diet, not only providing a source of metabolisable energy, but serving other important functions in hormone production, cellular signalling as well as providing structural components in biological membranes. To develop a better understanding of lipid metabolism in cheetahs, we compared the total serum fatty acid profiles of 35 captive cheetahs to those of 43 free-ranging individuals in Namibia using gas chromatography-mass spectrometry. The unsaturated fatty acid concentrations differed most remarkably between the groups, with all of the polyunsaturated and monounsaturated fatty acids, except arachidonic acid and hypogeic acid, detected at significantly lower concentrations in the serum of the free-ranging animals. The influence of age and sex on the individual fatty acid concentrations was less notable. This study represents the first evaluation of the serum fatty acids of free-ranging cheetahs, providing critical information on the normal fatty acid profiles of free-living, healthy individuals of this species. The results raise several important questions about the potential impact of dietary fatty acid composition on the health of cheetahs in captivity.

Serum lutein response is greater from free lutein than from esterified lutein during 4 weeks of supplementation in healthy adults.

Science.gov (United States)

Norkus, Edward P; Norkus, Katherine L; Dharmarajan, T S; Schierle, Joseph; Schalch, Wolfgang

2010-12-01

Current data suggest great variability in serum response following lutein ingestion from various sources. To compare the relative serum response during supplementation with free lutein (fL) and lutein esters (Le). 72 volunteers (23-52 years; body mass index [BMI] >20 and lutein lutein or 27 mg of lutein ester (equivalent to 13.5 mg free lutein), respectively. Fasting blood was obtained at baseline and after 7, 14, 21, and 28 days of supplementation. Supplements were consumed with standard portions of dry, ready-to-eat cereal and 2% cow's milk. Absolute changes in serum lutein, per mg daily dose, were significantly greater in fL vs. Le after 21 days (p  =  0.0012) and remained so after 28 days (p  =  0.0011) of supplementation. Serum lutein Area Under the Curve [AUC((day 0-28))] response was 17% greater for fL vs. Le (p  =  0.0187). Regression models were used and determined that (1) baseline serum lutein levels and (2) the form of lutein ingested (fL > Le) influence the serum lutein response during supplementation, while subject age, gender, BMI, and serum lipids do not affect serum response. These results suggest that the relative serum lutein response will be significantly greater from supplements containing free lutein than from supplements containing lutein esters. These findings should be useful for future clinical trials exploring the effectiveness of lutein supplementation in the prevention of or protection against age-related macular degeneration and/or cataracts.

Platelet lysate as a serum replacement for skin cell culture on biomimetic PCL nanofibers.

Science.gov (United States)

Sovkova, Vera; Vocetkova, Karolina; Rampichova, Michala; Mickova, Andrea; Buzgo, Matej; Lukasova, Vera; Dankova, Jana; Filova, Eva; Necas, Alois; Amler, Evzen

2018-06-01

Platelets are a popular source of native growth factors for tissue engineering applications. The aim of the study was to verify the use of platelet lysate as a fetal bovine serum (FBS) replacement for skin cell culture. The cytokine content of the platelet lysate was characterized using the Bio-Plex system. The cells (fibroblasts, melanocytes, and keratinocytes) were cultured on PCL nanofibrous scaffolds to mimic their natural microenvironment. The cytokine content of the platelet lysate was determined, and to the cells, a medium containing platelet lysate or platelet lysate in combination with FBS was added. The results showed that 7% (v/v) platelet lysate was sufficient to supplement 10% (v/v) FBS in the culture of fibroblasts and keratinocytes. The combination of platelet lysate and FBS had a rather inhibitory effect on fibroblasts, in contrary to keratinocytes, where the effect was synergic. Platelet lysate did not sufficiently promote proliferation in melanocytes; however, the combination of FBS and platelet lysate yielded a better outcome and resulted in bipolar morphology of the cultured melanocytes. The data indicated that platelet lysate improved cell proliferation and metabolic activity and may be used as an additive to the cell culture media.

Free insulin-like growth factor I serum levels in 1430 healthy children and adults, and its diagnostic value in patients suspected of growth hormone deficiency

DEFF Research Database (Denmark)

Juul, A; Holm, K; Kastrup, K W

1997-01-01

fraction of the total IGF-I circulates in its free form, which is believed to be the biologically active form. However, our knowledge of the clinical or physiological value of determination of free IGF-I in serum is limited at present. In adults, the diagnostic value of total IGF-I and IGFBP-3......, commercially available immunoradiometric assay (Diagnostic Systems Laboratories) to establish valid normative data for this analysis. We studied the diagnostic value of free IGF-I in relation to total IGF-I and IGFBP-3 determinations in adults who were suspected of GHD. A GH provocative test, using oral...... determinations in patients suspected of GHD has only been reported in a few studies, whereas no previous reports on the diagnostic value of free IGF-I levels in adults suspected of GHD exist. Serum levels of free IGF-I were determined in 1430 healthy children, adolescents, and adults by a newly developed...

Free insulin-like growth factor I serum levels in 1430 healthy children and adults, and its diagnostic value in patients suspected of growth hormone deficiency

DEFF Research Database (Denmark)

Juul, A; Holm, K; Kastrup, K W

1997-01-01

fraction of the total IGF-I circulates in its free form, which is believed to be the biologically active form. However, our knowledge of the clinical or physiological value of determination of free IGF-I in serum is limited at present. In adults, the diagnostic value of total IGF-I and IGFBP-3...... determinations in patients suspected of GHD has only been reported in a few studies, whereas no previous reports on the diagnostic value of free IGF-I levels in adults suspected of GHD exist. Serum levels of free IGF-I were determined in 1430 healthy children, adolescents, and adults by a newly developed......, commercially available immunoradiometric assay (Diagnostic Systems Laboratories) to establish valid normative data for this analysis. We studied the diagnostic value of free IGF-I in relation to total IGF-I and IGFBP-3 determinations in adults who were suspected of GHD. A GH provocative test, using oral...

[Diagnostic significance of serum free DNA human telomerase reverse transcriptase quantitative determination on spinal cord injury].

Science.gov (United States)

Yang, M K; Tang, J; Xiang, Z; Zhang, X; Wang, J; Li, Z; Li, Y; Sheng, W B

2018-02-06

Objective: To investigate the relationship between the content of human telomerase reverse transcriptase (hTERT) and its clinical features in serum free DNA in patients with different degree of spinal cord injury. Methods: From December 2013 to December 2016, inpatients of the Central Hospital of Bazhong, Sichuan Province were enrolledand divided into the experimental group, the disease control group and the negative control group. For the experimental group: 46 patients with spinal cord injury were graded according to the criteria of the American Association of Spinal Cord Injury (ASIA), including 12 cases of grade A, 10 cases of grade B, 10 cases of grade C, 7 cases of grade D and 7 cases of grade E; for the disease control group: 15 patients with spinal fractures (without spinal cord injury) at the same period were included; and for the negative control group: 20 healthy adult volunteers aged 18-50 years were selected.Real-time fluorescence quantitative PCR and immunoblotting were performed to detect the content of hTERT in serum free DNA both in patients and healthy controls and to compare the difference between them. The results of the somatosensory evoked potential (SEP) of all patients were compared and analyzed.The receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of hTERT content in serum free DNA in patients with spinal cord injury. Results: Comparison of serum free DNA hTERT content: in the experimental group, the serum free DNA hTERT content of grade A, B, C, D, E was (99.63±8.23), (76.24±4.37), (46.07±5.43), (16.30±0.95) and (15.74±1.12)μg/L, respectively.While it was (15.01±1.39)μg/L in the disease control group and (14.54±1.03)μg/L in the negative control group. The total difference was statistically significant between patients of each group and the control group ( F =857.917, P spinal cord injury has a certain guiding significance for the diagnosis of spinal cord injury and the degree of injury.

Oil-free culture system for in vitro bovine embryo production

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Paulo B.D. Gonçalves

2010-04-01

Full Text Available The use of oil to avoid water evaporation from cell culture has several disadvantages, amongst which there is the migration of compounds from media to oil and from oil to media. The aim of this study was to evaluate the osmolality of a culture system using four-well plates with water in the central hole as an alternative to in vitro bovine embryo production (IVP. In addition, the osmolality changes of the oocyte washing medium were assessed in 35mm dishes with or without 2 mL of silicon oil overlay. Osmolality of oocyte washing medium changed a great deal over time after 60 minutes on a 39°C heated plate (291 mOsm kg-1, which was not detected when the medium was overlaid with silicon oil (280 mOsm kg-1; P0.05. Blastocyst rates were higher when embryos were cultured in presence of water or oil (29.7 and 29.9% for water and 33% in oil conventional microdrop system, except in the group that oocytes were washed in hyperosmotic washing medium (15.1%; P<0.05. Groups cultured in absence of water in the central hole had lower blastocyst rates (P<0.05 independently of exposure (15.5% or not (16.2 and 16.8% to hyperosmotic washing medium. In conclusion, four-well plates with water in the central hole can be an alternative to replace oil overlay for bovine IVP, maintaining stable osmolality and embryo development rates.

Serum Levels Of Free And Total Insulin-Like Growth Factor (IGF)-1 And IGF Binding Protein-3 In Normal And Growth Hormone Deficient Children

International Nuclear Information System (INIS)

Shousha, M.A.; Soliman, S.E.T.; Hafez, M.H.

2006-01-01

Serum levels of total insulin-like growth factor-1 (IGF-1) and IGF-binding protein-3 (IGFBP-3) reflect the endogenous GH secretion in healthy children, which makes them good diagnostic markers for screening growth hormone deficiency (GHD) in short children, although some controversy still exists. Only a minor fraction of the total IGF-1 circulates in its free form, which is believed to be the biologically active form. Serum levels of free IGF-1, total IGF-I and IGFBP-3 were measured in 144 healthy children (72 boys and 72 girls, aged from 0 to 16 years) and in 12 pre-pubertal GH deficient (GHD) children to study the correlation between the age and free IGF-1, total IGF-1 and IGFBP-3 levels. In healthy subjects (both sexes), serum free IGF-1, total IGF-1 and IGFBP-3 levels were low in infancy, increasing during puberty and declining thereafter. Free IGF-1 in serum occupied about 0.97-1.45 % of the total IGF-1 values, and the ratios of free IGF-1 to total IGF-1 were significantly increased in the pubertal age groups than in the pre-pubertal age groups. Serum levels of free IGF-1 showed significant positive correlation with those of total IGF-I and IGFBP-3. Serum free IGF-1, total IGF-1 and IGFBP-3 levels in patients with GHD were decreased significantly with increasing the degree of hypopituitarism. These observations suggest that the increase in serum free IGF-1 level during puberty was caused by a dramatic increase in total IGF-1 rather than IGFBP-3. Also, high levels of these hormones may play an important role in pubertal growth spurt and may become a useful tool for diagnosing GHD and predicting growth response to long term GH therapy

Serum levels of free and total insulin-link growth factor (IGF)-1 and (IGF) binding protein-3 in normal and growth hormone deficient children

International Nuclear Information System (INIS)

Shousha, M.A.; Soliman, S.E.T.; Hafez, H.M.

2008-01-01

Serum levels of total insulin-like growth factor- 1 (IGF-1) and IGF-binding protein-3 (IGFBP-3) reflect endogenous GH secretion in healthy children, which makes them good diagnostic markers for screening GH deficiency (GHD) in short children, although some controversy still exists. Only a minor fraction of the total IGF-1 circulates in its free form, which is believed to be the biologically active form. Serum levels of free IGF-1, total IGF-I and IGFBP-3 were measured in 144 healthy children (72 boys and 72 girls, aged from 0 to 16 years) and in 12 prepubertal GH. deficient (GHD) children to study correlation between the age and free IGF-1, total IGF-1 and IGFBP-3 levels. In healthy subjects (both sexes), serum free IGF-1, total IGF-1 and IGFBP-3 levels were low in infancy, increasing during puberty and declining thereafter. Free IGF-1 in serum occupied about 0.97. 1.45 % of the total IGF-1 values, and the ratios of free IGF-1 to total IGF-1 were significantly increased in the pubertal age groups than in the prepubertal age groups. Serum levels of free IGF-1 showed significant positive correlation with those of total IGF-I and IGFBP-3. Serum free IGF-1, total IGF-1 and IGFBP-3 levels in patients with GHD decreased significantly with increasing degree of hypopituitarism. These observations suggest that the increase in serum free IGF-1 level during puberty was caused by a dramatic increase in total IGF-1 rather than IGFBP-3. Also, high levels of these hormones may play an important role in pubertal growth spurt and may become a useful tool for diagnosing GHD and predicting growth response to long term GH therapy

Prognostic Value of Serum Free Light Chain in Multiple Myeloma.

Science.gov (United States)

El Naggar, Amel A; El-Naggar, Mostafa; Mokhamer, El-Hassan; Avad, Mona W

2015-01-01

The measurement of serum free light chain (sFLC) has been shown to be valuable in screening for the presence of plasma cell dyscrasia as well as for baseline prognosis in newly diagnosed patients. The aim of the present work was to study the prognostic value of sFLC in multiple myeloma in relation to other serum biomarkers, response to therapy and survival. Forty five newly diagnosed patients with MM were included in the study. Patients were divided into responders and non-responders groups according to response to therapy. sFLC and serum Amyloid A (SAA) were measured by immunonephelometry. The non-responders group showed a statistically significant higher kappa/lambda or lambda/kappa ratio and higher β2 microglobulin level, but lower albumin level at presentation, as compared to the responders group (P < 0.001). However, no statistically significant difference was detected between the two groups regarding SA A or calcium levels. Comparison between sFLC ratio obtained before and after therapy revealed significant decrease after treatment in the responders group (P = 0.05). Survival was significantly inferior in patients with an FLC ratio of ≥ 2.6 or ≤ 0.56 compared with those with an FLC ratio that was between 0.56 and 2.6 (P = 0.002).

Characteristics of retinol accumulation from serum retinol-binding protein by cultured sertoli cells

International Nuclear Information System (INIS)

Shingleton, J.L.; Skinner, M.K.; Ong, D.E.

1989-01-01

The uptake of retinol was examined in cultured Sertoli cells when retinol was provided as a complex with the transport protein retinol-binding protein (RBP). Sertoil cells accumulated [ 3 H]retinol in a time- and temperature-dependent manner. The change in rate of retinol accumulation occurred when the cells had accumulated approximately 0.53 pmol of retinol/μg of cellular DNA. Extraction and HPLC analysis of the cell-associated radioactivity yielded retinol and retinyl esters, indicating that a significant proportion of the accumulated retinol was esterified. Excess unlabeled retinol-RBP competed with [ 3 H]retinol-RBP for [ 3 H]retinol delivery to the cells, indicating that RBP delivery of retinol was a saturable and competable process. However, free [ 3 H]retinol associated with Sertoli cells in a noncompetable manner. The transport constant for specific retinol accumulation from RBP was 3.0 μM. Neither iodinated nor reductively methylated RBP was accumulated by or tightly bound to Sertoli cells. Competition studies indicated, however, that protein recognition is important in the retinol uptake process. RBP, CRBP, and CRBP(II) competed with [ 3 H]retinol-RBP for [ 3 H]retinol accumulation, but free retinol, retinol-bovine serum albumin, and retinol-β-lactoglobulin did not. These studies indicated that Sertoli cell uptake of retinol involved recognition of the retinol-RBP complex at the cell surface with subsequent internalization of retinol, but not RBP

Comparative Serum Fatty Acid Profiles of Captive and Free-Ranging Cheetahs (Acinonyx jubatus) in Namibia

Science.gov (United States)

Wachter, Bettina; Heinrich, Sonja K.; Reyers, Fred; Mienie, Lodewyk J.

2016-01-01

Cheetahs (Acinonyx jubatus) are highly specialised large felids, currently listed as vulnerable on the IUCN red data list. In captivity, they are known to suffer from a range of chronic non-infectious diseases. Although low heterozygosity and the stress of captivity have been suggested as possible causal factors, recent studies have started to focus on the contribution of potential dietary factors in the pathogenesis of these diseases. Fatty acids are an important component of the diet, not only providing a source of metabolisable energy, but serving other important functions in hormone production, cellular signalling as well as providing structural components in biological membranes. To develop a better understanding of lipid metabolism in cheetahs, we compared the total serum fatty acid profiles of 35 captive cheetahs to those of 43 free-ranging individuals in Namibia using gas chromatography-mass spectrometry. The unsaturated fatty acid concentrations differed most remarkably between the groups, with all of the polyunsaturated and monounsaturated fatty acids, except arachidonic acid and hypogeic acid, detected at significantly lower concentrations in the serum of the free-ranging animals. The influence of age and sex on the individual fatty acid concentrations was less notable. This study represents the first evaluation of the serum fatty acids of free-ranging cheetahs, providing critical information on the normal fatty acid profiles of free-living, healthy individuals of this species. The results raise several important questions about the potential impact of dietary fatty acid composition on the health of cheetahs in captivity. PMID:27992457

Free software and free culture in Higher Education: pipelines and workflows for creative purposes

OpenAIRE

Gonçalves, Nelson; Figueiredo, Maria Pacheco

2015-01-01

OpenLab ESEV is a project of the School of Education of the Polytechnic Institute of Viseu (ESEV), Portugal, that aims to promote, foster and support the use of Free/Libre Software and Open Source Software, Open Educational Resources, Free Culture, Free file formats and more flexible copyright licenses for creative and educational purposes in the ESEV's domains of activity (education, arts, media). Most of the OpenLab ESEV activities are related to the teacher education and arts a...

Proposal of a candidate international conventional reference measurement procedure for free thyroxine in serum.

NARCIS (Netherlands)

Thienpont, L.M.; Beastall, G.H.; Christofides, N.D.; Faix, J.D.; Ieiri, T.; Jarrige, V.; Miller, W.G.; Miller, R.; Nelson, J.C.; Ronin, C.; Ross, H.A.; Rottmann, M.; Thijssen, J.H.; Toussaint, B.

2007-01-01

In the present paper the IFCC WG-STFT recommends and provides the rationale to establish metrological traceability of serum free thyroxine (FT4) measurements to a candidate international conventional reference measurement procedure. It is proposed that this procedure be based on equilibrium dialysis

Essential amino acids in the gluten-free diet and serum in relation to depression in patients with celiac disease.

Directory of Open Access Journals (Sweden)

Nathalie J M van Hees

Full Text Available Celiac disease (CD is associated with an increased risk of major depressive disorder, possibly due to deficiencies in micronutrients in the gluten-free diet. We aimed to investigate whether essential amino acids (i.e., the precursors of serotonin, dopamine and other neurotransmitters are depleted in the diet and serum of CD patients with major depressive disorder.In a cross-sectional study we assessed dietary intake of amino acids and serum levels of amino acids, in 77 CD patients on a gluten-free diet and in 33 healthy controls. Major depressive disorder was assessed with structured interviews (using the Mini International Neuropsychiatric Interview Plus. Dietary intake was assessed using a 203-item food frequency questionnaire.Participants had a mean age of 55 years and 74% were women. The intake of vegetable protein was significantly lower in CD patients than in healthy controls (mean difference of 7.8 g/d; 95% CI: 4.7-10.8, as were serum concentrations of tyrosine, phenylalanine and tryptophan (all p < 0.005. However, within the CD patient group, the presence of major depressive disorder (n = 42 was not associated with intake or serum levels of essential amino acids.Patients with CD on a long-term gluten-free diet, with good adherence, consume significantly less vegetable protein than controls, and their serum levels of several essential amino acids were also lower. Despite its potential adverse effect, intake and serum levels of essential amino acids were not related to major depression.

Analysis of Serum Total and Free PSA Using Immunoaffinity Depletion Coupled to SRM: Correlation with Clinical Immunoassay Tests

Science.gov (United States)

Liu, Tao; Hossain, Mahmud; Schepmoes, Athena A.; Fillmore, Thomas L.; Sokoll, Lori J.; Kronewitter, Scott R.; Izmirlian, Grant; Shi, Tujin; Qian, Wei-Jun; Leach, Robin J.; Thompson, Ian M.; Chan, Daniel W.; Smith, Richard D.; Kagan, Jacob; Srivastava, Sudhir; Rodland, Karin D.; Camp, David G.

2012-01-01

Recently, selected reaction monitoring mass spectrometry (SRM-MS) has been more frequently applied to measure low abundance biomarker candidates in tissues and biofluids, owing to its high sensitivity and specificity, simplicity of assay configuration, and exceptional multiplexing capability. In this study, we report for the first time the development of immunoaffinity depletion-based workflows and SRM-MS assays that enable sensitive and accurate quantification of total and free prostate-specific antigen (PSA) in serum without the requirement for specific PSA antibodies. Low ng/mL level detection of both total and free PSA was consistently achieved in both PSA-spiked female serum samples and actual patient serum samples. Moreover, comparison of the results obtained when SRM PSA assays and conventional immunoassays were applied to the same samples showed good correlation in several independent clinical serum sample sets. These results demonstrate that the workflows and SRM assays developed here provide an attractive alternative for reliably measuring candidate biomarkers in human blood, without the need to develop affinity reagents. Furthermore, the simultaneous measurement of multiple biomarkers, including the free and bound forms of PSA, can be performed in a single multiplexed analysis using high-resolution liquid chromatographic separation coupled with SRM-MS. PMID:22846433

Validity of Serum Testosterone, Free Androgen Index, and Calculated Free Testosterone in Women with Suspected Hyperandrogenism

Directory of Open Access Journals (Sweden)

Manal K. Al Kindi

2012-11-01

Full Text Available Objectives: There are technical limitations for the currently available methods of measuring serum total and free testosteronein females. The study objectives were to evaluate the usefulness of serum total testosterone, sex hormone-binding globulin (SHBG, free androgen index (FAI, and calculated free testosterone (CFT in the assessment of androgen status in women investigated for suspected hyperandrogenism.Methods: This is a case control study that was conducted during the period from 1st May 2011 to 31st October 2011 on 122 patients aged (18-45 years whom were referred to the Clinical Biochemistry Laboratory from the Endocrinology and Gynecology Clinics, Royal Hospital, Oman. Women with no clinical feature or laboratory data indicative of hormonal dysfunction and with midluteal progesterone >30 nmol/L were selected as controls (group 1; n=18. The patients were divided into subgroups based on the clinical/laboratory diagnosis of polycystic ovary syndrome (PCOS [group 2; n=19, hirsutism (group 3; n=18, menstrual disturbances (irregularities or infertility (group 4; n=49, as well as combination of PCOS or hirsutism and menstrual disturbances or infertility (group 5;n=18. Serum total testosterone and SHBG were measured, FAI was calculated as percentage ratio of total testosterone to SHBG values, and CFT was calculated according to Vermeulen equation.Results: There was a statistically significant difference in the mean levels of testosterone, FAI and CFT in each patient group compared with the control group. For diagnosing hyperandrogenism, each indicator was selected at the recommended cut-off: testosterone >3.0 nmol/L, SHBG 5%, and CFT >32 pmol/L. In group 2, 89.5% and 94.7% of the patients had increased FAI and CFT, respectively; compared with 36.4% for increased testosterone. In group 3, 88.9% and 88.9% of the patients had similarly increased FAI and CFT, respectively; compared with 66.7% for testosterone. In group 4, patients had 63.3% and 73

Thermometric enzyme linked immunosorbent assay in continuous flow system: optimization and evaluation using human serum albumin as a model system.

Science.gov (United States)

Borrebaeck, C; Börjeson, J; Mattiasson, B

1978-06-15

Thermometric enzyme-linked immunosorbent assay (TELISA) is described. After the procedure of optimization, human serum albumin was assayed using anti-human serum albumin bound to Sepharose CL 4-B in the enzyme thermistor unit and catalase as label on the free antigen. The model system was used for assays down to 10(-13)M and the preparation of immobilized antibodies was used repeatedly up to 100 times. Comparative studies of the TELISA technique with bromocresol green, immunoturbidimetric and rocket immunoelectrophoretic methods were carried out and showed that TELISA could be used as an alternative method.

Clonogenic growth of human breast cancer cells co-cultured in direct contact with serum-activated fibroblasts

International Nuclear Information System (INIS)

Samoszuk, Michael; Tan, Jenny; Chorn, Guillaume

2005-01-01

Accumulating evidence suggests that fibroblasts play a pivotal role in promoting the growth of breast cancer cells. The objective of the present study was to characterize and validate an in vitro model of the interaction between small numbers of human breast cancer cells and human fibroblasts. We measured the clonogenic growth of small numbers of human breast cancer cells co-cultured in direct contact with serum-activated, normal human fibroblasts. Using DNA microarrays, we also characterized the gene expression profile of the serum-activated fibroblasts. In order to validate the in vivo relevance of our experiments, we then analyzed clinical samples of metastatic breast cancer for the presence of myofibroblasts expressing α-smooth muscle actin. Clonogenic growth of human breast cancer cells obtained directly from in situ and invasive tumors was dramatically and consistently enhanced when the tumor cells were co-cultured in direct contact with serum-activated fibroblasts. This effect was abolished when the cells were co-cultured in transwells separated by permeable inserts. The fibroblasts in our experimental model exhibited a gene expression signature characteristic of 'serum response' (i.e. myofibroblasts). Immunostaining of human samples of metastatic breast cancer tissue confirmed that myofibroblasts are in direct contact with breast cancer cells. Serum-activated fibroblasts promote the clonogenic growth of human breast cancer cells in vitro through a mechanism that involves direct physical contact between the cells. This model shares many important molecular and phenotypic similarities with the fibroblasts that are naturally found in breast cancers

[Comparison of two types of cell cultures for preparation of sTNFRII-gAD fusion protein].

Science.gov (United States)

Huang, Shigao; Yin, Yuting; Xiong, Chunhui; Wang, Caihong; Lü, Jianxin; Gao, Jimin

2013-01-01

In this study we used two types of cell cultures, i.e., anchorage-dependent basket and full suspension batch cultures of sTNFRII-gAD-expressing CHO cells in the CelliGen 310 bioreactor (7.5 L) to compare their yields in order to optimize the culturing conditions for efficient expression of sTNFRII-gAD fusion protein consisting of soluble tumor necrosis factor receptor II and globular domain of adiponectin. The anchorage-dependent basket culture was performed in 4L 10% serum-containing medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 3 days, and then switched to 4 L serum-free LK021 medium to continue the culture for 4 days. The full suspension batch culture was carried out in the 4 L serum-free LK021 medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 7 days. The culturing conditions were monitored in real-time to maintain pH and dissolved oxygen stability through the whole process. The supernatants were collected by centrifuge, and the protein was concentrated through Pellicon flow ultrafiltration system and then purified by DEAE anion exchange. The results showed that the yields of sTNFRII-gAD fusion protein were 8.0 mg/L with 95% purity and 7.5 mg/L with 98% purity in the anchorage-dependent basket and the full suspension batch cultures, respectively. The study provided the framework for the pilot production of sTNFRII-gAD fusion protein.

Platelet-rich plasma can replace fetal bovine serum in human meniscus cell cultures

NARCIS (Netherlands)

Gonzales, V.K.; Mulder, E.L.W. de; Boer, T. den; Hannink, G.; Tienen, T.G. van; Heerde, W.L. van; Buma, P.

2013-01-01

Concerns over fetal bovine serum (FBS) limit the clinical application of cultured tissue-engineered constructs. Therefore, we investigated if platelet-rich plasma (PRP) can fully replace FBS for meniscus tissue engineering purposes. Human PRP and platelet-poor plasma (PPP) were isolated from three

Clinical significance of two-step magnetic radioimmunoassay for determining serum free T3 and free T4

International Nuclear Information System (INIS)

Chen Bing

1996-01-01

The concentrations of the serum free T 3 (FT 3 ), free T 4 (FT 4 ), total triodothyronine (TT 3 ), total thyroxine (TT 4 ) and thyrotropin (TSH) are determined for 355 cases of normal persons, pregnant women and various thyropathetic patients. The normal values of FT 3 and FT 4 are 2.0-8.5 pmol/l, and 9.5-26.5 pmol/l, respectively. Neither FT 3 nor FT 4 is affected by the thyroxine combined with globulin (TBG), which is of unique diagnostic value for those with variable TBG (such as pregnant women hyperthyroidism, hypothyroidism, etc.), FT 3 and FT 4 are the most sensitive indices for diagnosis of hyperthyroidism, and hypothyroidism, respectively. In addition, FT 3 and FT 4 can greatly contribute to the observation of curative effectiveness under treatment

The fluctuation of free amino acids in serum during acute ischemic stroke

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Szpetnar Maria

2016-12-01

Full Text Available Currently, little data exists regarding the involvement of free amino acids (AA in the pathogenesis of ischemic stroke (IS. Thus, our objective was to study the degree of the degree of fluctuation of free amino acids level in serum during the acute phase of IS. The study consisted of eighteen patients (female/male: 10/8; age: 73.1 ± 4.1 with acute IS that was confirmed by way of computed tomography, while twelve sex and age matched individuals were assigned as control group. During the study period, the patients did not receive any supplemental amino acids therapy that could affect the obtained results. The venous blood was obtained after >3 hours fasting at two time-points; time-point 1 – at admission to the hospital; time-point 2 – on day 5 from stroke onset. The blood for control purposes was collected only once, and the blood collection at time-point 1 was done before thrombolytic treatment (nine patients. The amino acids were identified using the Amino Acids Analyser (AAA 400 by INGOS Corp., Praha, Czech Republic. Our results revealed a statistically significant increase of glutamate, cystine and methionine on day 1 of stroke, in comparison to control, whereas, proline level was decreased on day 1 of stroke – in comparison to control serum. On comparing day 5 to the initial day of IS, elevation was observed of levels of asparagine, glycine, tyrosine, arginine, threonine, valine, leucine and phenylalanine. It can be said, then, that ischemic stroke induces both essential and nonessential amino acid fluctuations. Moreover, the decrease in proline and glutamine serum level with the simultaneous increase in the concentration of branch chain amino acids, Glu and Thr suggests a violent mobilization of the body’s proteins. Thus, a decrease of Pro and a simultaneous increase of Glu serum level could be considered as a marker of acute IS.

Systemic Analysis of the Cultural Production of a Virtual Learning Community

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Germán Alejandro Miranda Díaz

2013-04-01

Full Text Available This paper describes a systemic analysis from the standpoint of activity theory of the cultural emergence of a virtual learning community as a complex system. Three levels of analysis were employed: data mining, visualization of complex systems and analysis of discursive interactions, with the aim of understanding the emerging phenomena of online learning and in order to have the necessary elements to assist in planning the formation of virtual communities in formal settings. This was carried out using six years of activity from 3,324 people from different documentary sources: 9,871,531 CMS records; 1,371,907 from LMS; 67,828 IRC statements; and 27,798 online forum comments. In the process, we observed how action aimed at socialization and discussion of its object evolve into historical-cultural milestones such as the culture of merit as opposed to certification, the division of labor and the process of transition from free software to free culture.

Thymic epithelial cells. I. Expression of strong suppressive (veto) activity in mouse thymic epithelial cell cultures

DEFF Research Database (Denmark)

Claesson, Mogens Helweg; Ropke, C

1990-01-01

We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level. It is conclu......We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level...

Viability and proliferation of L929, tumour and hybridoma cells in the culture media containing sericin protein as a supplement or serum substitute.

Science.gov (United States)

Cao, Ting-Ting; Zhang, Yu-Qing

2015-09-01

Cell cultures often require the addition of animal serum and other supplements. In this study, silk sericin, a bioactive protein, recovered from the waste of silk floss production was hydrolysed into three pepsin-degraded sericin peptides with different ranges of molecular mass. Normal animal cells, tumour cells and hybridoma cells were cultured systematically in FBS culture media containing sericin as a supplement or serum substitute. The culture test and microscopic observation of L929 cells showed that the smaller molecular weight of the degraded sericin is most suitable for cell culture. The cell culture results showed that with the degradation of sericin, for normal mouse fibroblast L929 cells, addition of 0.75 % sericin into FBS culture medium yields cell viability that is superior to FBS culture medium alone. When all serum was replaced by sericin, cell viability in the sericin medium could reach about one half of that in FBS medium. When in a medium containing a mixture of FBS: sericin (6:4, v/v), the cell culture effect is about 80 %. For the cultures of four tumour and one hybridoma cells, regardless of the molecular weight range, these degraded sericin peptides could substitute all serum in FBS media. The cell viability and proliferation of these tumour and hybridoma cells are equivalent or superior to that in FBS medium. In other words, cell viability and proliferation of these tumour and hybridoma cells in sericin media are more preferable to serum media. The mechanism of the sericin protein to promote cell growth and proliferation will be further investigated later.

Direct, Label-Free, and Rapid Transistor-Based Immunodetection in Whole Serum.

Science.gov (United States)

Gutiérrez-Sanz, Óscar; Andoy, Nesha M; Filipiak, Marcin S; Haustein, Natalie; Tarasov, Alexey

2017-09-22

Transistor-based biosensors fulfill many requirements posed upon transducers for future point-of-care diagnostic devices such as scalable fabrication and label-free and real-time quantification of chemical and biological species with high sensitivity. However, the short Debye screening length in physiological samples (<1 nm) has been a major drawback so far, preventing direct measurements in serum. In this work, we demonstrate how tailoring the sensing surface with short specific biological receptors and a polymer polyethylene glycol (PEG) can strongly enhance the sensor response. In addition, the sensor performance can be dramatically improved if the measurements are performed at elevated temperatures (37 °C instead of 21 °C). With this novel approach, highly sensitive and selective detection of a representative immunosensing parameter-human thyroid-stimulating hormone-is shown over a wide measuring range with subpicomolar detection limits in whole serum. To the best of our knowledge, this is the first demonstration of direct immunodetection in whole serum using transistor-based biosensors, without the need for sample pretreatment, labeling, or washing steps. The presented sensor is low-cost, can be easily integrated into portable diagnostics devices, and offers a competitive performance compared to state-of-the-art central laboratory analyzers.

Hand-made cloned buffalo (Bubalus bubalis) embryos: comparison of different media and culture systems.

Science.gov (United States)

Shah, Riaz A; George, Aman; Singh, Manoj K; Kumar, Dharmendra; Chauhan, Manmohan S; Manik, Radhaysham; Palta, Prabhat; Singla, Suresh K

2008-12-01

Hand-made cloning (HMC) has proved to be an efficient alternative to the conventional micromanipulator-based technique in some domestic animal species. This study reports the development of an effective culture system for in vitro culture of zona-free cloned buffalo (Bubalus bubalis) embryos reconstructed using adult skin fibroblast cells as nucleus donor. Cleavage and blastocyst rates observed were 52 and 0% in modified Charles Rosenkrans 2 (mCR2), 61 and 4.6% in modified Synthetic Oviductal Fluid (mSOF), and 82 and 40.3% in Research Vitro Cleave (RVCL; Cook, Australia) medium, respectively. Similarly, higher blastocyst rates (24.5 +/- 4.1%) were observed when zona-free parthenotes were cultured in RVCL medium. Culturing zona-free cloned buffalo embryos on flat surfaces (FS) yielded significantly higher (p WOW) or microdrops (MD). Furthermore, development in WOW was found to be significantly better than MD culture. The quality of HMC blastocysts was examined using differential staining. This study establishes the application of zona-free nuclear transfer procedures for the production of hand-made cloned buffalo embryos and the development of efficient culture system and appropriate media requirements for enhancing their preimplantation development.

Changes of Serum Total and Free Testosterone Concentrations in Male Chronic Hemodialysis Patients with Secondary Hyperparathyroidism in Response to Cinacalcet Treatment

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Piotr Kuczera

2016-01-01

Full Text Available Background/Aims: Calcium sensing receptor (CaSR is expressed, among others also in testis. Cinacalcet binds to the CaSR, increases sensitivity of CaSR to serum calcium and is used in the treatment of secondary hyperparathyroidism (sHPT in chronic hemodialysis patients (HDP. In most of male HDP, serum testosterone concentration is lower than in healthy males. The aim of this study was to assess the influence of six-month treatment with cinacalcet on the serum total and free testosterone concentration in male HDP with sHPT. Methods: 38 male, hemodialysed CKD patients with sHPT (PTH>300 pg/ml were enrolled into the study. In each patient serum PTH, total testosterone (TT and free testosterone (FT concentrations were assessed before the first dose of cinacalcet and then after 3 and 6 months of treatment. The results are presented as means with 95% confidence interval. Results: In 33 patients who completed the study cinacalcet treatment caused significant decrease of serum PTH from 1143 pg/ml (828 - 1458 pg/ml at the baseline, to 809 pg/ml (487 - 1132pg/ml after 3 month of treatment (p = 0.002, and to 607 pg/ml (281 - 934pg/ml; p Conclusion: Treatment with cinacalcet decreases serum total and free testosterone concentration in male hemodialysed patients with chronic kidney disease and secondary hyperparathyroidism.

Culture of human mesenchymal stem cells using a candidate pharmaceutical grade xeno-free cell culture supplement derived from industrial human plasma pools.

Science.gov (United States)

Díez, José M; Bauman, Ewa; Gajardo, Rodrigo; Jorquera, Juan I

2015-03-13

Fetal bovine serum (FBS) is an animal product used as a medium supplement. The animal origin of FBS is a concern if cultured stem cells are to be utilized for human cell therapy. Therefore, a substitute for FBS is desirable. In this study, an industrial, xeno-free, pharmaceutical-grade supplement for cell culture (SCC) under development at Grifols was tested for growth of human mesenchymal stem cells (hMSCs), cell characterization, and differentiation capacity. SCC is a freeze-dried product obtained through cold-ethanol fractionation of industrial human plasma pools from healthy donors. Bone marrow-derived hMSC cell lines were obtained from two commercial suppliers. Cell growth was evaluated by culturing hMSCs with commercial media or media supplemented with SCC or FBS. Cell viability and cell yield were assessed with an automated cell counter. Cell surface markers were studied by indirect immunofluorescence assay. Cells were cultured then differentiated into adipocytes, chondrocytes, osteoblasts, and neurons, as assessed by specific staining and microscopy observation. SCC supported the growth of commercial hMSCs. Starting from the same number of seeded cells in two consecutive passages of culture with medium supplemented with SCC, hMSC yield and cell population doubling time were equivalent to the values obtained with the commercial medium and was consistent among lots. The viability of hMSCs was higher than 90%, while maintaining the characteristic phenotype of undifferentiated hMSCs (positive for CD29, CD44, CD90, CD105, CD146, CD166 and Stro-1; negative for CD14 and CD19). Cultured hMSCs maintained the potential for differentiation into adipocytes, chondrocytes, osteoblasts, and neurons. The tested human plasma-derived SCC sustains the adequate growth of hMSCs, while preserving their differentiation capacity. SCC can be a potential candidate for cell culture supplement in advanced cell therapies.

Analysis of Serum Total and Free PSA Using Immunoaffinity Depletion Coupled to SRM: Correlation with Clinical Immunoassay Tests

Energy Technology Data Exchange (ETDEWEB)

Liu, Tao; Hossain, Mahmud; Schepmoes, Athena A.; Fillmore, Thomas L.; Sokoll, Lori J.; Kronewitter, Scott R.; Izmirlian, Grant; Shi, Tujin; Qian, Weijun; Leach, Robin; Thompson, Ian M.; Chan, Daniel W.; Smith, Richard D.; Kagan, Jacob; Srinivastava, Sudhir; Rodland, Karin D.; Camp, David G.

2012-08-03

Sandwich immunoassay is the standard technique used in clinical labs for quantifying protein biomarkers for disease detection, monitoring and therapeutic intervention. Albeit highly sensitive, the development of a specific immunoassay is rather time-consuming and associated with extremely high cost due to the requirement for paired immunoaffinity reagents of high specificity. Recently, mass spectrometry-based methods, specifically selected reaction monitoring mass spectrometry (SRM-MS), have been increasingly applied to measure low abundance biomarker candidates in tissue and biofluids, owing to high sensitivity and specificity, simplicity of assay configuration, and great multiplexing capability. In this study, we report for the first time the development of immunoaffinity depletion-based workflows and SRM-MS assays that enable sensitive and accurate quantification of total and free prostate-specific antigen (PSA) in serum without the requirement for specific PSA antibodies. With stable isotope dilution and external calibration, low ng/mL level detection of both total and free PSA was consistently achieved in both PSA-spiked female serum samples and actual patient serum samples. Moreover, comparison of the results obtained when SRM PSA assays and conventional immunoassays were applied to the same samples showed very good correlation (R2 values ranging from 0.90 to 0.99) in several independent clinical serum sample sets, including a set of 33 samples assayed in a blinded test. These results demonstrate that the workflows and SRM assays developed here provide an attractive alternative for reliably measuring total and free PSA in human blood. Furthermore, simultaneous measurement of free and total PSA and many other biomarkers can be performed in a single analysis using high-resolution liquid chromatographic separation coupled with SRM-MS.

Formation of proteoglycan and collagen-rich scaffold-free stiff cartilaginous tissue using two-step culture methods with combinations of growth factors.

Science.gov (United States)

Miyazaki, Tatsuya; Miyauchi, Satoshi; Matsuzaka, Satoshi; Yamagishi, Chie; Kobayashi, Kohei

2010-05-01

Tissue-engineered cartilage may be expected to serve as an alternative to autologous chondrocyte transplantation treatment. Several methods for producing cartilaginous tissue have been reported. In this study, we describe the production of scaffold-free stiff cartilaginous tissue of pig and human, using allogeneic serum and growth factors. The tissue was formed in a mold using chondrocytes recovered from alginate bead culture and maintained in a medium with transforming growth factor-beta and several other additives. In the case of porcine tissue, the tear strength of the tissue and the contents of proteoglycan (PG) and collagen per unit of DNA increased dose-dependently with transforming growth factor-beta. The length of culture was significantly and positively correlated with thickness, tear strength, and PG and collagen contents. Tear strength showed positive high correlations with both PG and collagen contents. A positive correlation was also seen between PG content and collagen content. Similar results were obtained with human cartilaginous tissue formed from chondrocytes expanded in monolayer culture. Further, an in vivo pilot study using pig articular cartilage defect model demonstrated that the cartilaginous tissue was well integrated with surrounding tissue at 13 weeks after the implantation. In conclusion, we successfully produced implantable scaffold-free stiff cartilaginous tissue, which characterized high PG and collagen contents.

Enrichment of skin-derived neural precursor cells from dermal cell populations by altering culture conditions.

Science.gov (United States)

Bayati, Vahid; Gazor, Rohoullah; Nejatbakhsh, Reza; Negad Dehbashi, Fereshteh

2016-01-01

As stem cells play a critical role in tissue repair, their manipulation for being applied in regenerative medicine is of great importance. Skin-derived precursors (SKPs) may be good candidates for use in cell-based therapy as the only neural stem cells which can be isolated from an accessible tissue, skin. Herein, we presented a simple protocol to enrich neural SKPs by monolayer adherent cultivation to prove the efficacy of this method. To enrich neural SKPs from dermal cell populations, we have found that a monolayer adherent cultivation helps to increase the numbers of neural precursor cells. Indeed, we have cultured dermal cells as monolayer under serum-supplemented (control) and serum-supplemented culture, followed by serum free cultivation (test) and compared. Finally, protein markers of SKPs were assessed and compared in both experimental groups and differentiation potential was evaluated in enriched culture. The cells of enriched culture concurrently expressed fibronectin, vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors as compared to control culture. In addition, they possessed a multipotential capacity to differentiate into neurogenic, glial, adipogenic, osteogenic and skeletal myogenic cell lineages. It was concluded that serum-free adherent culture reinforced by growth factors have been shown to be effective on proliferation of skin-derived neural precursor cells (skin-NPCs) and drive their selective and rapid expansion.

Effects of age and health on the euthyroid reference ranges for serum free thyroxine and free triiodothyronine

International Nuclear Information System (INIS)

Midgley, J.E.M.

1985-01-01

Age-related trends in serum free thyroxine (FT 4 ) and free triiodothyronine (FT 3 ) concentrations were measured in 7248 euthyroid subjects (age-range 3 months to 106 years). 5700 were patients referred to hospitals for investigation of suspected thyroid dysfunction, but who were diagnosed euthyroid. 1548 were healthy blood donors (age-range 18-63 years) with no indication of thyroid dysfunction. FT 4 concentrations were little affected by the age, the sex or the state of health of the subjects in either group. Serum FT 3 concentrations were significantly affected by both age and health factors. The upper limit of the euthyroid reference range for young subjects up to 15 years was about 20% higher (10.4 pmol/l) than for adult subjects older than 25 years (8.8 pmol/l). The change in the upper limits typical of young subjects to that typical of adults occurred steadily over the decade 15-25 years. After this age, little further change occurred, especially in healthy subjects. Additionally, the lower limit of the euthyroid range for FT 3 was extended by the inclusion in the reference group of patients referred to hospitals. Compared with the lower limit of the FT 3 range for healthy subjects (5 pmol/l), the corresponding limit for referred subjects (young or adult) was 3.5-3.8 pmol/l. Broadening of the FT 3 reference range was probably brought about by a significant number of patients in the hospital-referred group with the 'low-T 3 syndrome' of mild non-thyroidal illness. Accordingly, FT 3 was inferior to FT 4 in the discrimination of hypothyroidism, as FT 4 was unaffected by this phenomenon. Effects of age and non-thyroidal illness on serum FT 3 concentrations require great care when selecting subjects for a laboratory euthyroid reference range typical of the routine workload. Constraints on the choice of subjects for FT 4 reference ranges are less stringent. (orig.) [de

Effect of different culture media and deswelling agents on survival of human corneal endothelial and epithelial cells in vitro.

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Valtink, Monika; Donath, Patricia; Engelmann, Katrin; Knels, Lilla

2016-02-01

To examine the effects of media and deswelling agents on human corneal endothelial and epithelial cell viability using a previously developed screening system. The human corneal endothelial cell line HCEC-12 and the human corneal epithelial cell line HCE-T were cultured in four different corneal organ culture media (serum-supplemented: MEM +2 % FCS, CorneaMax®/CorneaJet®, serum-free: Human Endothelial-SFM, Stemalpha-2 and -3) with and without 6 % dextran T500 or 7 % HES 130/0.4. Standard growth media F99HCEC and DMEM/F12HCE-T served as controls. In additional controls, the stress inducers staurosporine or hydrogen peroxide were added. After 5 days in the test media, cell viability was assessed by flow cytometrically quantifying apoptotic and necrotic cells (sub-G1 DNA content, vital staining with YO-PRO-1® and propidium iodide) and intracellular reactive oxygen species (ROS). The MEM-based media were unable to support HCEC-12 and HCE-T survival under stress conditions, resulting in significantly increased numbers of apoptotic and necrotic cells. HCEC-12 survival was markedly improved in SFM-based media even under staurosporine or hydrogen peroxide. Likewise, HCE-T survival was improved in SFM with or without dextran. The media CorneaMax®, CorneaJet®, and CorneaMax® with HES supported HCEC-12 survival better than MEM-based media, but less well than SFM-based media. HCE-T viability was also supported by CorneaJet®, but not by CorneaMax® with or without HES. Stemalpha-based media were not suitable for maintaining viability of HCEC-12 or HCE-T in the applied cell culture system. The use of serum-supplemented MEM-based media for corneal organ culture should be discontinued in favour of serum-free media like SFM.

[The value of serum free light chain in differential diagnosis of monoclonal gammopathy of renal significance].

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Li, C; Wen, Y B; Li, H; Su, W; Li, J; Cai, J F; Chen, L M; Li, X M; Li, X W

2017-08-08

Objective: To investigate the value of serum free light chain (FLC) in differential diagnosis of monoclonal gammopathy of renal significance (MGRS). Methods: Forty-nine hospitalized patients who underwent renal biopsy in Peking Union Medical College Hospital between January 2013 and December 2015 were included. Monoclonal gammopathy was detected by serum protein electrophoresis (SPE), serum immunofixation electrophoresis (IFE), urine IFE and serum FLC. All patients were classified as MGRS ( n =32) and monoclonal gammopathy of undetermined significance (MGUS) ( n =17). Results: Renal lesions in MGRS subgroup included light chain amyloidosis ( n =24, 75.0%), light chain deposition disease ( n =7, 21.9%), and fibrillary glomerulopathy ( n =1, 3.1%). Renal diseases in MGUS subgroup included membranous nephropathy ( n =10), focal segmental glomerulosclerosi (FSGS) ( n =3), diabetic glomerulopathy ( n =1), Henoch-Schonlein purpura nephritis ( n =1), anti-GBM disease concurrent with membranous nephropathy ( n =1) and glomerulomegaly ( n =1). Positive number of SPE, serum IFE, urine IFE and abnormal number of serum FLC ratio in MGRS subgroup were 12, 16, 23 and 30, respectively. Positive number of SPE, serum IFE, urine IFE and abnormal number of serum FLC ratio in MGUS subgroup were 11, 17, 6 and 3, respectively. MGRS and MGUS subgroups differed significantly in positive rate of serum IFE ( P value for MGRS, which was helpful for differential diagnosis of patients who had contraindication to renal biopsy.

Serum free light chain reference values: a critical approach.

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Altinier, Sara; Seguso, Mara; Zaninotto, Martina; Varagnolo, Mariacristina; Adami, Fausto; Angeli, Paolo; Plebani, Mario

2013-05-01

The clinical usefulness of serum free light chain (FLC) measurement in the management of patients with plasma cell proliferative disorders has been reported in several papers, and most clinical studies use the reference ranges declared by the manufacturer. The aim of the present study was to evaluate the reproducibility of FLCs immunoassay and to validate the reference range, before introducing it in routine setting. Internal quality control materials and a pool of fresh serum samples were used to evaluate imprecision; 162 fresh sera from healthy blood donors were analyzed to evaluate the reference range for FLCs. In order to verify the κ/λ FLC ratio, 43 sera from patients with polyclonal hypergammaglobulinemia were tested. The FLC immunoassay was performed using a nephelometer with the Freelite reagents. The imprecision studies performed using a serum pool tested with two different lots of reagents showed a mean CV of 16.09% for κFLC and of 16.72% for λFLC. Lower CV%s and different mean values were found by calculating the results from each specific lot separately, while different results were obtained using the control materials provided by the manufacturer. In reference subjects, the 2.5-97.5th percentiles were found to be 4.52-22.33 and 4.84-21.88mg/L for κFLC and λFLC, respectively. The range for κ/λ ratio (0.65-2.36) was validated with the values obtained from subjects with polyclonal hypergammaglobulinemia. In retesting 15 samples from blood donor subjects with a different lot of reagents, mean bias percentages of 17.60 for κFLC and 15.26 for λFLC were obtained. These findings confirm the lot-to-lot variability of the FLC assays also in the measurement of polyclonal light chains, as well as the need to carefully validate the reference values. Published by Elsevier Inc.

Serum vitamin D levels in free-ranging koalas (Phascolarctos cinereus).

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Pye, Geoffrey W; Ellis, William; FitzGibbon, Sean; Opitz, Brian; Keener, Laura; Hollis, Bruce W

2013-06-01

Due to climatic conditions in Northern America and Europe, koalas (Phascolarctos cinereus) are often housed indoors. Koala joeys raised in these environments are susceptible to the development of metabolic bone disease due to a lack of exposure to solar ultraviolet radiation to themselves and their dam. As an initial step toward describing vitamin D sufficiency and adequately measuring responses to supplementation, vitamin D values were calculated by using serum collected from 20 free-ranging koalas on St. Bees Island, Queensland, Australia. Vitamin D values ranged from 8.1 to 30.4 pg/ml (18.4 +/- 5.5 pg/ml) for 1, 25-hydroxyvitamin D, and from 1 to 14 nM/L (7.4 +/- 3.0 nM/L) for 25-hydroxyvitamin D. These koala serum vitamin D values are unusually low when compared with eutherian mammals. Although this study was limited in numbers and in the geographically range of the koalas sampled, it does suggest that the koala's requirement for vitamin D is low. Therefore, supplementation to prevent disease may be relatively easy to achieve because low doses will likely meet requirements. Caution should be taken to avoid intoxication if supplementing vitamin D in koalas.

Robust and highly-efficient differentiation of functional monocytic cells from human pluripotent stem cells under serum- and feeder cell-free conditions.

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Masakatsu D Yanagimachi

Full Text Available Monocytic lineage cells (monocytes, macrophages and dendritic cells play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells from human embryonic stem cells (ESCs and induced pluripotent stem cells (iPSCs is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established, these methods depend on the use of xenogeneic materials and, therefore, have a relatively poor-reproducibility. Here, we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3 × 10(6 ± 0.3 × 10(6 floating monocytes from approximately 30 clusters of ESCs/iPSCs 5-6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine, disease-specific iPSC studies and drug discovery.

Application of cell co-culture system to study fat and muscle cells.

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Pandurangan, Muthuraman; Hwang, Inho

2014-09-01

Animal cell culture is a highly complex process, in which cells are grown under specific conditions. The growth and development of these cells is a highly unnatural process in vitro condition. Cells are removed from animal tissues and artificially cultured in various culture vessels. Vitamins, minerals, and serum growth factors are supplied to maintain cell viability. Obtaining result homogeneity of in vitro and in vivo experiments is rare, because their structure and function are different. Living tissues have highly ordered complex architecture and are three-dimensional (3D) in structure. The interaction between adjacent cell types is quite distinct from the in vitro cell culture, which is usually two-dimensional (2D). Co-culture systems are studied to analyze the interactions between the two different cell types. The muscle and fat co-culture system is useful in addressing several questions related to muscle modeling, muscle degeneration, apoptosis, and muscle regeneration. Co-culture of C2C12 and 3T3-L1 cells could be a useful diagnostic tool to understand the muscle and fat formation in animals. Even though, co-culture systems have certain limitations, they provide a more realistic 3D view and information than the individual cell culture system. It is suggested that co-culture systems are useful in evaluating the intercellular communication and composition of two different cell types.

Adhesion of cultured human endothelial cells onto methacrylate polymers with varying surface wettability and charge

NARCIS (Netherlands)

van Wachem, P.B.; Hogt, A.H.; Beugeling, T.; Feijen, Jan; Bantjes, A.; Detmers, J.P.; van Aken, W.G.

1987-01-01

The adhesion of human endothelial cells (HEC) onto a series of well-characterized methacrylate polymer surfaces with varying wettabilities and surface charges was studied either in serum-containing (CMS) or in serum-free (CM) culture medium. HEC adhesion in CMS onto (co)polymers * of hydroxyethyl

RNA synthesis in primary cultures of adult rat hepatocytes

International Nuclear Information System (INIS)

Fugassa, E.; Gallo, G.; Voci, A.; Cordone, A.

1983-01-01

The ability of hepatocyte monolayers to synthesize RNA was investigated by measuring [3H]orotic acid incorporation into RNA and the total nuclear RNA polymerase activity as a function of the time in culture. The results demonstrate that primary cultures of hepatocytes maintained in a chemically defined serum- and hormone-free medium are able to synthesize RNA actively. This ability increases within the first 2 d of culture, despite the concomitant decrease in [3H]orotic acid uptake, and decreases only after 3 d. Factors such as serum, insulin, and dexamethasone, known to improve maintenance of functional hepatocytes, markedly stimulate the uptake of labeled precursor without apparently affecting the rate of RNA synthesis by cultured cells. It is suggested that the culture of adult rat hepatocytes provides a useful experimental model for the studies of hormonal regulation of transcription in liver

The comparison of free androgen index and serum free testosterone levels in women with hirsutism or polycystic ovary syndrome

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M. Metin Yıldırımkaya

2011-06-01

Full Text Available In many laboratories free testosterone can not be measured, so that free androgen index is suggested instead. The aim of this study was to compare free androgen index and serum free testosterone levels measured by radioimmunoassay in women with hirsutism or polycystic ovary syndrome.Materials and methods: Totally 94 women referred to the polyclinics of Ankara Numune Hospital were retrospectively included. Three patient groups were composed; 55 of hirsutism, 20 of polycystic ovary syndrome and 19 of both hirsutism and polycystic ovary syndrome. Total testosterone and sex hormone binding globuline levels were measured by chemiluminescence method and free testosterone levels were measured by radioimmunoassay. Free androgen index was calculated from total testosterone and sex hormone binding globuline.Results: There was a significant positive correlation between free testosterone and free androgen index in patients with hirsutism, in patients with polycystic ovary syndrome, in patients with hirsutism and polycystic ovary syndrome, and in total patient group [r(hirsutism=0,597, r(PCOS=0,617, r(hirsutism and PCOS=0,779, r(total patient group=0,649, P<0,01].Receiver operating characteristics curves were drawn to assess the diagnostic power of parameters for all patient groups [For hirsutism (n=55 auROC (FT=0,431 auROC (FAI=0,485] [For PCOS (n=20 auROC (FT=0,431 auROC (FAI=0,359] [For hirsutism and PCOS (n=19 auROC (FT=0,676 auROC (FAI=0,669]. In our study, free testosterone and free androgen index were found useful to diagnose ‘hirsutism and polycystic ovary syndrome’ but not others.Conclusion: Free androgen index can be used instead of free testosterone in hirsutism and polycystic ovary syndrome for diagnosis. J Clin Exp Invest 2011;2(2:152-6

Selection of osteoprogenitors from the jaw periosteum by a specific animal-free culture medium.

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Dorothea Alexander

Full Text Available The goal of our research work is to establish mesenchymal osteoprogenitors derived from human jaw periosteum for tissue engineering applications in oral and maxillofacial surgery. For future autologous and/or allogeneic transplantations, some issues must be addressed. On the one hand, animal-free culture conditions have yet to be established. On the other hand, attempts should be undertaken to shorten the in vitro culturing process efficiently. The aim of the present study is to compare and analyze the phenotype of osteoprogenitors from the jaw periosteum under normal FCS-containing and animal-free culture conditions. Therefore, we analyzed the proliferation rates of MesenCult-XF medium (MC- in comparison to DMEM-cultured JPCs. Whereas jaw periosteal cells (JPCs show relatively slow proliferation rates and a fibroblastoid shape under DMEM culture conditions, MC-cultured JPCs diminished their cell size significantly and proliferated rapidly. By live-monitoring measurements of adhesion and proliferation, we made an interesting observation: whereas the proliferation of the MSCA-1(+ subpopulation and the unseparated cell fraction were favored by the animal-free culture medium, the proliferation of the MSCA-1(- subpopulation seemed to be repressed under these conditions. The alkaline phosphatase expression pattern showed similar results under both culture conditions. Comparison of the mineralization capacity revealed an earlier formation of calcium-phosphate precipitates under MC culture conditions; however, the mineralization capacity of the DMEM-cultured cells seemed to be higher. We conclude that the tested animal-free medium is suitable for the in vitro expansion and even for the specific selection of osteoprogenitor cells derived from the jaw periosteum.

Cytoarchitecture in cultured rat neocortex explants

NARCIS (Netherlands)

de Jong, B. M.; Ruijter, J. M.; Romijn, H. J.

1988-01-01

Neocortex explants obtained from 6-day-old rat pups and cultured in a serum-free medium from 5 hr to 13 days in vitro (DIV) show preservation of cytoarchitectural characteristics. Major changes in the size of the explants and their layers occur during the first 2 DIV. A radial arrangement of neurons

Serum Free Light Chains in Neoplastic Monoclonal Gammopathies: Relative Under-Detection of Lambda Dominant Kappa/Lambda Ratio, and Underproduction of Free Lambda Light Chains, as Compared to Kappa Light Chains, in Patients With Neoplastic Monoclonal Gammopathies.

Science.gov (United States)

Lee, Won Sok; Singh, Gurmukh

2018-07-01

Quantitative evaluation of serum free light chains is recommended for the work up of monoclonal gammopathies. Immunoglobulin light chains are generally produced in excess of heavy chains. In patients with monoclonal gammopathy, κ/λ ratio is abnormal less frequently with lambda chain lesions. This study was undertaken to ascertain if the levels of overproduction of the two light chain types and their detection rates are different in patients with neoplastic monoclonal gammopathies. Results of serum protein electrophoresis (SPEP), serum protein immunofixation electrophoresis (SIFE), urine protein electrophoresis (UPEP), urine protein immunofixation electrophoresis (UIFE), and serum free light chain assay (SFLCA) in patients with monoclonal gammopathies were examined retrospectively. The κ/λ ratios were appropriately abnormal more often in kappa chain lesions. Ratios of κ/λ were normal in about 25% of patients with lambda chain lesions in whom free homogenous lambda light chains were detectable in urine. An illustrative case suggests underproduction of free lambda light chains, in some instances. The lower prevalence of lambda dominant κ/λ ratio in lesions with lambda light chains is estimated to be due to relative under-detection of lambda dominant κ/λ ratio in about 25% of the patients and because lambda chains are not produced in as much excess of heavy chains as are kappa chains, in about 5% of the patients. The results question the medical necessity and clinical usefulness of the serum free light chain assay. UPEP/UIFE is under-utilized.

Smart management of sample dilution using an artificial neural network to achieve streamlined processes and saving resources: the automated nephelometric testing of serum free light chain as case study.

Science.gov (United States)

Ialongo, Cristiano; Pieri, Massimo; Bernardini, Sergio

2017-02-01

Saving resources is a paramount issue for the modern laboratory, and new trainable as well as smart technologies can be used to allow the automated instrumentation to manage samples more efficiently in order to achieve streamlined processes. In this regard the serum free light chain (sFLC) testing represents an interesting challenge, as it usually causes using a number of assays before achieving an acceptable result within the analytical range. An artificial neural network based on the multi-layer perceptron (MLP-ANN) was used to infer the starting dilution status of sFLC samples based on the information available through the laboratory information system (LIS). After the learning phase, the MLP-ANN simulation was applied to the nephelometric testing routinely performed in our laboratory on a BN ProSpec® System analyzer (Siemens Helathcare) using the N Latex FLC kit. The MLP-ANN reduced the serum kappa free light chain (κ-FLC) and serum lambda free light chain (λ-FLC) wasted tests by 69.4% and 70.8% with respect to the naïve stepwise dilution scheme used by the automated analyzer, and by 64.9% and 66.9% compared to a "rational" dilution scheme based on a 4-step dilution. Although it was restricted to follow-up samples, the MLP-ANN showed good predictive performance, which alongside the possibility to implement it in any automated system, made it a suitable solution for achieving streamlined laboratory processes and saving resources.

The Effect of Calcusol™ to the Plasma Free Radical and Serum Creatinin in Mus Musculus Nephrolithiasis Model

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A’liyatur Rosyidah

2013-09-01

Full Text Available Calcusol™ is a traditional medicine (jamu made from Tempuyung (Sonchus arvensis extract and is usually used for cure kidney stone disease. However, there has not been any studies which investigate the its mechanism. The aim of this study is to know the effect of Calcusol™ to the plasma free radical and serum creatinin of Mus musculus model for renal calcium-oxalate accumulation. This study is carried out by administration of Porang (Amorphophallus muelleri tuber flour for 3 months with the doses 6 mg/100g BW to induce renal calcium oxalate accumulation and Calcusol™ treatment for 7 days with the doses of 3.3mg/g BW. Group I was used as a control group. Group II was only given porang every day for 3 months. Group III was only given Calcusol™ for 7 days. Group IV were given porang for 3 months then given Calcusol™ for 7 days. Group V were given porang and Calcusol™ simultaneously for 3 months. Porang and CalcusolTM is administrated orally. Blood was collected from the tail of the animal for serum creatinin test and plasma free radical test using TBARS method. The data was analyzed using ANOVA followed by Tukey HSD to compare the means employing SPSS 16.0 for windows. The result of the research shows that the treatment Calcusol™ on mice model for renal calcium oxalate accumulation. The content of MDA at group I, group II, group III, group IV and group V, respectivelyis 0.81±0.5 mg/ml; 2.63±0.8 mg/ml; 0.56±0.5 mg/ml; 2.09±0.9 mg/ml and 0.17±0.17 mg/ml. The content of serum creatinine are 0.15±0.04 mg/dL; 0.13±0.03 mg/dL; 0.12±0.08 mg/dL; 0.11±0.016 mg/dL and 0.14±0.015 mg/dL at group I, group II, group III, Group IV, and group V respectively. This indicates that Calcusol™ decreases plasma free radical production during renal stone formation, while serum creatinin reduced but not significantly changed. Calcusol™ has an effect to decrease free radical during renalstone formation inmice model for calcium oxalate accumulation

The influence of parenteral nitrogen feeding on free amino acid composition of blood serum and hepatic tissue of irradiated rats

International Nuclear Information System (INIS)

Mil'ko, V.I.; Kirichenko, A.V.; Chalaya, L.A.

1985-01-01

A considerable change in the free am ino acid composition of blood serum and hepatic tissued was noted on the 7th and 14th days following total-body X-irradiation of rats with a dose of 2.9 Gy. The total free amino acid content of blood serum increased and that of hepatic tissue decreased by 85% (on an average) as compared to the intact controls. Quantitative changes in the content of individual amino acids were analysed. Polyamine injected enterally for 7 days and parenterally for 3 days after irradiation a the elimination of the postirradiation changes in the amino acid balance

Animal cell cultures: recent achievements and perspectives in the production of biopharmaceuticals.

Science.gov (United States)

Butler, Michael

2005-08-01

There has been a rapid increase in the number and demand for approved biopharmaceuticals produced from animal cell culture processes over the last few years. In part, this has been due to the efficacy of several humanized monoclonal antibodies that are required at large doses for therapeutic use. There have also been several identifiable advances in animal cell technology that has enabled efficient biomanufacture of these products. Gene vector systems allow high specific protein expression and some minimize the undesirable process of gene silencing that may occur in prolonged culture. Characterization of cellular metabolism and physiology has enabled the design of fed-batch and perfusion bioreactor processes that has allowed a significant improvement in product yield, some of which are now approaching 5 g/L. Many of these processes are now being designed in serum-free and animal-component-free media to ensure that products are not contaminated with the adventitious agents found in bovine serum. There are several areas that can be identified that could lead to further improvement in cell culture systems. This includes the down-regulation of apoptosis to enable prolonged cell survival under potentially adverse conditions. The characterization of the critical parameters of glycosylation should enable process control to reduce the heterogeneity of glycoforms so that production processes are consistent. Further improvement may also be made by the identification of glycoforms with enhanced biological activity to enhance clinical efficacy. The ability to produce the ever-increasing number of biopharmaceuticals by animal cell culture is dependent on sufficient bioreactor capacity in the industry. A recent shortfall in available worldwide culture capacity has encouraged commercial activity in contract manufacturing operations. However, some analysts indicate that this still may not be enough and that future manufacturing demand may exceed production capacity as the number

Elevated Concentrations of Serum Immunoglobulin Free Light Chains in Systemic Lupus Erythematosus Patients in Relation to Disease Activity, Inflammatory Status, B Cell Activity and Epstein-Barr Virus Antibodies.

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Anette H Draborg

Full Text Available In this study, we examined the concentration of serum immunoglobulin free light chains (FLCs in systemic lupus erythematosus (SLE patients and investigated its association with various disease parameters in order to evaluate the role of FLCs as a potential biomarker in SLE. Furthermore, FLCs' association with Epstein-Barr virus (EBV antibodies was examined.Using a nephelometric assay, κFLC and λFLC concentrations were quantified in sera from 45 SLE patients and 40 healthy controls. SLE patients with renal insufficiency were excluded in order to preclude high concentrations of serum FLCs due to decreased clearance.Serum FLC concentrations were significantly elevated in SLE patients compared to healthy controls (p<0.0001 also after adjusting for Ig levels (p<0.0001. The concentration of serum FLCs correlated with a global disease activity (SLE disease activity index (SLEDAI score of the SLE patients (r = 0.399, p = 0.007. Furthermore, concentrations of FLCs correlated with titers of dsDNA antibodies (r = 0.383, p = 0.009, and FLC levels and SLEDAI scores correlated in the anti-dsDNA-positive SLE patients, but not in anti-dsDNA-negative SLE patients. Total immunoglobulin (IgG and IgA concentrations correlated with FLC concentrations and elevated FLC levels were additionally shown to associate with the inflammatory marker C-reactive protein and also with complement consumption determined by low C4 in SLE patients. Collectively, results indicated that elevated serum FLCs reflects increased B cell activity in relation to inflammation. SLE patients had an increased seropositivity of EBV-directed antibodies that did not associate with elevated FLC concentrations. An explanation for this could be that serum FLC concentrations reflect the current EBV activity (reactivation whereas EBV-directed antibodies reflect the extent of previous infection/reactivations.SLE patients have elevated concentrations of serum FLCs that correlate with global disease

Micro-bead injection spectroscopy for label-free automated determination of immunoglobulin G in human serum.

Science.gov (United States)

Ramos, Inês I; Magalhães, Luís M; Barreiros, Luisa; Reis, Salette; Lima, José L F C; Segundo, Marcela A

2018-01-01

Immunoglobulin G (IgG) represents the major fraction of antibodies in healthy adult human serum, and deviations from physiological levels are a generic marker of disease corresponding to different pathologies. Therefore, screening methods for IgG evaluation are a valuable aid to diagnostics. The present work proposes a rapid, automatic, and miniaturized method based on UV-vis micro-bead injection spectroscopy (μ-BIS) for the real-time determination of human serum IgG with label-free detection. Relying on attachment of IgG in rec-protein G immobilized in Sepharose 4B, a bioaffinity column is automatically assembled, where IgG is selectively retained and determined by on-column optical density measurement. A "dilution-and-shoot" approach (50 to 200 times) was implemented without further sample treatment because interferences were flushed out of the column upon sample loading, with minimization of carryover and cross-contamination by automatically discarding the sorbent (0.2 mg) after each determination. No interference from human serum albumin at 60 mg mL -1 in undiluted sample was found. The method allowed IgG determination in the range 100-300 μg mL -1 (corresponding to 5.0-60 mg mL -1 in undiluted samples), with a detection limit of 33 μg mL -1 (1.7 mg mL -1 for samples, dilution factor of 50). RSD values were time-to-result decreased from several hours to times, showing the potential of the proposed approach as a point-of-care method. Graphical abstract Micro-Bead Injection Spectroscopy method for real time, automated and label-free determination of total serum human Immunoglobulin G (IgG). The method was designed for Lab-on-Valve (LOV) platforms using a miniaturised protein G bioaffinity separative approach. IgG are separated from serum matrix components upon quantification with low non-specific binding in less than 5 min.

Development of microLIPS (Luciferase Immunoprecipitation Systems): a novel microfluidic assay for rapid serum antibody detection

Science.gov (United States)

Chandrangsu, Matt; Burbelo, Peter D.; Iadarola, Michael J.; Smith, Paul D.; Morgan, Nicole Y.

2012-06-01

There is considerable interest in the development of rapid, point-of-care antibody detection for the diagnosis of infectious and auto-immune diseases. In this paper, we present work on the development of a self-contained microfluidic format for the Luciferase Immunoprecipitation Systems (LIPS) assay. Whereas the majority of immunoassays for antigen-specific antibodies employ either bacteria- or yeast-expressed proteins and require the use of secondary antibodies, the LIPS technique uses a fusion protein comprised of a Renilla luciferase reporter and the antigen of interest produced via mammalian cell culture, ensuring the addition of mammalian post-translational modifications. Patient serum is mixed with the fusion protein and passed over immobilized Protein A/G; after washing, the only remaining luciferase-tagged antigens are those retained by specific antibodies. These can be quantitatively measured using chemiluminescence upon the introduction of coelenterazine. The assay has been successfully employed for a wide variety of diseases in a microwell format. We report on a recent demonstration of rapid HSV-2 diagnosis with the LIPS assay in a microfluidic format, using one microliter of serum and obtaining results in under ten minutes. We will also discuss recent progress on two fronts, both aimed at the deployment of this technology in the field: first, simplifying assay operation through the automation of flow control using power-free means; and second, efforts to increase signal levels, primarily through strategies to increase antibody binding capacity, in order to move towards portable battery powered electronics.

Agency Beliefs Over Time and Across Cultures: Free Will Beliefs Predict Higher Job Satisfaction

Science.gov (United States)

Feldman, Gilad; Farh, Jiing-Lih; Wong, Kin Fai Ellick

2017-01-01

In three studies, we examined the relationship between free will beliefs and job satisfaction over time and across cultures. Study 1 examined 252 Taiwanese real-estate agents over a 3-months period. Study 2 examined job satisfaction for 137 American workers on an online labor market over a 6-months period. Study 3 extended to a large sample of 14,062 employees from 16 countries and examined country-level moderators. We found a consistent positive relationship between the belief in free will and job satisfaction. The relationship was above and beyond other agency constructs (Study 2), mediated by perceived autonomy (Studies 2-3), and stronger in countries with a higher national endorsement of the belief in free will (Study 3). We conclude that free-will beliefs predict outcomes over time and across cultures beyond other agency constructs. We call for more cross-cultural and longitudinal studies examining free-will beliefs as predictors of real-life outcomes. PMID:29191084

Effects of rearing systems on laying performance, egg quality, and serum biochemistry of Xianju chickens in summer.

Science.gov (United States)

Dong, X Y; Yin, Z Z; Ma, Y Z; Cao, H Y; Dong, D J

2017-09-01

The objective of this study was to compare the laying performance, egg quality, and serum biochemistry of hens maintained in conventional cage rearing system (CRS), flat net-rearing system (NRS), and free range system (FRS) under summer conditions. Indigenous Xianju chickens (n = 540) were randomly allocated into cages or pens of rearing system groups, within each system there were 5 replicates with 36 hens in each replicate. The experiment lasted between 21 and 29 wk of age. Hen-day egg production (P = 0.00) and egg mass (P = 0.00) were higher in the CRS but were similar in the NRS and FRS. Lowest egg weight (P = 0.02), yolk weight (P = 0.00) and yolk ratio (P = 0.01), and feed intake (P = 0.01) were observed from the FRS, whereas lowest feed conversion ratio (FCR) was recorded from the CRS (P = 0.01). Rearing systems had negligible effect on egg quality. Serum Ca (P = 0.04) and total protein (P = 0.03) levels were found to be higher in the CRS but were lower in the FRS. Serum levels of glucose (P = 0.01), cholesterol (P = 0.00), and triglyceride (P = 0.00) in the CRS increased compared with the NRS and FRS groups, whereas serum levels of high density lipoprotein cholesterol (HDL-C; P = 0.01) in the CRS decreased. It can be concluded that under summer conditions, Xianju chickens from CRS had an advantage in terms of productivity parameters, but exhibited higher levels of serum lipids and glucose. © 2017 Poultry Science Association Inc.

Differentially expressed genes: OCT-4, SOX2, STAT3, CDH1 and CDH2, in cultured mesenchymal stem cells challenged with serum of women with endometriosis

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Ehab Salama

2018-06-01

Full Text Available Endometriosis is a common chronic gynecological disorder defined as the presence of ectopic functional endometrial tissues, outside uterine cavity, primarily on the pelvic peritoneum and the ovaries. Several studies revealed a correlation between aberrant stem-cell activity in the endometrium and endometriosis. Yet the molecular and cellular behaviors of mesnchymal stem cells in development of endometriosis are hampered by lack of invitro experiments. Our aim was to explore morphological and molecular changes associated with mesenchymal stem cells (MSCs exposition to serum derived from women with severe endometriosis. Two cell cultures of MSCs isolated from endometrial tissues of two endometriosis-free women. Each cell culture was treated individually with the serum of women with endometriosis (experimental group/n = 7, and serum of women without endometriosis (control group/ n = 4 for 14 days. Quantitative Real-Time PCR was performed later to reveal expression of OCT-4, CDH1 and CDH2, STAT3 and SOX2 genes. Morphologically, cells showed no significant changes. However from molecular point of view, we found increased expression in OCT-4, CDH1 and CDH2. For STAT3 and SOX2 we did not find a significant difference. This study shows that endometriosis serum induced molecular changes in human endometrial MSCs (EnMSCs that might be related to altered cell behavior which may be a step in differentiation that may be completed invivo by other factors to complete the process of transition. Further researches are needed for optimization to reach differentiation. Keywords: Endometriosis, Mesnchymal stem cells, OCT-4, SOX2, STAT3, E-cadherin, N-cadherin

Changes during hibernation in different phospholipid and free and esterified cholesterol serum levels in black bears

Science.gov (United States)

Chauhan, V.; Sheikh, A.; Chauhan, A.; Tsiouris, J.; Malik, M.; Vaughan, M.

2002-01-01

During hibernation, fat is known to be the preferred source of energy. A detailed analysis of different phospholipids, as well as free and esterified cholesterol, was conducted to investigate lipid abnormalities during hibernation. The levels of total phospholipids and total cholesterol in the serum of black bears were found to increase significantly in hibernation as compared with the active state. Both free and esterified cholesterol were increased in the hibernating state in comparison with the active state (P biologie mole??culaire. All rights reserved.

Create, share and learn. Experiences with free software, free culture and collaboration in formal and non formal education in Colombia

OpenAIRE

Medina Cardona, Luis Fernando

2012-01-01

New media technologies, specially software have been of great impact in modern society.The combination of computer/software/networks as creative machines is present in everyday life. This article is focused in this interactions specially from the perspective of free open source software (FOSS). In doing so, the influence of its values is traced from the original hacker culture ethics to the free software four freedoms, showing a software explained culture as in the software studies discipline...

Development of bovine embryos cultured in CR1aa and IVD101 media using different oxygen tensions and culture systems.

Science.gov (United States)

Somfai, Tamás; Inaba, Yasushi; Aikawa, Yoshio; Ohtake, Masaki; Kobayashi, Shuji; Konishi, Kazuyuki; Nagai, Takashi; Imai, Kei

2010-12-01

The aim of the present study was to optimise the culture conditions for the in vitro production of bovine embryos. The development of in vitro fertilised bovine oocytes in CR1aa supplemented with 5% calf serum and IVD101 culture media were compared using traditional microdrops and Well of the Well (WOW) culture systems either under 5% or 20% oxygen tension. After 7 days of culture, a significantly higher blastocyst formation rate was obtained for embryos cultured in CR1aa medium compared to those cultured in IVD101, irrespective of O2 tensions and culture systems. The blastocyst formation in IVD101 was suppressed under 20% O2 compared to 5% O2 . Despite their similar total cell numbers, higher rates of inner cell mass (ICM) cells were observed in blastocysts developed in IVD101 medium than in those developed in CR1aa, irrespective of O2 tensions. There was no significant difference in blastocyst formation, total, ICM and trophectoderm (TE) cell numbers between embryos obtained by microdrop and WOW culture systems irrespective of the culture media and O2 tensions used. In conclusion, CR1aa resulted in higher blastocyst formation rates irrespective of O2 tension, whereas IVD101 supported blastocyst formation only under low O2 levels but enhanced the proliferation of ICM cells.

Effects of culture media conditions on production of eggs fertilized in vitro of embryos derived from ovary of high grade Hanwoo

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Jun Young Lee

2016-03-01

Full Text Available Abstract Background This study was investigated the effects of culture media conditions on production of eggs fertilized in vitro of embryos from ovaries of high grade Korean native cow, Hanwoo. Methods The IVMD 101 and IVF 100 were used for in vitro maturation of selected Hanwoo oocytes and In vitro embryo culture after in vitro fertilization, respectively. The IVMD 101 and IVD 101 were used for in vitro culture and completely free of serum. Results The cleavage rates of 2-cell embryos in reference to Hanwoo oocytes were 86.7, 92.9 , and 90.1 % in the control group, IVDM101 medium and IVD101 medium, respectively which indicates that the IVDM101 medium and IVD101 medium may result favorable outcomes. The in vitro development rates of blastocysts were 12.4, 38.4 and 32.4 % in the control group, serum free IVMD101 medium and IVD101 medium, respectively. For hatched blastocysts, it was 5.3, 33.9, and 28.6 % in the control group, serum free IVMD101 medium and IVD101 medium, respectively. Hence, more favorable results were expected for the hatched blastocysts in which the IVMD101 medium and IVD101 medium were used than the control group. Average cell numbers of blastocysts were 128.3, 165.7, and 163.6 in the groups of TCM-199 + 10 % FBS medium, IVMD 101 medium, and IVD 101 medium, respectively which clearly show that the IVMD 101 and IVD 101 medium consequence significantly higher cell numbers compared to the control group (i.e., TCM-199 + 10 % FBS medium. Pregnancy rate after embryo transfer was 39.6 % when the serum free medium was used which is higher than that of the medium supplemented with serum (32.8 %. In addition, stillbirth rates were 4.9 % in the group of serum free medium whereas it was 13.6 % in the serum supplemented medium (13.6 %. Conclusions Taken altogether, serum free media, the IVMD 101 and IVD 101 represented more favorable results in the embryo development rate of embryos, cell numbers of blastocyst, and

Enhanced production and isotope enrichment of recombinant glycoproteins produced in cultured mammalian cells

Energy Technology Data Exchange (ETDEWEB)

Skelton, David; Goodyear, Abbey [Florida State University, Department of Chemistry and Biochemistry (United States); Ni, DaQun; Walton, Wendy J.; Rolle, Myron; Hare, Joan T. [Florida State University, Institute of Molecular Biophysics (United States); Logan, Timothy M., E-mail: tlogan@fsu.ed [Florida State University, Department of Chemistry and Biochemistry (United States)

2010-10-15

NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant protein yield, and isotope enrichment. We demonstrate the method by producing partially enriched recombinant Thy1 glycoprotein from Lec1 Chinese hamster ovary (CHO) cells using U-{sup 13}C-glucose and {sup 15}N-glutamate as labeled precursors. This study suggests that uniformly {sup 15}N,{sup 13}C-labeled recombinant proteins may be produced in cultured mammalian cells starting from a mixture of labeled essential amino acids, glucose, and glutamate.

Enhanced production and isotope enrichment of recombinant glycoproteins produced in cultured mammalian cells

International Nuclear Information System (INIS)

Skelton, David; Goodyear, Abbey; Ni, DaQun; Walton, Wendy J.; Rolle, Myron; Hare, Joan T.; Logan, Timothy M.

2010-01-01

NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant protein yield, and isotope enrichment. We demonstrate the method by producing partially enriched recombinant Thy1 glycoprotein from Lec1 Chinese hamster ovary (CHO) cells using U- 13 C-glucose and 15 N-glutamate as labeled precursors. This study suggests that uniformly 15 N, 13 C-labeled recombinant proteins may be produced in cultured mammalian cells starting from a mixture of labeled essential amino acids, glucose, and glutamate.

Detection of atomic scale changes in the free volume void size of three-dimensional colorectal cancer cell culture using positron annihilation lifetime spectroscopy.

Science.gov (United States)

Axpe, Eneko; Lopez-Euba, Tamara; Castellanos-Rubio, Ainara; Merida, David; Garcia, Jose Angel; Plaza-Izurieta, Leticia; Fernandez-Jimenez, Nora; Plazaola, Fernando; Bilbao, Jose Ramon

2014-01-01

Positron annihilation lifetime spectroscopy (PALS) provides a direct measurement of the free volume void sizes in polymers and biological systems. This free volume is critical in explaining and understanding physical and mechanical properties of polymers. Moreover, PALS has been recently proposed as a potential tool in detecting cancer at early stages, probing the differences in the subnanometer scale free volume voids between cancerous/healthy skin samples of the same patient. Despite several investigations on free volume in complex cancerous tissues, no positron annihilation studies of living cancer cell cultures have been reported. We demonstrate that PALS can be applied to the study in human living 3D cell cultures. The technique is also capable to detect atomic scale changes in the size of the free volume voids due to the biological responses to TGF-β. PALS may be developed to characterize the effect of different culture conditions in the free volume voids of cells grown in vitro.

Serum Hydroxyl Radical Scavenging Capacity as Quantified with Iron-Free Hydroxyl Radical Source

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Endo, Nobuyuki; Oowada, Shigeru; Sueishi, Yoshimi; Shimmei, Masashi; Makino, Keisuke; Fujii, Hirotada; Kotake, Yashige

2009-01-01

We have developed a simple ESR spin trapping based method for hydroxyl (OH) radical scavenging-capacity determination, using iron-free OH radical source. Instead of the widely used Fenton reaction, a short (typically 5 seconds) in situ UV-photolysis of a dilute hydrogen peroxide aqueous solution was employed to generate reproducible amounts of OH radicals. ESR spin trapping was applied to quantify OH radicals; the decrease in the OH radical level due to the specimen’s scavenging activity was converted into the OH radical scavenging capacity (rate). The validity of the method was confirmed in pure antioxidants, and the agreement with the previous data was satisfactory. In the second half of this work, the new method was applied to the sera of chronic renal failure (CRF) patients. We show for the first time that after hemodialysis, OH radical scavenging capacity of the CRF serum was restored to the level of healthy control. This method is simple and rapid, and the low concentration hydrogen peroxide is the only chemical added to the system, that could eliminate the complexity of iron-involved Fenton reactions or the use of the pulse-radiolysis system. PMID:19794928

Evaluation of in vitro culture systems for the maintenance of microfilariae and infective larvae of Loa loa.

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Zofou, Denis; Fombad, Fanny Fri; Gandjui, Narcisse V T; Njouendou, Abdel Jelil; Kengne-Ouafo, Arnaud Jonas; Chounna Ndongmo, Patrick W; Datchoua-Poutcheu, Fabrice R; Enyong, Peter A; Bita, Dizzle Tayong; Taylor, Mark J; Turner, Joseph D; Wanji, Samuel

2018-05-02

Suitable and scalable in vitro culture conditions for parasite maintenance are needed to foster drug research for loiasis, one of the neglected tropical diseases which has attracted only limited attention over recent years, despite having important public health impacts. The present work aims to develop adequate in vitro culture systems for drug screening against both microfilariae (mf) and infective third-stage larvae (L3) of Loa loa. In vitro culture conditions were evaluated by varying three basic culture media: Roswell Park Memorial Institute (RPMI-1640), Dulbecco's modified Eagle's medium (DMEM) and Iscove's modified Dulbecco's medium (IMDM); four sera/proteins: newborn calf serum (NCS), foetal bovine serum (FBS), bovine serum albumin (BSA) and the lipid-enriched BSA (AlbuMax® II, ALB); and co-culture with the Monkey Kidney Epithelial Cell line (LLC-MK2) as a feeder layer. The various culture systems were tested on both mf and L3, using survival (% motile), motility (T 90 = mean duration (days) at which at least 90% of parasites were fully active) and moulting rates of L3 as the major criteria. The general linear model regression analysis was performed to assess the contribution of each variable on the viability of Loa loa L3 and microfilarie. All statistical tests were performed at 95% confidence interval. Of the three different media tested, DMEM and IMDM were the most suitable sustaining the maintenance of both L. loa L3 and mf. IMDM alone could sustain L3 for more than 5 days (T 90 = 6.5 ± 1.1 day). Serum supplements and LLC-MK2 co-cultures significantly improved the survival of parasites in DMEM and IMDM. In co-cultures with LLC-MK2 cells, L. loa mf were maintained in each of the three basic media (T 90 of 16.4-19.5 days) without any serum supplement. The most effective culture systems promoting significant moulting rate of L3 into L4 (at least 25%) with substantial maintenance time were: DMEM + BSA, DMEM + NCS, DMEM-AlbuMax®II, DMEM + FBS all in co-culture

Diagnostic significance of the serum thyroid hormone indicies in various thyroid diseases

International Nuclear Information System (INIS)

Han, B.H.; Ko, S.M.; Yoon, S.R.; Ro, H.K.

1980-01-01

In an attempt to evaluate the diagnostic significance of the serum thyroid hormones in various thyroid function states, the author measured serum T 3 uptake, serum T 3 , serum T 4 , serum free T 4 and free T 4 index in 27 cases of normal subjects, 11 cases of hypothyroidism, 152 cases of euthyroidism and 81 cases of hyperthyroidism by the radioimmunoassay method. The results were as follows: 1) The ranges of serum thyroid hormones in normal subjects were, serum T 3 uptake; 27.4-42.1%, serum T 3 ; 93-245 ng/dl, serum T 4 ; 4.08-12.9 ng/dl and serum free T 4 ; 0.57-1.53 ng/dl (M+-2 S.D.). 2) Free T 4 index and serum T 4 show relatively high diagnostic value in euthyroidism group, and serum T 3 and T 4 in hypothyroidism group, while serum T 3 , free T 4 and T 4 show relatively high diagnostic value in hyperthyroidism group. 3) There were significant correlation between free T 4 index and serum T 4 (r=0.68) and between free T 4 index and serum free T 4 (r=0.67) in hyperthyroidism group. (author)

Differentiating Graves' disease from subacute thyroiditis using ratio of serum free triiodothyronine to free thyroxine.

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Sriphrapradang, Chutintorn; Bhasipol, Adikan

2016-09-01

The measurement of free thyroid hormone, instead of the total form, is more commonly used in current practice. We aimed to evaluate the usefulness of the ratio of serum free triiodothyronine (FT3, pg/mL) to free thyroxine (FT4, ng/dL) for differentiating Graves' disease from subacute thyroiditis. Medical records of thyrotoxic patients aged >15 years who had measurement of FT3, FT4 and thyrotropin on the first diagnosis of thyrotoxicosis before initiating treatment were retrospectively reviewed. Data were collected from all clinics, and were not limited to the endocrine clinic. Pregnant women were excluded. A total of 548 patients (468 with Graves' disease, 40 with subacute thyroiditis and 40 with toxic adenoma/multinodular goiter) were recruited. Mean age was 43.9 ± 15.4 years. Most were female 434 (79.2%), and goiter was present in 55.3%. Prevalence of T3-toxicosis and T4-toxicosis were 5.6% and 6.6%, respectively. Mean FT3/FT4 ratios were 4.62 ± 2 (10(-2) pg/ng) in patients with Graves' disease and 2.73 ± 0.5 in subacute thyroiditis. The area under the ROC curve of the FT3/FT4 ratio for diagnosis of Graves' disease was 0.83 (95%CI, 0.76-0.91). Cutoff level of this ratio >4.4 offered sensitivity of 47.2% and specificity of 92.8%. FT3/FT4 ratio of >4.4 (10(-2) pg/ng) may help in differentiating the cause of thyrotoxicosis.

Production of virus-free orchid Cymbidium aloifolium (L.) Sw. by various tissue culture techniques.

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Pradhan, Shreeti; Regmi, Tripti; Ranjit, Mukunda; Pant, Bijaya

2016-10-01

Orchids are affected by many viruses resulting in poor growth, yield and quality, and an overall decline in population. Cymbidium mosaic virus (CymMV) is one of the common orchid viruses found in Cymbidium species but it infects different orchid genera. In this study Cymbidium aloifolium was propagated in vitro using MS medium at different strength (1.0, ½, and ¼) with or without 0.5 mg/l BAP (6-benzylaminopurine) and 0.5 mg/l NAA (Naphthalene acetic acid). To provide disease-free planting material, source plant for in vitro propagation needs to be screened for pathogenic viruses. In the present study, in vivo -grown source (mother) plants and tissue culture-derived plants of C. aloifolium were tested for CymMV virus using Double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA). All the tissue cultured plants were found to be 100% virus-free whereas the in vivo grown source plants were highly affected by CymMV virus (83.33%). The virus-free in vitro plantlets were multiplied in large scale and then acclimatized on earthen pot containing a mixture of cocopeat, litter and clay in the ratio of 3:2:1. Eighty five percent of acclimatized plantlets survived making this method an efficient mass production system for high quality virus-free C. aloifolium for commercial floriculture and germplasm preservation.

Production of virus-free orchid Cymbidium aloifolium (L. Sw. by various tissue culture techniques

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Shreeti Pradhan

2016-10-01

Full Text Available Orchids are affected by many viruses resulting in poor growth, yield and quality, and an overall decline in population. Cymbidium mosaic virus (CymMV is one of the common orchid viruses found in Cymbidium species but it infects different orchid genera. In this study Cymbidium aloifolium was propagated in vitro using MS medium at different strength (1.0, ½, and ¼ with or without 0.5 mg/l BAP (6-benzylaminopurine and 0.5 mg/l NAA (Naphthalene acetic acid. To provide disease-free planting material, source plant for in vitro propagation needs to be screened for pathogenic viruses. In the present study, in vivo-grown source (mother plants and tissue culture-derived plants of C. aloifolium were tested for CymMV virus using Double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA. All the tissue cultured plants were found to be 100% virus-free whereas the in vivo grown source plants were highly affected by CymMV virus (83.33%. The virus-free in vitro plantlets were multiplied in large scale and then acclimatized on earthen pot containing a mixture of cocopeat, litter and clay in the ratio of 3:2:1. Eighty five percent of acclimatized plantlets survived making this method an efficient mass production system for high quality virus-free C. aloifolium for commercial floriculture and germplasm preservation. Keywords: Biological sciences, Plant biology

Weights, hematology and serum chemistry of seven species of free-ranging tropical pelagic seabirds

Science.gov (United States)

Work, Thierry M.

1996-01-01

I established reference values for weight, hematology, and serum chemistry for seven species of free-ranging Hawaiian tropical pelagic seabirds comprising three orders (Procellariiformes, Pelecaniformes, Charadriiformes) and six families (Procellariidae, Phaethontidae, Diomedeidae, Sulidae, Fregatidae, and Laridae). Species examined included 84 Hawaiian dark-rumped petrels (Pterodoma phaeopygia), 90 wedge-tailed shearwaters (Puffinus pacificus), 151 Laysan albatrosses (Diomedea immutabilis), 69 red-footed boobies (Sula sula), 154 red-tailed tropicbirds (Phaeton rubricauda), 90 great frigatebirds (Fregata minor), and 72 sooty terns (Sterna fuscata). Hematocrit, total plasma solids, total and differential white cell counts, serum glucose, calcium, phosphorus, uric acid, total protein, albumin, globulin, aspartate aminotransferase and creatinine phosphokinase were analyzed. Among and within species, hematology and chemistry values varied with age, sex, season, and island of collection. Despite this variation, order-wide trends were observed.

Enacting cultural boundaries in French and German diphtheria serum research.

Science.gov (United States)

Klöppel, Ulrike

2008-06-01

The experimental development of a therapeutic serum against diphtheria between 1891 and 1894 was characterized by a scientific competition that pitted Emil Behring from the Institute for Infectious Diseases in Berlin against Emile Roux and Elie Metschnikoff from the Pasteur Institute in Paris. In general, their competition can be regarded as an extension of the fundamental differences that separated the research schools of Robert Koch and Louis Pasteur. However, to characterize the competition for a diphtheria-serum as "national rivalry" fails to account adequately for the mutual adoption of experimental practices by the Berlin and Parisian protagonists, whose contributions to the development of a therapeutic serum were intertwined in complex ways. Nor can it be characterized as "cooperation," given their fierce public disputes over scientific concepts and the fact that these disputes also shaped the peculiarities of the experimental procedures in Berlin and Paris. A close analysis reveals a complex picture of the dynamic interaction between the conceptual and experimental activities of Behring, Roux, and Metschnikoff- interaction that defined as well as bridged the "French" and "Prussian" experimental systems of diphtheria-serum research.

Interrelationships of serum testosterone and free testosterone index with FFM and strength in aging men.

Science.gov (United States)

Roy, Tracey Ann; Blackman, Marc R; Harman, S Mitchell; Tobin, Jordan D; Schrager, Matthew; Metter, E Jeffery

2002-08-01

Muscle mass and strength losses during aging may be associated with declining levels of serum testosterone (T) in men. Few studies have shown a direct relationship between T and muscle mass and strength. Subjects were 262 men, aged 24-90 yr, from the Baltimore Longitudinal Study of Aging, who had T and sex hormone-binding globulin sex hormone-binding globulin (SHBG) measurements, from which the free T index (FTI) was calculated (T/SHBG) from serum samples collected longitudinally since 1963, total body fat mass and arm and leg fat-free mass (FFM) by dual-energy X-ray absorptiometry and arm and leg strength by dynanomometry. Mixed-effects models estimated T and FTI at the time of mass and strength measurements. Age, total body fat, arm and leg FFM, T, and FTI were significantly associated with concentric and eccentric strength. FTI, not T, was modestly, but directly, related to arm and leg strength after fat, arm and leg FFM, height, and age were accounted for and indirectly through body mass. FTI is a better predictor of arm and leg strength than T in aging men.

Mesenchymal Stromal Cells Cultured in Serum from Heart Failure Patients Are More Resistant to Simulated Chronic and Acute Stress

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Timo Z. Nazari-Shafti

2018-01-01

Full Text Available Despite regulatory issues surrounding the use of animal-derived cell culture supplements, most clinical cardiac cell therapy trials using mesenchymal stromal cells (MSCs still rely on fetal bovine serum (FBS for cell expansion before transplantation. We sought to investigate the effect of human serum from heart failure patients (HFS on cord blood MSCs (CB-MSCs during short-term culture under regular conditions and during simulated acute and chronic stress. Cell survival, proliferation, metabolic activity, and apoptosis were quantified, and gene expression profiles of selected apoptosis and cell cycle regulators were determined. Compared to FBS, HFS and serum from healthy donors (CS showed similar effects by substantially increasing cell survival during chronic and acute stress and by increasing cell yields 5 days after acute stress. Shortly after the termination of acute stress, both HFS and CS resulted in a marked decrease in apoptotic cells. Transcriptome analysis suggested a decrease in TNF-mediated induction of caspases and decreased activation of mitochondrial apoptosis. Our data confirm that human serum from both healthy donors and heart failure patients results in increased cell yields and increased resistance to cellular stress signals. Therefore, we consider autologous serum a valid alternative to FBS in cell-based therapies addressing severe heart disease.

Serum Iron Protects from Renal Postischemic Injury.

Science.gov (United States)

Vaugier, Céline; Amano, Mariane T; Chemouny, Jonathan M; Dussiot, Michael; Berrou, Claire; Matignon, Marie; Ben Mkaddem, Sanae; Wang, Pamella H M; Fricot, Aurélie; Maciel, Thiago T; Grapton, Damien; Mathieu, Jacques R R; Beaumont, Carole; Peraldi, Marie-Noëlle; Peyssonnaux, Carole; Mesnard, Laurent; Daugas, Eric; Vrtovsnik, François; Monteiro, Renato C; Hermine, Olivier; Ginzburg, Yelena Z; Benhamou, Marc; Camara, Niels O S; Flamant, Martin; Moura, Ivan C

2017-12-01

Renal transplants remain a medical challenge, because the parameters governing allograft outcome are incompletely identified. Here, we investigated the role of serum iron in the sterile inflammation that follows kidney ischemia-reperfusion injury. In a retrospective cohort study of renal allograft recipients ( n =169), increased baseline levels of serum ferritin reliably predicted a positive outcome for allografts, particularly in elderly patients. In mice, systemic iron overload protected against renal ischemia-reperfusion injury-associated sterile inflammation. Furthermore, chronic iron injection in mice prevented macrophage recruitment after inflammatory stimuli. Macrophages cultured in high-iron conditions had reduced responses to Toll-like receptor-2, -3, and -4 agonists, which associated with decreased reactive oxygen species production, increased nuclear localization of the NRF2 transcription factor, increased expression of the NRF2-related antioxidant response genes, and limited NF- κ B and proinflammatory signaling. In macrophage-depleted animals, the infusion of macrophages cultured in high-iron conditions did not reconstitute AKI after ischemia-reperfusion, whereas macrophages cultured in physiologic iron conditions did. These findings identify serum iron as a critical protective factor in renal allograft outcome. Increasing serum iron levels in patients may thus improve prognosis of renal transplants. Copyright © 2017 by the American Society of Nephrology.

Phylogenetically distinct bacteria involve extensive dechlorination of aroclor 1260 in sediment-free cultures.

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Shanquan Wang

Full Text Available Microbial reductive dechlorination of the persistent polychlorinated biphenyls (PCBs is attracting much attention in cleanup of the contaminated environment. Nevertheless, most PCB dechlorinating cultures require presence of sediment or sediment substitutes to maintain their dechlorination activities which hinders subsequent bacterial enrichment and isolation processes. The information on enriching sediment-free PCB dechlorinating cultures is still limited. In this study, 18 microcosms established with soils and sediments were screened for their dechlorination activities on a PCB mixture - Aroclor 1260. After one year of incubation, 10 out of 18 microcosms showed significant PCB dechlorination with distinct dechlorination patterns (e.g., Process H, N and T classified based on profiles of PCB congeners loss and new congeners formation. Through serial transfers in defined medium, six sediment-free PCB dechlorinating cultures (i.e., CW-4, CG-1, CG-3, CG-4, CG-5 and SG-1 were obtained without amending any sediment or sediment-substitutes. PCB dechlorination Process H was the most frequently observed dechlorination pattern, which was found in four sediment-free cultures (CW-4, CG-3, CG-4 and SG-1. Sediment-free culture CG-5 showed the most extensive PCB dechlorination among the six cultures, which was mediated by Process N, resulting in the accumulation of penta- (e.g., 236-24-CB and tetra-chlorobiphenyls (tetra-CBs (e.g., 24-24-CB, 24-25-CB, 24-26-CB and 25-26-CB via dechlorinating 30.44% hepta-CBs and 59.12% hexa-CBs after three months of incubation. For culture CG-1, dechlorinators mainly attacked double flanked meta-chlorines and partially ortho-chlorines, which might represent a novel dechlorination pattern. Phylogenetic analysis showed distinct affiliation of PCB dechlorinators in the microcosms, including Dehalogenimonas and Dehalococcoides species. This study broadens our knowledge in microbial reductive dechlorination of PCBs, and provides

Nutritional manipulation of primate retinas, I: effects of lutein or zeaxanthin supplements on serum and macular pigment in xanthophyll-free rhesus monkeys.

Science.gov (United States)

Neuringer, Martha; Sandstrom, Marita M; Johnson, Elizabeth J; Snodderly, D Max

2004-09-01

The xanthophylls lutein (L) and zeaxanthin (Z) are the primary components of macular pigment (MP) and may protect the macula from age-related degeneration (AMD). In this study, L or Z was fed to rhesus monkeys reared on xanthophyll-free diets to follow the accumulation of serum carotenoids and MP over time. Eighteen rhesus monkeys were fed xanthophyll-free semipurified diets from birth until 7 to 16 years. The diets of six were then supplemented with pure L and six with pure Z at 3.9 micromol/kg per day (2.2 mg/kg per day) for 24 to 56 weeks. At baseline and 4- to 12-week intervals during supplementation, serum carotenoids were measured by HPLC, and MP density was estimated by two-wavelength reflectometry. Serum carotenoids and MP were also measured in monkeys fed a stock diet. Monkeys fed xanthophyll-free diets had no L or Z in serum and no detectable MP. During supplementation, serum L or Z increased rapidly over the first 4 weeks and from 16 weeks onward maintained similar levels, both several times higher than in stock-diet-fed monkeys. The central peak of MP optical density increased to a relatively steady level by 24 to 32 weeks in both L- and Z-fed groups. Rhesus monkeys fed a stock diet had lower blood concentrations of L than those found in humans and other nonhuman primates. Rhesus monkeys respond to either dietary L or Z supplementation with increases in serum xanthophylls and MP, even after life-long xanthophyll deficiency. These animals provide a potential model to study mechanisms of protection from AMD. Copyright Association for Research in Vision and Ophthalmology

Serum complexed and free prostate-specific antigen (PSA) for the diagnosis of the polycystic ovarian syndrome (PCOS).

Science.gov (United States)

Diamandis, Eleftherios P; Stanczyk, Frank Z; Wheeler, Sarah; Mathew, Anu; Stengelin, Martin; Nikolenko, Galina; Glezer, Eli N; Brown, Marshall D; Zheng, Yingye; Chen, Yen-Hao; Wu, Hsiao-Li; Azziz, Ricardo

2017-10-26

Polycystic ovarian syndrome (PCOS) is a common cause of reproductive and metabolic dysfunction. We hypothesized that serum prostate-specific antigen (PSA) may constitute a new biomarker for hyperandrogenism in PCOS. We conducted a cross-sectional study of 45 women with PCOS and 40 controls. Serum from these women was analyzed for androgenic steroids and for complexed PSA (cPSA) and free PSA (fPSA) with a novel fifth- generation assay with a sensitivity of ~10 fg/mL for cPSA and 140 fg/mL for fPSA. cPSA and fPSA levels were about three times higher in PCOS compared to controls. However, in PCOS, cPSA and fPSA did not differ according to waist-to-hip ratio, Ferriman-Gallwey score, or degree of hyperandrogenemia or oligo-ovulation. In PCOS and control women, serum cPSA and fPSA levels were highly correlated with each other, and with free and total testosterone levels, but not with other hormones. Adjusting for age, body mass index (BMI) and race, cPSA was significantly associated with PCOS, with an odds ratio (OR) of 5.67 (95% confidence interval [CI]: 1.86, 22.0). The OR of PCOS for fPSA was 7.04 (95% CI: 1.65, 40.4). A multivariate model that included age, BMI, race and cPSA yielded an area-under-the-receiver-operating-characteristic curve of 0.89. Serum cPSA and fPSA are novel biomarkers for hyperandrogenism in PCOS and may have value for disease diagnosis.

Human platelet lysate is a successful alternative serum supplement for propagation of monocyte-derived dendritic cells.

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Švajger, Urban

2017-04-01

Clinical protocols for dendritic cell (DC) generation from monocytes require the use of animal serum-free supplements. Serum-free media can also require up to 1% of serum supplementation. In addition, recommendations based on the 3Rs (Refinement, Reduction, Replacement) principle also recommend the use of non-animal sera in in vitro studies. The aim of this study was to explore the potential use of platelet lysate (PL) for generation of optimally differentiated DCs from monocytes. Cells were isolated from buffy coats from healthy volunteers using immunomagnetic selection. DCs were differentiated in RPMI1640 supplemented with either 10% fetal bovine serum (FBS), 10% AB serum or 10% PL with the addition of granulocyte monocyte colony stimulating factor and interleukin-4. Generated DCs were assessed for their morphology, viability, endocytotic capacity, surface phenotype (immature, mature and tolerogenic DCs) and activation of important signaling pathways. DC function was evaluated on the basis of their allostimulatory capacity, cytokine profile and ability to induce different T-helper subsets. DCs generated with PL displayed normal viability, morphology and endocytotic capacity. Their differentiation and maturation phenotype was comparable to FBS-cultured DCs. They showed functional plasticity and up-regulated tolerogenic markers in response to their environment. PL-cultured mature DCs displayed unhindered allostimulatory potential and the capacity to induce Th1 responses. The use of PL allowed for activation of crucial signaling proteins associated with DC differentiation and maturation. This study demonstrates for the first time that human PL represents a successful alternative to FBS in differentiation of DCs from monocytes. DCs display the major phenotypic and functional characteristics compared with existing culture protocols. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Non-Mulberry and Mulberry Silk Protein Sericins as Potential Media Supplement for Animal Cell Culture

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Neety Sahu

2016-01-01

Full Text Available Silk protein sericins, in the recent years, find application in cosmetics and pharmaceuticals and as biomaterials. We investigate the potential of sericin, extracted from both mulberry Bombyx mori and different non-mulberry sources, namely, tropical tasar, Antheraea mylitta; muga, Antheraea assama; and eri, Samia ricini, as growth supplement in serum-free culture medium. Sericin supplemented media containing different concentrations of sericins from the different species are examined for attachment, growth, proliferation, and morphology of fibrosarcoma cells. The optimum sericin supplementation seems to vary with the source of sericins. The results indicate that all the sericins promote the growth of L929 cells in serum-free culture media; however, S. ricini sericin seems to promote better growth of cells amongst other non-mulberry sericins.

Quantification of Maternal Serum Cell-Free Fetal DNA in Early-Onset Preeclampsia

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Mulan Ren

2013-04-01

Full Text Available The aim of this study was to determine whether the increased serum cell-free fetal DNA (cffDNA level of gravidas developed into early-onset preeclampsia (EOPE subsequently in the early second trimesters is related to prenatal screening markers. Serum was collected from 1011 gravidas. The level of cffDNA and prenatal screening markers were analyzed in 20 cases with EOPE and 20 controls. All fetuses were male. The maternal serum cffDNA level was assessed by amplification of the Y chromosome specific gene. Correlations between the variables were examined. (Logged cffDNA in EOPE (median, 3.08; interquartile range, 2.93–3.68 was higher than controls (median, 1.79; interquartile range, 1.46–2.53. The increased level of (logged cffDNA was correlated significantly with the increased human chorionic gonadotropin (HCG level (r = 0.628, p < 0.001. Significant reciprocal correlations between cffDNA and babies’ birth weight as well as gestation weeks at delivery were noted (r = −0.516, p = 0.001; r = −0.623, p < 0.001, respectively. The sensitivity and specificity of cffDNA to discriminate between the EOPE cases and the controls were 90% and 85%, respectively. CffDNA is a potential marker for EOPE, which had a significant reciprocal correlation with babies’ birth weight and gestation weeks at delivery. Moreover, it may help in indicating the underlying hypoxic condition in the placenta.

Bipotential precursors of putative fibrous astrocytes and oligodendrocytes in rat cerebellar cultures express distinct surface features and neuron-like γ-aminobutyric acid transport

International Nuclear Information System (INIS)

Levi, G.; Gallo, V.; Ciotti, T.

1986-01-01

When postnatal rat cerebellar cells were cultured in a chemically defined, serum-free medium, the only type of astrocyte present was unable to accumulate γ-[ 3 H]aminobutyric acid (GABA), did not express surface antigens recognized by two monoclonal antibodies, A2B5 and LB1, and showed minimal proliferation. In these cultures, nonneuronal A2B5 + , LB1 + stellate cells exhibiting neuron-like [ 3 H]GABA uptake formed cell colonies of increasing size and were GFAP - . After about one week of culturing, the A2B5 + , LB1 + , GABA-uptake positive cell groups became galactocerebroside (GalCer) positive. Immunocytolysis of the A2B5 + cells at 3 and 4 days in vitro prevented the appearance of the A2B5 + , LB1 + , GABA-uptake positive cell colonies, and also of the GalCer + cell groups. If 10% (vol/vol) fetal calf serum was added to 6-day cultures, the A2B5 + , LB1 + , GABA-uptake positive cell groups expressed GFAP and not GalCer. If the serum was added to the cultures 2 days after lysing the A2B5 + cells, only A2B5 - , LB1 - , GABA-uptake negative astrocytes proliferated. It is concluded that the putative fibrous astrocytes previously described in serum-containing cultures derive from bipotential precursors that differentiate into oligodendrocytes (GalCer + ) in serum-free medium or into astrocytes (GFAP + ) in the presence of serum, while the epithelioid A2B5 - , LB1 - , GABA-uptake negative astrocytes originate from a different precursor not yet identified

The presence of serum alters the properties of iron oxide nanoparticles and lowers their accumulation by cultured brain astrocytes

International Nuclear Information System (INIS)

Geppert, Mark; Petters, Charlotte; Thiel, Karsten; Dringen, Ralf

2013-01-01

Iron oxide nanoparticles (IONPs) are considered for various diagnostic and therapeutic applications. Such particles are able to cross the blood–brain barrier and are taken up into brain cells. To test whether serum components affect the properties of IONPs and/or their uptake into brain cells, we have incubated dimercaptosuccinate-coated magnetic IONPs without and with fetal calf serum (FCS) and have exposed cultured brain astrocytes with IONPs in the absence or presence of FCS. Incubation with FCS caused a concentration-dependent increase in the average hydrodynamic diameter of the particles and of their zeta-potential. In the presence of 10 % FCS, the diameter of the IONPs increased from 57 ± 2 to 107 ± 6 nm and the zeta-potential of the particles from −22 ± 5 to −9 ± 1 mV. FCS affected also strongly the uptake of IONPs by cultured astrocytes. The efficient time- and temperature-dependent cellular accumulation of IONPs was lowered with increasing concentration of FCS by up to 90 %. In addition, in the absence of serum, endocytosis inhibitors did not alter the IONP accumulation by astrocytes, while chlorpromazine or wortmannin lowered significantly the accumulation of IONPs in the presence of FCS, suggesting that clathrin-mediated endocytosis and macropinocytosis are involved in astrocytic IONP uptake from serum-containing medium. These data demonstrate that the presence of FCS strongly affects the properties of IONPs as well as their accumulation by cultured brain cells.

Serum thyroid stimulating hormone, total and free T4 during the neonatal period: Establishing regional reference intervals

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Sara Sheikhbahaei

2014-01-01

Full Text Available Context: Congenital hypothyroidism (CH, the most common etiology of preventable mental retardation in children, is estimated to be more prevalent among Asian population. Aims: Since thyroid function tests (TFTs varied among different ages and geographical regions, in this study, the neonatal thyroid reference intervals in a healthy neonatal population is determined for the first time in Iran. Settings and Design: A cross-sectional study performed on 246 healthy term newborns aged between 2 days and 1 month. Materials and Methods: Blood samples were obtained by venipuncture from all subjects. The median, 2.5 th , 5 th , 95 th , and 97.5 th percentile of serum thyroid-stimulating hormone (TSH, as well as the total and free T4 were assessed among different age groups. Statistical Analysis Used: Predictive Analytics Software (PASW Statistics 18 was used for the analysis. Results: Serum TSH, total and free T4 concentration peaked in 5 th to 7 th days of life, continued over 2 weeks, then decreased and started reaching to adult reference range. A significant negative correlation between age and serum concentration of TSH (P = 0.02, total T4 (P = 0.01 and free T4 (P = 0.01 was found. Conclusion: This study yielded fairly different values for TFTs compared compared values found in other countries and also different from values reported for laboratory kits we used. These differences were assumed to be due to variations in ethnicity, age, and laboratory methods used. Due to the lack of international standardization, conducting multicenter studies helps in making a more precise evaluation of thyroid status in neonates.

Serum amyloid A is a marker for pulmonary involvement in systemic sclerosis.

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Katja Lakota

Full Text Available Inflammation in systemic sclerosis (SSc is a prominent, but incompletely characterized feature in early stages of the disease. The goal of these studies was to determine the circulating levels, clinical correlates and biological effects of the acute phase protein serum amyloid A (SAA, a marker of inflammation, in patients with SSc. Circulating levels of SAA were determined by multiplex assays in serum from 129 SSc patients and 98 healthy controls. Correlations between SAA levels and clinical and laboratory features of disease were analyzed. The effects of SAA on human pulmonary fibroblasts were studied ex vivo. Elevated levels of SAA were found in 25% of SSc patients, with the highest levels in those with early-stage disease and diffuse cutaneous involvement. Significant negative correlations of SAA were found with forced vital capacity and diffusion capacity for carbon monoxide. Patients with elevated SAA had greater dyspnea and more frequent interstitial lung disease, and had worse scores on patient-reported outcome measures. Incubation with recombinant SAA induced dose-dependent stimulation of IL-6 and IL-8 in normal lung fibroblasts in culture. Serum levels of the inflammatory marker SAA are elevated in patients with early diffuse cutaneous SSc, and correlate with pulmonary involvement. In lung fibroblasts, SAA acts as a direct stimulus for increased cytokine production. These findings suggest that systemic inflammation in SSc may be linked to lung involvement and SAA could serve as a potential biomarker for this complication.

Gender and cultural issues in psychiatric nosological classification systems.

Science.gov (United States)

van de Water, Tanya; Suliman, Sharain; Seedat, Soraya

2016-08-01

Much has changed since the two dominant mental health nosological systems, the International Classification of Diseases (ICD) and the Diagnostic and Statistical Manual of Mental Disorders (DSM), were first published in 1900 and 1952, respectively. Despite numerous modifications to stay up to date with scientific and cultural changes (eg, exclusion of homosexuality as a disorder) and to improve the cultural sensitivity of psychiatric diagnoses, the ICD and DSM have only recently renewed attempts at harmonization. Previous nosological iterations demonstrate the oscillation in the importance placed on the biological focus, highlighting the tension between a gender- and culture-free nosology (solely biological) and a contextually relevant understanding of mental illness. In light of the release of the DSM 5, future nosological systems, such as the ICD 11, scheduled for release in 2017, and the Research Development Criteria (RDoC), can learn from history and apply critiques. This article aims to critically consider gender and culture in previous editions of the ICD and DSM to inform forthcoming classifications.

Serum total anti-oxidant capacity of some Nigerian cigarette smokers

African Journals Online (AJOL)

Abstract. Cigarette smoke has been reported to contain free radicals. The interaction of these free radicals with the body defense system and associated health risk among Nigerian smokers have remained scarcely investigated despite the high numbers of smokers in our society. This study thus, investigates the serum total ...

PRGF exerts more potent proliferative and anti-inflammatory effects than autologous serum on a cell culture inflammatory model.

Science.gov (United States)

Anitua, E; Muruzabal, F; de la Fuente, M; Riestra, A; Merayo-Lloves, J; Orive, G

2016-10-01

Ocular graft versus host disease (oGVHD) is part of a systemic inflammatory disease that usually affects ocular surface tissues manifesting as a dry eye syndrome. Current treatments provide unsatisfactory results. Blood-derived products, like plasma rich in growth factors (PRGF) emerge as a potential therapy for this disease. The purpose of this study was to evaluate the tissue regeneration and anti-inflammatory capability of PRGF, an autologous platelet enriched plasma eye-drop, compared to autologous serum (AS) obtained from oGVHD patients on ocular surface cells cultured in a pro-inflammatory environment. PRGF and AS were obtained from four GVHD patients. Cell proliferation and inflammation markers, intercellular adhesion molecule-1 (ICAM-1) and cyclooxygenase-2 (COX-2), were measured in corneal and conjunctival fibroblastic cells cultured under pro-inflammatory conditions and after treatment with PRGF or AS eye drops. Moreover, cell proliferation increased after treatment with PRGF and AS, though this enhancement in the case of keratocytes was significantly higher with PRGF. PRGF eye drops showed a significant reduction of both inflammatory markers with respect to the initial inflammatory situation and to the AS treatment. Our results concluded that PRGF exerts more potent regenerative and anti-inflammatory effects than autologous serum on ocular surface fibroblasts treated with pro-inflammatory IL-1β and TNFα. Copyright © 2016 Elsevier Ltd. All rights reserved.

Changes in responsiveness of rat tracheal epithelial cells to growth factors during preneoplastic transformation in cell culture

International Nuclear Information System (INIS)

Thomassen, D.G.

1988-01-01

Preneoplastic rat tracheal epithelial (RTE) cell lines require fewer growth factors for clonal proliferation in culture than normal cells. Serum-free media missing various combinations of growth factors (e.g., cholera toxin, serum albumin, epidermal growth factor, hydrocortisone) required for proliferation of normal, but not preneoplastic, RTE cells can be used to select for carcinogen-induced preneoplastic variants having an increased proliferative potential in culture. These results suggest that reductions in growth factor requirements are primary events in the carcinogenic process. (author)

The relationship between serum total testosterone and free testosterone levels with serum hemoglobin and hematocrit levels: a study in 1221 men.

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Shin, Yu Seob; You, Jae Hyung; Cha, Jai Seong; Park, Jong Kwan

2016-12-01

To investigate the relationship between serum total testosterone (TT) and free testosterone (FT) levels in men with anemia. We reviewed the records of 1221 subjects between March 2009 and December 2014. All the subjects' blood samples were drawn for TT and FT assays. Their serum hemoglobin (Hb) and serum hematocrit (Hct) levels were measured. The primary objective of our study was to investigate the association between TT and FT levels with Hb and Hct levels. The mean age was 59.82 ± 12.71 years. The mean TT and FT levels were 4.54 ± 2.02 ng/mL and 10.63 ± 3.69 pg/mL, respectively. The mean Hb and Hct levels were 14.72 ± 1.34 g/dL and 43.11 ± 3.75%, respectively. Subjects with low TT (<2.35 ng/mL) had low Hb and Hct levels (p < 0.001, p < 0.001, respectively). TT was positively associated with FT, Hb, and Hct. TT and FT levels were significantly lower in older men. Subjects with low TT and FT levels had low Hb and Hct levels. This suggests that TT and FT play a significant role in erythropoiesis. Testosterone replacement therapy may be effective in men with hypogonadism to reduce the incidence of anemia.

Critical evaluation of gamma-irradiated serum used as feeder in the culture and demonstration of putative nanobacteria and calcifying nanoparticles.

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Jan Martel

Full Text Available The culture and demonstration of putative nanobacteria (NB and calcifying nanoparticles (CNP from human and animal tissues has relied primarily on the use of a culture supplement consisting of FBS that had been gamma-irradiated at a dose of 30 kGy (gamma-FBS. The use of gamma-FBS is based on the assumption that this sterilized fluid has been rid entirely of any residual NB/CNP, while it continues to promote the slow growth in culture of NB/CNP from human/animal tissues. We show here that gamma-irradiation (5-50 kGy produces extensive dose-dependent serum protein breakdown as demonstrated through UV and visible light spectrophotometry, fluorometry, Fourier-transformed infrared spectroscopy, and gel electrophoresis. Yet, both gamma-FBS and gamma-irradiated human serum (gamma-HS produce NB/CNP in cell culture conditions that are morphologically and chemically indistinguishable from their normal serum counterparts. Contrary to earlier claims, gamma-FBS does not enhance the formation of NB/CNP from several human body fluids (saliva, urine, ascites, and synovial fluid tested. In the presence of additional precipitating ions, both gamma-irradiated serum (FBS and HS and gamma-irradiated proteins (albumin and fetuin-A retain the inherent dual NB inhibitory and seeding capabilities seen also with their untreated counterparts. By gel electrophoresis, the particles formed from both gamma-FBS and gamma-HS are seen to have assimilated into their scaffold the same smeared protein profiles found in the gamma-irradiated sera. However, their protein compositions as identified by proteomics are virtually identical to those seen with particles formed from untreated serum. Moreover, particles derived from human fluids and cultured in the presence of gamma-FBS contain proteins derived from both gamma-FBS and the human fluid under investigation-a confusing and unprecedented scenario indicating that these particles harbor proteins from both the host tissue and the FBS

Simple suspension culture system of human iPS cells maintaining their pluripotency for cardiac cell sheet engineering.

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Haraguchi, Yuji; Matsuura, Katsuhisa; Shimizu, Tatsuya; Yamato, Masayuki; Okano, Teruo

2015-12-01

In this study, a simple three-dimensional (3D) suspension culture method for the expansion and cardiac differentiation of human induced pluripotent stem cells (hiPSCs) is reported. The culture methods were easily adapted from two-dimensional (2D) to 3D culture without any additional manipulations. When hiPSCs were directly applied to 3D culture from 2D in a single-cell suspension, only a few aggregated cells were observed. However, after 3 days, culture of the small hiPSC aggregates in a spinner flask at the optimal agitation rate created aggregates which were capable of cell passages from the single-cell suspension. Cell numbers increased to approximately 10-fold after 12 days of culture. The undifferentiated state of expanded hiPSCs was confirmed by flow cytometry, immunocytochemistry and quantitative RT-PCR, and the hiPSCs differentiated into three germ layers. When the hiPSCs were subsequently cultured in a flask using cardiac differentiation medium, expression of cardiac cell-specific genes and beating cardiomyocytes were observed. Furthermore, the culture of hiPSCs on Matrigel-coated dishes with serum-free medium containing activin A, BMP4 and FGF-2 enabled it to generate robust spontaneous beating cardiomyocytes and these cells expressed several cardiac cell-related genes, including HCN4, MLC-2a and MLC-2v. This suggests that the expanded hiPSCs might maintain the potential to differentiate into several types of cardiomyocytes, including pacemakers. Moreover, when cardiac cell sheets were fabricated using differentiated cardiomyocytes, they beat spontaneously and synchronously, indicating electrically communicative tissue. This simple culture system might enable the generation of sufficient amounts of beating cardiomyocytes for use in cardiac regenerative medicine and tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.

The role of elevated serum procalcitonin in neuroendocrine neoplasms of digestive system.

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Chen, Luohai; Zhang, Yu; Lin, Yuan; Deng, Langhui; Feng, Shiting; Chen, Minhu; Chen, Jie

2017-12-01

Elevated serum procalcitonin (PCT) was reported in patients with certain type of neuroendocrine neoplasms (NENs). The aim of this study was to assess the role of elevated serum PCT in NENs from digestive system. Serum PCT and serum CgA level were measured in 155 patients with NENs from digestive system. Elevated serum PCT was found in 63 patients (40.6%). Grade 3 disease was a significant factor associated with elevated serum PCT (OR, 9.24; 95%CI, 3.04-28.08; Pdigestive system, especially in patients with grade 3 disease. Serum PCT level can help evaluate treatment response and its elevation indicates poor prognosis. Combination of serum PCT and CgA can improve outcome prediction. Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Two different serum-free media and osmolality effect upon human 293 cell growth and adenovirus production.

Science.gov (United States)

Ferreira, Tiago B; Ferreira, Ana L; Carrondo, Manuel J T; Alves, Paula M

2005-11-01

Adenoviruses are promising vectors for gene therapy and vaccination protocols. Consequently, the market demands for adenovirus are increasing, driving the search for new methodologies for large-scale production of concentrated vectors with warranted purity and efficacy, in a cost-effective way. Nevertheless, the production of adenovirus is currently limited by the so-called 'cell density effect', i.e. a drop in cell specific productivity concomitant with increased cell concentration at infection. Of two different serum-free culture media (CD293 and EX-Cell), evaluated for their effect on human 293 cells growth and adenovirus production at cell densities higher than 1x10(6) cells/ml, EX-Cell proved the better medium for cell growth. Although adenovirus production was equivalent in both media when the infection was performed at 1x10(6) cells/ml, at 3x10(6) cells/ml CD293 was the better. This result related to the high ammonia content in EX-Cell medium at the highest cell concentration at infection. Besides this, the large-scale production of these vectors at high cell densities often requires re-feed strategies, which increase medium osmolality. While a negative effect on cell growth was observed with increasing osmolalities, adenovirus productivity was only affected for osmolalities higher than 430 mOsm.

Comparison of real-time reverse transcriptase polymerase chain reaction of peripheral blood mononuclear cells, serum and cell-free body cavity effusion for the diagnosis of feline infectious peritonitis.

Science.gov (United States)

Doenges, Stephanie J; Weber, Karin; Dorsch, Roswitha; Fux, Robert; Hartmann, Katrin

2017-04-01

Objectives Diagnosis of feline infectious peritonitis (FIP) remains challenging, especially in cats without effusions. The objective of this study was to evaluate the sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction (RT-PCR) detecting feline coronavirus (FCoV) RNA in peripheral blood mononuclear cells (PBMCs) and serum in comparison with the same real-time RT-PCR in cell-free body cavity effusion. Methods This prospective case-control study included 92 cats. Forty-three cats had a definitive diagnosis of FIP, established either by histopathological examination (n = 28) or by positive immunofluorescence staining of FCoV antigen in macrophages of effusions (n = 11), or by both methods (n = 4). Forty-nine control cats had other diseases but similar clinical signs. Real-time RT-PCR was performed on PBMCs of 37 cats (21 cats with FIP, 16 controls), on serum of 51 cats (26 cats with FIP, 25 controls) and on cell-free body cavity effusion of 69 cats (36 cats with FIP, 33 controls). Sensitivity, specificity, positive and negative predictive value, including 95% confidence intervals (CI), were calculated. Results Real-time RT-PCR of PBMCs, serum and cell-free body cavity effusion showed a specificity of 100% (95% CI 79.4-100% in PBMCs, 86.3-100% in serum, 89.4-100% in cell-free body cavity effusion) and a sensitivity of 28.6% (95% CI 11.3-52.2%) in PBMCs, 15.4% (95% CI 4.4-34.9%) in serum and 88.9% (95% CI 73.9-96.9%) in cell-free body cavity effusion to diagnose FIP. Conclusions and relevance Although it is known that RT-PCR can often provide false-positive results in healthy cats, this real-time RT-PCR was shown to be a specific tool for the diagnosis of FIP when applied in a clinical setting. Sensitivity in cell-free body cavity effusion was high but low in PBMCs and serum. PBMC samples showed a higher sensitivity than serum samples, and are therefore a better choice if no effusion is present.

Serum osteopontin and vitronectin levels in systemic sclerosis.

Science.gov (United States)

Gundogdu, Baris; Yolbas, Servet; Yilmaz, Musa; Aydin, Suleyman; Koca, Sulayman Serdar

2017-11-01

Osteopontin a matricellular protein has pro-fibrotic effects and binds integrin such as αvβ1 and αvβ3. Vitronectin is one of the integrin αvβ3 ligands and is a multifunctional glycoprotein. The aim of the present study was to evaluate serum osteopontin and vitronectin levels in a cohort of patients with systemic sclerosis (SSc). Eighty-six patients with SSc, 46 patients with systemic lupus erythematosus (SLE), and 38 healthy controls (HC) were enrolled in the study. Serum osteopontin, vitronectin, IL-6, and TGF-β levels were analyzed. Serum osteopontin levels were higher in the SSc and SLE groups compared to the HC group (p < 0.01 and p < 0.001, respectively). However, it was not correlated with disease activity and severity scores in the SSc group. On the other hand, serum vitronectin levels were lower in the SSc group than in the SLE and HC groups (p < 0.001 for both). These results may suggest that osteopontin levels may be increased due to the inflammatory process and osteopontin has not a specific role on fibrosis in SSc. On the other hand, serum vitronectin levels decrease in SSc in contrast to SLE. It may be concluded that the one cause of decreased serum vitronectin levels in SSc may be its accumulation in fibrotic area.

Evaluation of performance of bacterial culture of feces and serum ELISA across stages of Johne's disease in cattle using a Bayesian latent class model.

Science.gov (United States)

Espejo, L A; Zagmutt, F J; Groenendaal, H; Muñoz-Zanzi, C; Wells, S J

2015-11-01

The objective of this study was to evaluate the performance of bacterial culture of feces and serum ELISA to correctly identify cows with Mycobacterium avium ssp. paratuberculosis (MAP) at heavy, light, and non-fecal-shedding levels. A total of 29,785 parallel test results from bacterial culture of feces and serum ELISA were collected from 17 dairy herds in Minnesota, Pennsylvania, and Colorado. Samples were obtained from adult cows from dairy herds enrolled for up to 10 yr in the National Johne's Disease Demonstration Herd Project. A Bayesian latent class model was fitted to estimate the probabilities that bacterial culture of feces (using 72-h sedimentation or 30-min centrifugation methods) and serum ELISA results correctly identified cows as high positive, low positive, or negative given that cows were heavy, light, and non-shedders, respectively. The model assumed that no gold standard test was available and conditional independency existed between diagnostic tests. The estimated conditional probabilities that bacterial culture of feces correctly identified heavy shedders, light shedders, and non-shedders were 70.9, 32.0, and 98.5%, respectively. The same values for the serum ELISA were 60.6, 18.7, and 99.5%, respectively. Differences in diagnostic test performance were observed among states. These results improve the interpretation of results from bacterial culture of feces and serum ELISA for detection of MAP and MAP antibody (respectively), which can support on-farm infection control decisions and can be used to evaluate disease-testing strategies, taking into account the accuracy of these tests. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Efficient generation of patient-matched malignant and normal primary cell cultures from clear cell renal cell carcinoma patients: clinically relevant models for research and personalized medicine

International Nuclear Information System (INIS)

Lobo, Nazleen C.; Gedye, Craig; Apostoli, Anthony J.; Brown, Kevin R.; Paterson, Joshua; Stickle, Natalie; Robinette, Michael; Fleshner, Neil; Hamilton, Robert J.; Kulkarni, Girish; Zlotta, Alexandre; Evans, Andrew; Finelli, Antonio; Moffat, Jason; Jewett, Michael A. S.; Ailles, Laurie

2016-01-01

Patients with clear cell renal cell carcinoma (ccRCC) have few therapeutic options, as ccRCC is unresponsive to chemotherapy and is highly resistant to radiation. Recently targeted therapies have extended progression-free survival, but responses are variable and no significant overall survival benefit has been achieved. Commercial ccRCC cell lines are often used as model systems to develop novel therapeutic approaches, but these do not accurately recapitulate primary ccRCC tumors at the genomic and transcriptional levels. Furthermore, ccRCC exhibits significant intertumor genetic heterogeneity, and the limited cell lines available fail to represent this aspect of ccRCC. Our objective was to generate accurate preclinical in vitro models of ccRCC using tumor tissues from ccRCC patients. ccRCC primary single cell suspensions were cultured in fetal bovine serum (FBS)-containing media or defined serum-free media. Established cultures were characterized by genomic verification of mutations present in the primary tumors, expression of renal epithelial markers, and transcriptional profiling. The apparent efficiency of primary cell culture establishment was high in both culture conditions, but genotyping revealed that the majority of cultures contained normal, not cancer cells. ccRCC characteristically shows biallelic loss of the von Hippel Lindau (VHL) gene, leading to accumulation of hypoxia-inducible factor (HIF) and expression of HIF target genes. Purification of cells based on expression of carbonic anhydrase IX (CA9), a cell surface HIF target, followed by culture in FBS enabled establishment of ccRCC cell cultures with an efficiency of >80 %. Culture in serum-free conditions selected for growth of normal renal proximal tubule epithelial cells. Transcriptional profiling of ccRCC and matched normal cell cultures identified up- and down-regulated networks in ccRCC and comparison to The Cancer Genome Atlas confirmed the clinical validity of our cell cultures. The ability

Comparative studies of types 1 and 2 herpes simplex virus infection of cultured normal keratinocytes.

OpenAIRE

Su, S J; Wu, H H; Lin, Y H; Lin, H Y

1995-01-01

AIMS--To investigate the differences in biological properties, multiplication patterns, and cytopathic effects between type 1 and type 2 herpes simplex virus (HSV) through the replication of HSV in cultured normal human keratinocytes. METHODS--Keratinocytes were obtained from surgical specimens of normal gingiva, cervix, trunk skin, and newborn foreskin. They were cultured in serum free, chemically defined, culture medium and infected with a pool of HSV collected from clinical specimens. RESU...

Quantification of circulating cell-free DNA in the serum of patients with obstructive sleep apnea-hypopnea syndrome.

Science.gov (United States)

Ye, Liang; Ma, Guan-Hua; Chen, Ling; Li, Min; Liu, Jia-Lin; Yang, Kun; Li, Qing-Yun; Li, Ning; Wan, Huan-Ying

2010-12-01

Serum cell-free DNA concentrations have been reported to increase in many acute diseases as well as in some chronic conditions such as cancer and autoimmune diseases. The aim of this study was to examine whether serum DNA concentrations were elevated in patients with obstructive sleep apnea-hypopnea syndrome (OSAHS). The effects of nasal continuous positive airway pressure (nCPAP) on serum DNA were also investigated. One hundred twenty-seven people diagnosed with OSAHS by polysomnography (PSG) were admitted into the OSAHS group, and 52 subjects without OSAHS were recruited for the control group. The OSAHS group was further divided into mild, moderate, and severe OSAHS subgroups based on their apnea-hypopnea index (AHI) during sleep. Ten patients with moderate and severe OSAHS were treated with nCPAP. Serum DNA, interleukin-6 (IL-6), and malonaldehyde (MDA) concentrations were measured and were found to be significantly higher in patients with moderate and severe OSAHS groups than those in the mild OSAHS and control groups (p DNA correlated positively with AHI, oxygen desaturation index (ODI), IL-6, and MDA, and negatively correlated with minimal oxygen saturation (miniSaO(2)) (all p DNA concentrations. After 6 months of nCPAP therapy, serum concentrations of DNA, IL-6, and MDA were significantly decreased (p DNA in patients with OSAHS was positively correlated with disease severity. Serum DNA may become an important parameter for monitoring the severity of OSAHS and effectiveness of therapy.

Cultured rat and purified human Pneumocystis carinii stimulate intra- but not extracellular free radical production in human neutrophils

DEFF Research Database (Denmark)

Jensen, T; Aliouat, E M; Lundgren, B

1998-01-01

The production of free radicals in human neutrophils was studied in both Pneumocystis carinii derived from cultures of L2 rat lung epithelial-like cells and Pneumocystis carinii purified from human lung. Using the cytochrome C technique, which selectively measured extracellular superoxide...... generation, hardly any free radical production was observed after stimulation with cultured rat-derived P. carinii. A chemiluminescence technique, which separately measured intra- and extracellular free radical production, was subsequently employed to differentiate the free radical generation....... It was established that 1) P. carinii stimulated intra- but not extracellular free radical production in human neutrophils, 2) opsonized cultured rat-derived P. carinii stimulated human neutrophils to a strong intracellular response of superoxide production, and 3) opsonized P. carinii, purified from human lung also...

Correlation of serum androgens and pituitary hormone levels with serum PSA less than 2.5 ng/ml.

Science.gov (United States)

Sofikerim, Mustafa; Oruç, Ozgür; Eskicorapci, Sadettin; Guliyev, Fuat; Ozen, Haluk

2007-07-27

The aim of this clinical study was to determine whether there is a relationship between total serum testosterone, free testosterone, FSH (Follicle-Stimulating Hormone), LH (Luteinizing Hormone) and serum prostate specific antigen (PSA) levels. We postulated that such a correlation existed then the use of hormone specific reference ranges might enhance the usefullness of PSA concentrations 40 years of age visiting our urology outpatient clinics. PSA was correlated to age (r = 0.23, p = 0.019), but there none between serum testosterone and age. No significant correlation was noted between testosterone or free testosterone and serum PSA levels, and none between serum FSH or LH and PSA. In age specific reference groups (41-49; 50-59; 60-69 years), we found no significant correlation between PSA and hormone concentrations. In this population of eugonadal men with serum PSA values less than 2.5 ng/ml, serum androgens and pituitary hormones do not appear to correlate with serum PSA.

Examining the sources of variability in cell culture media used for biopharmaceutical production.

Science.gov (United States)

McGillicuddy, Nicola; Floris, Patrick; Albrecht, Simone; Bones, Jonathan

2018-01-01

Raw materials, in particular cell culture media, represent a significant source of variability to biopharmaceutical manufacturing processes that can detrimentally affect cellular growth, viability and specific productivity or alter the quality profile of the expressed therapeutic protein. The continual expansion of the biopharmaceutical industry is creating an increasing demand on the production and supply chain consistency for cell culture media, especially as companies embrace intensive continuous processing. Here, we provide a historical perspective regarding the transition from serum containing to serum-free media, the development of chemically-defined cell culture media for biopharmaceutical production using industrial scale bioprocesses and review production mechanisms for liquid and powder culture media. An overview and critique of analytical approaches used for the characterisation of cell culture media and the identification of root causes of variability are also provided, including in-depth liquid phase separations, mass spectrometry and spectroscopic methods.

Prediabetes and prehypertension in disease free obese adults correlate with an exacerbated systemic proinflammatory milieu

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Johnson William D

2010-07-01

Full Text Available Abstract Background Obesity is a pro-inflammatory state frequently associated with widespread metabolic alterations that include insulin resistance and deregulation of blood pressure (BP. This cascade of events in some measure explains the susceptibility of obese adults for co-morbid conditions like diabetes mellitus and hypertension. Hypothesis We hypothesized that an elevated systemic proinflammatory burden correlates with dysglycemia and deregulated blood pressure. Methods We analyzed the screening anthropometric and laboratory measures from healthy disease free obese adults (n = 35; women (W 27, men (M 8 in a weight loss study. Results Healthy obese normoglycemic (fasting serum glucose: FSG Conclusions In otherwise healthy disease free obese adults, a higher degree of systemic inflammation is associated with prediabetes and prehypertension.

Development of an automated chip culture system with integrated on-line monitoring for maturation culture of retinal pigment epithelial cells

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Mee-Hae Kim

2017-10-01

Full Text Available In cell manufacturing, the establishment of a fully automated, microfluidic, cell culture system that can be used for long-term cell cultures, as well as for process optimization is highly desirable. This study reports the development of a novel chip bioreactor system that can be used for automated long-term maturation cultures of retinal pigment epithelial (RPE cells. The system consists of an incubation unit, a medium supply unit, a culture observation unit, and a control unit. In the incubation unit, the chip contains a closed culture vessel (2.5 mm diameter, working volume 9.1 μL, which can be set to 37 °C and 5% CO2, and uses a gas-permeable resin (poly- dimethylsiloxane as the vessel wall. RPE cells were seeded at 5.0 × 104 cells/cm2 and the medium was changed every day by introducing fresh medium using the medium supply unit. Culture solutions were stored either in the refrigerator or the freezer, and fresh medium was prepared before any medium change by warming to 37 °C and mixing. Automated culture was allowed to continue for 30 days to allow maturation of the RPE cells. This chip culture system allows for the long-term, bubble-free, culture of RPE cells, while also being able to observe cells in order to elucidate their cell morphology or show the presence of tight junctions. This culture system, along with an integrated on-line monitoring system, can therefore be applied to long-term cultures of RPE cells, and should contribute to process control in RPE cell manufacturing.

Metabolic system alterations in pancreatic cancer patient serum: potential for early detection

International Nuclear Information System (INIS)

Ritchie, Shawn A; Jin, Wei; Sajobi, Tolulope T; Jayasinghe, Dushmanthi; Chitou, Bassirou; Yamazaki, Yasuyo; White, Thayer; Goodenowe, Dayan B; Akita, Hirofumi; Takemasa, Ichiro; Eguchi, Hidetoshi; Pastural, Elodie; Nagano, Hiroaki; Monden, Morito; Doki, Yuichiro; Mori, Masaki

2013-01-01

The prognosis of pancreatic cancer (PC) is one of the poorest among all cancers, due largely to the lack of methods for screening and early detection. New biomarkers for identifying high-risk or early-stage subjects could significantly impact PC mortality. The goal of this study was to find metabolic biomarkers associated with PC by using a comprehensive metabolomics technology to compare serum profiles of PC patients to healthy control subjects. A non-targeted metabolomics approach based on high-resolution, flow-injection Fourier transform ion cyclotron resonance mass spectrometry (FI-FTICR-MS) was used to generate comprehensive metabolomic profiles containing 2478 accurate mass measurements from the serum of Japanese PC patients (n=40) and disease-free subjects (n=50). Targeted flow-injection tandem mass spectrometry (FI-MS/MS) assays for specific metabolic systems were developed and used to validate the FI-FTICR-MS results. A FI-MS/MS assay for the most discriminating metabolite discovered by FI-FTICR-MS (PC-594) was further validated in two USA Caucasian populations; one comprised 14 PCs, six intraductal papillary mucinous neoplasims (IPMN) and 40 controls, and a second comprised 1000 reference subjects aged 30 to 80, which was used to create a distribution of PC-594 levels among the general population. FI-FTICR-MS metabolomic analysis showed significant reductions in the serum levels of metabolites belonging to five systems in PC patients compared to controls (all p<0.000025). The metabolic systems included 36-carbon ultra long-chain fatty acids, multiple choline-related systems including phosphatidylcholines, lysophosphatidylcholines and sphingomyelins, as well as vinyl ether-containing plasmalogen ethanolamines. ROC-AUCs based on FI-MS/MS of selected markers from each system ranged between 0.93 ±0.03 and 0.97 ±0.02. No significant correlations between any of the systems and disease-stage, gender, or treatment were observed. Biomarker PC-594 (an ultra long

Neuroprotective effect of the endogenous neural peptide apelin in cultured mouse cortical neurons

International Nuclear Information System (INIS)

Zeng, Xiang Jun; Yu, Shan Ping; Zhang, Like; Wei, Ling

2010-01-01

The adipocytokine apelin and its G protein-coupled APJ receptor were initially isolated from a bovine stomach and have been detected in the brain and cardiovascular system. Recent studies suggest that apelin can protect cardiomyocytes from ischemic injury. Here, we investigated the effect of apelin on apoptosis in mouse primary cultures of cortical neurons. Exposure of the cortical cultures to a serum-free medium for 24 h induced nuclear fragmentation and apoptotic death; apelin-13 (1.0-5.0 nM) markedly prevented the neuronal apoptosis. Apelin neuroprotective effects were mediated by multiple mechanisms. Apelin-13 reduced serum deprivation (SD)-induced ROS generation, mitochondria depolarization, cytochrome c release and activation of caspase-3. Apelin-13 prevented SD-induced changes in phosphorylation status of Akt and ERK1/2. In addition, apelin-13 attenuated NMDA-induced intracellular Ca 2+ accumulation. These results indicate that apelin is an endogenous neuroprotective adipocytokine that may block apoptosis and excitotoxic death via cellular and molecular mechanisms. It is suggested that apelins may be further explored as a potential neuroprotective reagent for ischemia-induced brain damage.

Culture Medium Supplements Derived from Human Platelet and Plasma: Cell Commitment and Proliferation Support

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Anita Muraglia

2017-11-01

Full Text Available Present cell culture medium supplements, in most cases based on animal sera, are not fully satisfactory especially for the in vitro expansion of cells intended for human cell therapy. This paper refers to (i an heparin-free human platelet lysate (PL devoid of serum or plasma components (v-PL and (ii an heparin-free human serum derived from plasma devoid of PL components (Pl-s and to their use as single components or in combination in primary or cell line cultures. Human mesenchymal stem cells (MSC primary cultures were obtained from adipose tissue, bone marrow, and umbilical cord. Human chondrocytes were obtained from articular cartilage biopsies. In general, MSC expanded in the presence of Pl-s alone showed a low or no proliferation in comparison to cells grown with the combination of Pl-s and v-PL. Confluent, growth-arrested cells, either human MSC or human articular chondrocytes, treated with v-PL resumed proliferation, whereas control cultures, not supplemented with v-PL, remained quiescent and did not proliferate. Interestingly, signal transduction pathways distinctive of proliferation were activated also in cells treated with v-PL in the absence of serum, when cell proliferation did not occur, indicating that v-PL could induce the cell re-entry in the cell cycle (cell commitment, but the presence of serum proteins was an absolute requirement for cell proliferation to happen. Indeed, Pl-s alone supported cell growth in constitutively activated cell lines (U-937, HeLa, HaCaT, and V-79 regardless of the co-presence of v-PL. Plasma- and plasma-derived serum were equally able to sustain cell proliferation although, for cells cultured in adhesion, the Pl-s was more efficient than the plasma from which it was derived. In conclusion, the cells expanded in the presence of the new additives maintained their differentiation potential and did not show alterations in their karyotype.

Serum levels of beta2-microglobulin and free light chains of immunoglobulins are associated with systemic disease activity in primary Sjögren's syndrome. Data at enrollment in the prospective ASSESS cohort.

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Jacques-Eric Gottenberg

Full Text Available To analyze the clinical and immunological characteristics at enrollment in a large prospective cohort of patients with primary Sjögren's syndrome (pSS and to investigate the association between serum BAFF, beta2-microglobulin and free light chains of immunoglobulins and systemic disease activity at enrollment.Three hundred and ninety five patients with pSS according to American-European Consensus Criteria were included from fifteen centers of Rheumatology and Internal Medicine in the "Assessment of Systemic Signs and Evolution of Sjögren's Syndrome" (ASSESS 5-year prospective cohort. At enrollment, serum markers were assessed as well as activity of the disease measured with the EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI.Patient median age was 58 (25(th-75(th: 51-67 and median disease duration was 5 (2-9 years. Median ESSDAI at enrollment was 2 (0-7 with 30.9% of patients having features of systemic involvement. Patients with elevated BAFF, beta2-microglobulin and kappa, lambda FLCS had higher ESSDAI scores at enrollment (4 [2]-[11] vs 2 [0-7], P = 0.03; 4 [1]-[11] vs 2 [0-7], P< 0.0001; 4 [2]-[10] vs 2 [0-6.6], P< 0.0001 and 4 [2-8.2] vs 2 [0-7.0], P = 0.02, respectively. In multivariate analysis, increased beta2-microglobulin, kappa and lambda FLCs were associated with a higher ESSDAI score. Median BAFF and beta2-microglobulin were higher in the 16 patients with history of lymphoma (1173.3(873.1-3665.5 vs 898.9 (715.9-1187.2 pg/ml, P = 0.01 and 2.6 (2.2-2.9 vs 2.1 (1.8-2.6 mg/l, P = 0.04, respectively.In pSS, higher levels of beta2-microglobulin and free light chains of immunoglobulins are associated with increased systemic disease activity.

Cell-free DNA in a three-dimensional spheroid cell culture model

DEFF Research Database (Denmark)

Aucamp, Janine; Calitz, Carlemi; Bronkhorst, Abel J.

2017-01-01

Background Investigating the biological functions of cell-free DNA (cfDNA) is limited by the interference of vast numbers of putative sources and causes of DNA release into circulation. Utilization of three-dimensional (3D) spheroid cell cultures, models with characteristics closer to the in vivo...... cultures can serve as effective, simplified in vivo-simulating “closed-circuit” models since putative sources of cfDNA are limited to only the targeted cells. In addition, cfDNA can also serve as an alternative or auxiliary marker for tracking spheroid growth, development and culture stability. Biological...... significance 3D cell cultures can be used to translate “closed-circuit” in vitro model research into data that is relevant for in vivo studies and clinical applications. In turn, the utilization of cfDNA during 3D culture research can optimize sample collection without affecting the stability of the growth...

Influence of starter culture on total free aminoacids concentration during ripening of Krk cheese

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Biljana Radeljević

2013-03-01

Full Text Available The aim of this study was to determine the influence of microbial (commercial starter culture on concentration of total free amino groups (amino acids in cheeses in different ripening stages. Free amino groups were determined by reaction with ninhydrin with cadmium (Cd in the water soluble cheese extract, and were expressed as the concentration of leucine in cheese dry matter. Changes in concentration of total free amino acids during cheese ripening (0th, 30th, 60th, 90th and 120th day were monitored. In water soluble extracts of cheese, the presence of free NH2 groups in all ripening stages was detected, which means smaller peptides and amino acids, whose concentration significantly (P<0.01 increased during ripening. Cheeses produced with and without microbial culture resulted in statistically significant differences (P<0.01 in content amino acids free on the 90th and 120th day of ripening. Cd - ninhydrin method was found to be suitable for cheese ripening monitoring, as well as for determination of the differences in mature characteristics of cheeses, depending on the production process.

Reassessing the Role of the Active TGF-β1 as a Biomarker in Systemic Sclerosis: Association of Serum Levels with Clinical Manifestations

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Andréa Tavares Dantas

2016-01-01

Full Text Available Objective. To determine active TGF-β1 (aTGF-β1 levels in serum, skin, and peripheral blood mononuclear cell (PBMC culture supernatants and to understand their associations with clinical parameters in systemic sclerosis (SSc patients. Methods. We evaluated serum samples from 56 SSc patients and 24 healthy controls (HC. In 20 SSc patients, we quantified spontaneous or anti-CD3/CD28 stimulated production of aTGF-β1 by PBMC. The aTGF-β1 levels were measured by ELISA. Skin biopsies were obtained from 13 SSc patients and six HC, and TGFB1 expression was analyzed by RT-PCR. Results. TGF-β1 serum levels were significantly higher in SSc patients than in HC (p < 0.0001. Patients with increased TGF-β1 serum levels were more likely to have diffuse subset (p = 0.02, digital ulcers (p = 0.02, lung fibrosis (p < 0.0001, positive antitopoisomerase I (p = 0.03, and higher modified Rodnan score (p = 0.046. Most of our culture supernatant samples had undetectable levels of TGF-β1. No significant difference in TGFB1 expression was observed in the SSc skin compared with HC skin. Conclusion. Raised active TGF-β1 serum levels and their association with clinical manifestations in scleroderma patients suggest that this cytokine could be a marker of fibrotic and vascular involvement in SSc.

SERPINA3K plays antioxidant roles in cultured pterygial epithelial cells through regulating ROS system.

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Chengpeng Zhu

Full Text Available We recently demonstrated that SERPINA3K, a serine proteinase inhibitor, has antioxidant activity in the cornea. Here we investigated the antioxidant effects of SERPINA3K on the pterygial, which is partially caused by oxidative stress in pathogenesis. The head part of primary pterygial tissue was dissected and then cultured in keratinocyte serum-free defined medium (KSFM. The cultured pterygial epithelial cells (PECs were treated with SERPINA3K. The cell proliferation and migration of PECs were measured and analyzed. Western blot and quantitative real-time polymerase chain reaction (PCR assay were performed. It showed that SERPINA3K significantly suppressed the cell proliferation of PECs in a concentration-dependent manner, compared with cultured human conjunctival epithelial cells. SERPINA3K also inhibited the cell migration of PECs. Towards its underlying mechanism, SERPINA3K had antioxidant activities on the PECs by significantly inhibiting NADPH oxidase 4 (NOX4, which is an important enzyme of ROS generation, and by elevating the levels of key antioxidant factors of ROS: such as NAD(PH dehydrogenase (quinone 1 (NQO1, NF-E2-related factor-2 (NRF2 and superoxide dismutases (SOD2. Meanwhile, SERPINA3K down-regulated the key effectors of Wnt signaling pathway: β-catenin, nonphospho-β-catenin, and low-density lipoprotein receptor-related protein 6 (LRP6. We provided novel evidence that SERPINA3K had inhibitory effects on pterygium and SERPINA3K played antioxidant role via regulating the ROS system and antioxidants.

Diagnostic value of serum free PSA and the ratio of free to total PSA in the diagnosis of prostate cancer

International Nuclear Information System (INIS)

Qiu Ningyan; Zhang Jingxin; Wu Jinchang; Gong Yiming; Li Huiping

2005-01-01

In order to evaluate the value of free prostate specific antigen (FPSA) and F/T PSA ratio in differential diagnosis of benign prostate hyperplasia (BPH) from prostate cancer (PC), serum FPSA and TPSA levels were measured in 85 patients with PC, 97 BPH and 89 healthy volunteers by chemiluminescence enzyme immunoassay (CLEIA), and the ratio of F/T PSA was calculated. The results showed that serum FPSA and TPSA levels were increased in healthy volunteers of 41-88 years old and were significantly higher in healthy volunteers of 61-88 years old than that in 20-40 gear old (P 10.0 μg/L were 65.0%, 30.9% and 4.1%, respectively, while they were 5.9%, 20.0% and 74.1% in PC patients (P<0.01). When the TPSA value was between 4.0-10.0 μg/L and the ratio of F/T PSA was at 0.10 and below, the probability of PC was larger(88.9%). But the ratio of F/T PSA was at 0.25 and above, the probability of PC was smaller(6.20%). Serum FPSA and TPSA both increased with age in healthy volunteers of 41-88 years old and were positively correlated with age. There were about 30.9% of BPH and 20.0% of PC patients with overlapping of TPSA level. Our conclusion is that the F/T PSA ratio can significantly enhance the specificity for PC diagnosis, especially when the TPSA is within the diagnostic gray zone. (authors)

Elevated Adiponectin Serum Levels in Women with Systemic Autoimmune Diseases

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Éric Toussirot

2010-01-01

Full Text Available Adipose tissue produces a wide range of proteins that may influence the immune system. In this study, we assessed the serum levels of leptin, adiponectin, and ghrelin, in association with the measurements of body composition, in 15 female patients with various autoimmune diseases (systemic lupus erythematosus, primary Sjögren's syndrome, sarcoidosis, mixed connective tissue disease, vasculitis, CREST syndrome, and polymyositis and in 15 healthy female controls. There were no statistically significant differences between the patients and controls with regard to serum leptin, serum ghrelin, global fat mass, adiposity, and fat mass in the android or gynoid regions, whereas serum adiponectin levels were higher in patients than controls (16.3±1.6 μg/mL versus 9.7±0.6 μg/mL; =.01. As adiponectin is known to exhibit potent anti-inflammatory properties, a high adiponectinemia in patients with systemic autoimmune disease may mitigate the inflammatory response. However, the precise consequences of these elevated serum adiponectin levels on the metabolic syndrome development and atherosclerotic cardiovascular risk in this patient population still needs to be determined.

Reference intervals for selected serum biochemistry analytes in cheetahs Acinonyx jubatus.

Science.gov (United States)

Hudson-Lamb, Gavin C; Schoeman, Johan P; Hooijberg, Emma H; Heinrich, Sonja K; Tordiffe, Adrian S W

2016-02-26

Published haematologic and serum biochemistry reference intervals are very scarce for captive cheetahs and even more for free-ranging cheetahs. The current study was performed to establish reference intervals for selected serum biochemistry analytes in cheetahs. Baseline serum biochemistry analytes were analysed from 66 healthy Namibian cheetahs. Samples were collected from 30 captive cheetahs at the AfriCat Foundation and 36 free-ranging cheetahs from central Namibia. The effects of captivity-status, age, sex and haemolysis score on the tested serum analytes were investigated. The biochemistry analytes that were measured were sodium, potassium, magnesium, chloride, urea and creatinine. The 90% confidence interval of the reference limits was obtained using the non-parametric bootstrap method. Reference intervals were preferentially determined by the non-parametric method and were as follows: sodium (128 mmol/L - 166 mmol/L), potassium (3.9 mmol/L - 5.2 mmol/L), magnesium (0.8 mmol/L - 1.2 mmol/L), chloride (97 mmol/L - 130 mmol/L), urea (8.2 mmol/L - 25.1 mmol/L) and creatinine (88 µmol/L - 288 µmol/L). Reference intervals from the current study were compared with International Species Information System values for cheetahs and found to be narrower. Moreover, age, sex and haemolysis score had no significant effect on the serum analytes in this study. Separate reference intervals for captive and free-ranging cheetahs were also determined. Captive cheetahs had higher urea values, most likely due to dietary factors. This study is the first to establish reference intervals for serum biochemistry analytes in cheetahs according to international guidelines. These results can be used for future health and disease assessments in both captive and free-ranging cheetahs.

Serum Free Light Chains

Science.gov (United States)

... lab's website in order to provide you with background information about the test(s) you had performed. You will need to return ... the free light chains have a much shorter half-life (3-5 hours) than ... cell disorders, the test is also used for assessing response and minimal ...

Assessment of genetic and epigenetic changes in virus-free garlic (Allium sativum L.) plants obtained by meristem culture followed by in vitro propagation.

Science.gov (United States)

Gimenez, Magalí Diana; Yañez-Santos, Anahí Mara; Paz, Rosalía Cristina; Quiroga, Mariana Paola; Marfil, Carlos Federico; Conci, Vilma Cecilia; García-Lampasona, Sandra Claudia

2016-01-01

This is the first report assessing epigenetic variation in garlic. High genetic and epigenetic polymorphism during in vitro culture was detected.Sequencing of MSAP fragments revealed homology with ESTs. Garlic (Allium sativum) is a worldwide crop of economic importance susceptible to viral infections that can cause significant yield losses. Meristem tissue culture is the most employed method to sanitize elite cultivars.Often the virus-free garlic plants obtained are multiplied in vitro (micro propagation). However, it was reported that micro-propagation frequently produces somaclonal variation at the phenotypic level, which is an undesirable trait when breeders are seeking to maintain varietal stability. We employed amplification fragment length polymorphism and methylation sensitive amplified polymorphism (MSAP) methodologies to assess genetic and epigenetic modifications in two culture systems: virus-free plants obtained by meristem culture followed by in vitro multiplication and field culture. Our results suggest that garlic exhibits genetic and epigenetic polymorphism under field growing conditions. However, during in vitro culture system both kinds of polymorphisms intensify indicating that this system induces somaclonal variation. Furthermore, while genetic changes accumulated along the time of in vitro culture, epigenetic polymorphism reached the major variation at 6 months and then stabilize, being demethylation and CG methylation the principal conversions.Cloning and sequencing differentially methylated MSAP fragments allowed us to identify coding and unknown sequences of A. sativum, including sequences belonging to LTR Gypsy retrotransposons. Together, our results highlight that main changes occur in the initial 6 months of micro propagation. For the best of our knowledge, this is the first report on epigenetic assessment in garlic.

Development of humanized culture medium with plant-derived serum replacement for human pluripotent stem cells

Czech Academy of Sciences Publication Activity Database

Kunová, M.; Matulka, K.; Eiselleová, L.; Trčková, P.; Hampl, Aleš; Dvořák, Petr

2010-01-01

Roč. 21, - (2010), s. 676-686 ISSN 1472-6483 Grant - others:GA MŠk(CZ) LC06077; EC FP6(XE) LSHG-CT-2006-018739 Program:LC Institutional research plan: CEZ:AV0Z50390512; CEZ:AV0Z50390703 Keywords : animal protein-free culture * high-density culture * human embryonic stem cells Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.285, year: 2010

Establishment of a continuous culture system for Entamoeba muris and analysis of the small subunit rRNA gene

OpenAIRE

Kobayashi S.; Suzuki J.; Takeuchi T.

2009-01-01

We established a culture system for Entamoeba muris (MG-EM-01 strain isolated from a Mongolian gerbil) using a modified Balamuth’s egg yolk infusion medium supplemented with 4% adult bovine serum and Bacteroides fragilis cocultured with Escherichia coli. Further, encystation was observed in the culture medium. The morphological characteristics of E. muris are similar to those of Entamoeba coli (E. coli); moreover, the malic isoenzyme electrophoretic band, which shows species-specific electrop...

3D+time acquisitions of 3D cell culture by means of lens-free tomographic microscopy

Science.gov (United States)

Berdeu, Anthony; Laperrousaz, Bastien; Bordy, Thomas; Morales, S.; Gidrol, Xavier; Picollet-D'hahan, Nathalie; Allier, Cédric

2018-02-01

We propose a three-dimensional (3D) imaging platform based on lens-free microscopy to perform multi-angle acquisitions on 3D cell cultures embedded in extracellular matrix (ECM). We developed algorithms based on the Fourier diffraction theorem to perform fully 3D reconstructions of biological samples and we adapted the lens-free microscope to incubator conditions. Here we demonstrate for the first time, 3D+time lens-free acquisitions of 3D cell culture over 8 days directly into the incubator. The 3D reconstructed volume is as large as 5 mm3 and provides a unique way to observe in the same 3D cell culture experiment multiple cell migration strategies. Namely, in a 3D cell culture of prostate epithelial cells embedded within a Matrigel® matrix, we are able to distinguish single cell 'leaders', migration of cell clusters, migration of large aggregates of cells, and also close-gap and large-scale branching. In addition, we observe long-scale 3D deformations of the ECM that modify the geometry of the 3D cell culture. Interestingly, we also observed the opposite, i.e. we found that large aggregates of cells may deform the ECM by generating traction forces over very long distances. In sum we put forward a novel 3D lens-free microscopy tomographic technique to study the single and collective cell migrations, the cell-to-cell interactions and the cell-to-matrix interactions.

Effects of a honeybee sting on the serum free amino acid profile in humans.

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Jan Matysiak

Full Text Available The aim of this study was to assess the response to a honeybee venom by analyzing serum levels of 34 free amino acids. Another goal of this study was to apply complex analytic-bioinformatic-clinical strategy based on up-to-date achievements of mass spectrometry in metabolomic profiling. The amino acid profiles were determined using hybrid triple quadrupole/linear ion trap mass spectrometer coupled with a liquid chromatography instrument. Serum samples were collected from 27 beekeepers within 3 hours after they were stung and after a minimum of 6 weeks following the last sting. The differences in amino acid profiles were evaluated using MetaboAnalyst and ROCCET web portals. Chemometric tests showed statistically significant differences in the levels of L-glutamine (Gln, L-glutamic acid (Glu, L-methionine (Met and 3-methyl-L-histidine (3MHis between the two analyzed groups of serum samples. Gln and Glu appeared to be the most important metabolites for distinguishing the beekeepers tested shortly after a bee sting from those tested at least 6 weeks later. The role of some amino acids in the response of an organism to the honeybee sting was also discussed. This study indicated that proposed methodology may allow to identify the individuals just after the sting and those who were stung at least 6 weeks earlier. The results we obtained will contribute to better understanding of the human body response to the honeybee sting.

Effect of operating conditions in production of diagnostic Salmonella Enteritidis O-antigen-specific monoclonal antibody in different bioreactor systems.

Science.gov (United States)

Ayyildiz-Tamis, Duygu; Nalbantsoy, Ayse; Elibol, Murat; Deliloglu-Gurhan, Saime Ismet

2014-01-01

In this study, different cultivation systems such as roller bottles (RB), 5-L stirred-tank bioreactor (STR), and disposable bioreactors were used to cultivate hybridoma for lab-scale production of Salmonella Enteritidis O-antigen-specific monoclonal antibody (MAb). Hybridoma cell line was cultivated in either serum-containing or serum-free medium (SFM) culture conditions. In STR, MAb production scaled up to 4 L, and production capabilities of the cells were also evaluated in different featured production systems. Moreover, the growth parameters of the cells in all production systems such as glucose consumption, lactate and ammonia production, and also MAb productivities were determined. Collected supernatants from the reactors were concentrated by a cross-flow filtration system. In conclusion, cells were not adapted to SFM in RB and STR. Therefore, less MAb titer in both STR and RB systems with SFM was observed compared to the cultures containing fetal bovine serum-supplemented medium. A higher MAb titer was gained in the membrane-aerated system compared to those in STR and RB. Although the highest MAb titer was obtained in the static membrane bioreactor system, the highest productivity was obtained in STR operated in semicontinuous mode with overlay aeration.

Label-free silicon photonic biosensor system with integrated detector array

Science.gov (United States)

Yan, Rongjin; Mestas, Santano P.; Yuan, Guangwei; Safaisini, Rashid; Dandy, David S.

2010-01-01

An integrated, inexpensive, label-free photonic waveguide biosensor system with multi-analyte capability has been implemented on a silicon photonics integrated circuit from a commercial CMOS line and tested with nanofilms. The local evanescent array coupled (LEAC) biosensor is based on a new physical phenomenon that is fundamentally different from the mechanisms of other evanescent field sensors. Increased local refractive index at the waveguide’s upper surface due to the formation of a biological nanofilm causes local modulation of the evanescent field coupled into an array of photodetectors buried under the waveguide. The planar optical waveguide biosensor system exhibits sensitivity of 20%/nm photocurrent modulation in response to adsorbed bovine serum albumin (BSA) layers less than 3 nm thick. In addition to response to BSA, an experiment with patterned photoresist as well as beam propagation method simulations support the evanescent field shift principle. The sensing mechanism enables the integration of all optical and electronic components for a multi-analyte biosensor system on a chip. PMID:19606292

Label-free silicon photonic biosensor system with integrated detector array.

Science.gov (United States)

Yan, Rongjin; Mestas, Santano P; Yuan, Guangwei; Safaisini, Rashid; Dandy, David S; Lear, Kevin L

2009-08-07

An integrated, inexpensive, label-free photonic waveguide biosensor system with multi-analyte capability has been implemented on a silicon photonics integrated circuit from a commercial CMOS line and tested with nanofilms. The local evanescent array coupled (LEAC) biosensor is based on a new physical phenomenon that is fundamentally different from the mechanisms of other evanescent field sensors. Increased local refractive index at the waveguide's upper surface due to the formation of a biological nanofilm causes local modulation of the evanescent field coupled into an array of photodetectors buried under the waveguide. The planar optical waveguide biosensor system exhibits sensitivity of 20%/nm photocurrent modulation in response to adsorbed bovine serum albumin (BSA) layers less than 3 nm thick. In addition to response to BSA, an experiment with patterned photoresist as well as beam propagation method simulations support the evanescent field shift principle. The sensing mechanism enables the integration of all optical and electronic components for a multi-analyte biosensor system on a chip.

Optimized conditions for primary culture of pituitary cells from the Atlantic cod (Gadus morhua). The importance of osmolality, pCO₂, and pH.

Science.gov (United States)

Hodne, Kjetil; von Krogh, Kristine; Weltzien, Finn-Arne; Sand, Olav; Haug, Trude M

2012-09-01

Protocols for primary cultures of teleost cells are commonly only moderately adjusted from similar protocols for mammalian cells, the main adjustment often being of temperature. Because aquatic habitats are in general colder than mammalian body temperatures and teleosts have gills in direct contact with water, pH and buffer capacity of blood and extracellular fluid are different in fish and mammals. Plasma osmolality is generally higher in marine teleosts than in mammals. Using Atlantic cod (Gadus morhua) as a model, we have optimized these physiological parameters to maintain primary pituitary cells in culture for an extended period without loosing key properties. L-15 medium with adjusted osmolality, adapted to low pCO(2) (3.8mm Hg) and temperature (12°C), and with pH 7.85, maintained the cells in a physiologically sounder state than traditional culture medium, significantly improving cell viability compared to the initial protocol. In the optimized culture medium, resting membrane potential and response to releasing hormone were stable for at least two weeks, and the proportion of cells firing action potentials during spawning season was about seven times higher than in the original culture medium. The cells were moderately more viable when the modified medium was supplemented with newborn calf serum or artificial serum substitute. Compared to serum-free L-15 medium, expression of key genes (lhb, fshb, and gnrhr2a) was better maintained in medium containing SSR, whereas NCS tended to decrease the expression level. Although serum-free medium is adequate for many applications, serum supplement may be preferable for experiments dependent on membrane integrity. Copyright © 2012 Elsevier Inc. All rights reserved.

Multiplexed activity-based protein profiling of the human pathogen Aspergillus fumigatus reveals large functional changes upon exposure to human serum.

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Wiedner, Susan D; Burnum, Kristin E; Pederson, LeeAnna M; Anderson, Lindsey N; Fortuin, Suereta; Chauvigné-Hines, Lacie M; Shukla, Anil K; Ansong, Charles; Panisko, Ellen A; Smith, Richard D; Wright, Aaron T

2012-09-28

Environmental adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised host lung. We hypothesized that exposure of the fungal pathogen to human serum would lead to significant alterations to the organism's physiology, including metabolic activity and stress response. Shifts in functional pathway and corresponding enzyme reactivity of A. fumigatus upon exposure to the human host may represent much needed prognostic indicators of fungal infection. To address this, we employed a multiplexed activity-based protein profiling (ABPP) approach coupled to quantitative mass spectrometry-based proteomics to measure broad enzyme reactivity of the fungus cultured with and without human serum. ABPP showed a shift from aerobic respiration to ethanol fermentation and utilization over time in the presence of human serum, which was not observed in serum-free culture. Our approach provides direct insight into this pathogen's ability to survive, adapt, and proliferate. Additionally, our multiplexed ABPP approach captured a broad swath of enzyme reactivity and functional pathways and provides a method for rapid assessment of the A. fumigatus response to external stimuli.

Multiplexed Activity-based Protein Profiling of the Human Pathogen Aspergillus fumigatus Reveals Large Functional Changes upon Exposure to Human Serum*

Science.gov (United States)

Wiedner, Susan D.; Burnum, Kristin E.; Pederson, LeeAnna M.; Anderson, Lindsey N.; Fortuin, Suereta; Chauvigné-Hines, Lacie M.; Shukla, Anil K.; Ansong, Charles; Panisko, Ellen A.; Smith, Richard D.; Wright, Aaron T.

2012-01-01

Environmental adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised host lung. We hypothesized that exposure of the fungal pathogen to human serum would lead to significant alterations to the organism's physiology, including metabolic activity and stress response. Shifts in functional pathway and corresponding enzyme reactivity of A. fumigatus upon exposure to the human host may represent much needed prognostic indicators of fungal infection. To address this, we employed a multiplexed activity-based protein profiling (ABPP) approach coupled to quantitative mass spectrometry-based proteomics to measure broad enzyme reactivity of the fungus cultured with and without human serum. ABPP showed a shift from aerobic respiration to ethanol fermentation and utilization over time in the presence of human serum, which was not observed in serum-free culture. Our approach provides direct insight into this pathogen's ability to survive, adapt, and proliferate. Additionally, our multiplexed ABPP approach captured a broad swath of enzyme reactivity and functional pathways and provides a method for rapid assessment of the A. fumigatus response to external stimuli. PMID:22865858

A thin-layer liquid culture technique for the growth of Helicobacter pylori.

Science.gov (United States)

Joo, Jung-Soo; Park, Kyung-Chul; Song, Jae-Young; Kim, Dong-Hyun; Lee, Kyung-Ja; Kwon, Young-Cheol; Kim, Jung-Min; Kim, Kyung-Mi; Youn, Hee-Shang; Kang, Hyung-Lyun; Baik, Seung-Chul; Lee, Woo-Kon; Cho, Myung-Je; Rhee, Kwang-Ho

2010-08-01

Several attempts have been successful in liquid cultivation of Helicobaccter pylori. However, there is a need to improve the growth of H. pylori in liquid media in order to get affluent growth and a simple approach for examining bacterial properties. We introduce here a thin-layer liquid culture technique for the growth of H. pylori. A thin-layer liquid culture system was established by adding liquid media to a 90-mm diameter Petri dish. Optimal conditions for bacterial growth were investigated and then viability, growth curve, and released proteins were examined. Maximal growth of H. pylori was obtained by adding 3 mL of brucella broth supplemented with 10% horse to a Petri dish. H. pylori grew in both DMEM and RPMI-1640 supplemented with 10% fetal bovine serum and 0.5% yeast extract. Serum-free RPMI-1640 supported the growth of H. pylori when supplemented with dimethyl-beta-cyclodextrin (200 microg/mL) and 1% yeast extract. Under optimal growth, H. pylori grew exponentially for 28 hours, reaching a density of 3.4 OD(600) with a generation time of 3.3 hours. After 24 hours, cultures at a cell density of 1.0 OD(600) contained 1.3 +/- 0.1 x 10(9 )CFU/mL. gamma-Glutamyl transpeptidase, nuclease, superoxide dismutase, and urease were not detected in culture supernatants at 24 hours in thin-layer liquid culture, but were present at 48 hours, whereas alcohol dehydrogenase, alkylhydroperoxide reductase, catalase, and vacuolating cytotoxin were detected at 24 hours. Thin-layer liquid culture technique is feasible, and can serve as a versatile liquid culture technique for investigating bacterial properties of H. pylori.

Reference intervals for selected serum biochemistry analytes in cheetahs (Acinonyx jubatus

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Gavin C. Hudson-Lamb

2016-02-01

Full Text Available Published haematologic and serum biochemistry reference intervals are very scarce for captive cheetahs and even more for free-ranging cheetahs. The current study was performed to establish reference intervals for selected serum biochemistry analytes in cheetahs. Baseline serum biochemistry analytes were analysed from 66 healthy Namibian cheetahs. Samples were collected from 30 captive cheetahs at the AfriCat Foundation and 36 free-ranging cheetahs from central Namibia. The effects of captivity-status, age, sex and haemolysis score on the tested serum analytes were investigated. The biochemistry analytes that were measured were sodium, potassium, magnesium, chloride, urea and creatinine. The 90% confidence interval of the reference limits was obtained using the non-parametric bootstrap method. Reference intervals were preferentially determined by the non-parametric method and were as follows: sodium (128 mmol/L – 166 mmol/L, potassium (3.9 mmol/L – 5.2 mmol/L, magnesium (0.8 mmol/L – 1.2 mmol/L, chloride (97 mmol/L – 130 mmol/L, urea (8.2 mmol/L – 25.1 mmol/L and creatinine (88 µmol/L – 288 µmol/L. Reference intervals from the current study were compared with International Species Information System values for cheetahs and found to be narrower. Moreover, age, sex and haemolysis score had no significant effect on the serum analytes in this study. Separate reference intervals for captive and free-ranging cheetahs were also determined. Captive cheetahs had higher urea values, most likely due to dietary factors. This study is the first to establish reference intervals for serum biochemistry analytes in cheetahs according to international guidelines. These results can be used for future health and disease assessments in both captive and free-ranging cheetahs.

Chitosan-catechol: a writable bioink under serum culture media.

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Lee, Daiheon; Park, Joseph P; Koh, Mi-Young; Kim, Pureum; Lee, Junhee; Shin, Mikyung; Lee, Haeshin

2018-05-01

Mussel-inspired adhesive coatings on biomedical devices have attracted significant interest due to their unique properties such as substrate independency and high efficiency. The key molecules for mussel-inspired adhesive coatings are catechol and amine groups. Along with the understanding of catechol chemistry, chitosan-catechol has also been developed as a representative mussel-inpired adhesive polymer that contains catechol and amine groups for adhesiveness. Herein, we demonstrated the direct writability of chitosan-catechol as a bioink for 3D printing, one of the additive techniques. The use of chitosan-catechol bioink results in the formation of 3D constructs in normal culture media via rapid complexation of this bioink with serum proteins; in addition, the metal/catechol combination containing tiny amounts of vanadyl ions, in which the ratio of metal to catechol is 0.0005, dramatically enhances the mechanical strength and printability of the cell-encapsulated inks, showing a cell viability of approximately 90%. These findings for mussel-inspired bioinks will be a promising way to design a biocompatible 3D bioink cross-linked without any external stimuli.

Raman Spectroscopic Analyses of Jaw Periosteal Cell Mineralization

Directory of Open Access Journals (Sweden)

Eva Brauchle

2017-01-01

Full Text Available To achieve safer patient treatments, serum-free cell culture conditions have to be established for cell therapies. In previous studies, we demonstrated that serum-free culture favored the proliferation of MSCA-1+ osteoprogenitors derived from the jaw periosteum. In this study, the in vitro formation of bone-specific matrix by MSCA-1+ jaw periosteal cells (JPCs, 3 donors was assessed and compared under serum-free and serum-containing media conditions using the marker-free Raman spectroscopy. Based on a standard fluorescence assay, JPCs from one patient were not able to mineralize under serum-containing culture conditions, whereas the other cells showed similar mineralization levels under both conditions. Raman spectra from mineralizing MSCA-1+ JPCs revealed higher levels of hydroxyapatite formation and higher mineral to matrix ratios under serum-free culture conditions. Higher carbonate to phosphate ratios and higher crystallinity in JPCs cultured under serum-containing conditions indicated immature bone formation. Due to reduced collagen production under serum-free conditions, we obtained significant differences in collagen maturity and proline to hydroxyproline ratios compared to serum-free conditions. We conclude that Raman spectroscopy is a useful tool for the assessment and noninvasive monitoring of in vitro mineralization of osteoprogenitor cells. Further studies should extend this knowledge and improve JPC mineralization by optimizing culture conditions.

A knowledge-based theory of rising scores on "culture-free" tests.

Science.gov (United States)

Fox, Mark C; Mitchum, Ainsley L

2013-08-01

Secular gains in intelligence test scores have perplexed researchers since they were documented by Flynn (1984, 1987). Gains are most pronounced on abstract, so-called culture-free tests, prompting Flynn (2007) to attribute them to problem-solving skills availed by scientifically advanced cultures. We propose that recent-born individuals have adopted an approach to analogy that enables them to infer higher level relations requiring roles that are not intrinsic to the objects that constitute initial representations of items. This proposal is translated into item-specific predictions about differences between cohorts in pass rates and item-response patterns on the Raven's Matrices (Flynn, 1987), a seemingly culture-free test that registers the largest Flynn effect. Consistent with predictions, archival data reveal that individuals born around 1940 are less able to map objects at higher levels of relational abstraction than individuals born around 1990. Polytomous Rasch models verify predicted violations of measurement invariance, as raw scores are found to underestimate the number of analogical rules inferred by members of the earlier cohort relative to members of the later cohort who achieve the same overall score. The work provides a plausible cognitive account of the Flynn effect, furthers understanding of the cognition of matrix reasoning, and underscores the need to consider how test-takers select item responses. PsycINFO Database Record (c) 2013 APA, all rights reserved.

Serum Thyroxine to Thyroxine-Binding Globulin Ratio in Pregnancy and Newborn

International Nuclear Information System (INIS)

Kim, Ji Yeul

1982-01-01

To evaluate the diagnostic value of the ratio of serum thyroxine(T 4 ) /thyroxine-binding globulin (TBG) for the thyroid status in pregnancy and newborn serum thyroxine, TBG, triiodothyronine, and free thyroxine levels were radioimmunoassayed in normal pregnant women at each of the trimesters, and the calculated serum T 4 /TBG ratios were compared with other parameters such as T 3 /TBG ratio and free T 4 /TBG ratio and free T 4 /TBG ratio. Serum T 4 levels were elevated with the proportionate increase in TBG levels during pregnancy, leading to the nearly constant value of serum T 4 /TBG ratios as in normal non-pregnant controls. In contrast, serum T 3 /TBG and free T 4 /TBG ratios varied considerably during pregnancy. In newborn, T 4 levels were nearly not changed with compared non-pregnant control value and TBG levels were elevated. The results indicate that serum T 4 /TBG ratio is a better parameter than others in evaluating the thyroid status during pregnancy and but newborn is a no better.

Fasting induces the generation of serum thyronine-binding globulin in Zucker rats

International Nuclear Information System (INIS)

Young, R.A.; Rajatanavin, R.; Moring, A.F.; Braverman, L.E.

1985-01-01

Five-month-old lean and obese Zucker rats were fasted for up to 7 days (lean rats) or 28 days (obese rats), and serum total and free T4 and T3 concentrations, percent free T4 and T3 by equilibrium dialysis, and the binding of [ 125 I] T4 to serum proteins by gel electrophoresis were measured. In the lean rats, a 4- or 7-day fast resulted in significant decreases in serum total and free T4 and T3 concentrations. There was a decrease in the percent free T3 after 7 days of starvation. In contrast, a 4- or 7-day fast did not alter any of these variables in the obese rats. However, after 14 or more days of starvation, serum total T4 and T3 concentrations increased, and the percent free T4 and T3 decreased, resulting in no change in the serum free T4 or T3 concentrations in the obese rats. The percent of [ 125 I]T4 bound to serum thyronine-binding globulin increased and the percent bound to thyronine-binding prealbumin decreased with the duration of the fast in both the lean and obese rats. The increase in serum thyronine-binding globulin binding of T4 can explain the increase in serum total T4 and T3 concentrations, the decrease in percent free T4 and T3, and the normal free hormone concentration in the long term fasted obese rats. The findings in the lean rats appear to be due to a combination of the known central hypothyroidism that occurs during 4-7 days of fasting and the fasting-induced changes in T4 binding in serum. Changes in T4 and T3 binding in serum during fasting in the rat must be considered when the effects of fasting on serum concentrations of the thyroid hormones, thyroid hormone kinetics, and the peripheral action of the thyroid hormones are evaluated

Solid phase radioimmunoassay for detection of serum autoantibodies in systemic lupus erythematosus

Energy Technology Data Exchange (ETDEWEB)

Smith, K O; Harrington, J T; Gehle, W D

1977-03-01

Solid phase radioimmunoassay (RIA) methods for measuring autoantibodies in systemic lupus erythematosus (SLE) patients' serum were developed to improve the sensitvity and quantitative precision of the determinations. Two mechanical systems were studied: (1) acetone fixed cell monolayers in glass tubes and (2) antigen coated plastic beads. Both systems were senssitive and reproducible. The most sensitive, versatile system involves the coating of the plastic beads with nuclear antigen(s), incubation overnight with sera and labelling with /sup 125/I conjugated antihuman globulin. Linear binding of this radioactive tag is obtained over a wide range of SLE serum dilutions and the slopes of the serum dilution titration curves are almost identical for all SLE patients' sera tested. Therefore, a standard titration curve can be constructed from the results with a positive serum, and end-point dilutions of unknown sera estimated from results obtained with a single serum dilution. Alternatively, binding ratios of unknown sera can be usefully compared at fixed dilutions with standard positive and negative sera. For example, high binding ratios (>3.0) were obtained with 19/20 SLE sera and 0/20 control sera. Antigens used in these systems include crude, whole-cell lysates and lysates from purified nuclei.

Enhanced engraftment of mesenchymal stem cells in a cutaneous wound model by culture in allogenic species-specific serum and administration in fibrin constructs.

Science.gov (United States)

Gregory, Carl A; Reyes, Emigdio; Whitney, Mandolin J; Spees, Jeffrey L

2006-10-01

Human mesenchymal stem cells (hMSCs), also referred to as multipotent stromal cells, are currently being applied in clinical trials for bone diseases, graft versus host disease, and myocardial infarction. However, the standard growth medium for hMSCs contains 10%-20% fetal calf serum (FCS), and FCS is strongly immunogenic in both rodents and humans. Previously, we reported that by a sensitive fluorescence-based assay, 7-30 mg of internalized FCS is associated with 10(8) hMSCs, a dosage that will probably be needed for most therapies. We also found that a brief culture in medium containing autologous 20% adult human serum (AHS) or autologous 10% AHS supplemented with growth factors (AHS(+)) reduced the contamination by more than 99.9%. We have now extensively characterized the culture conditions and shown that hMSC expansion is possible using heterologous 20% AHS or heterologous 10% AHS(+). The uptake of FCS is an active process that acts to concentrate contamination in the cells even under low serum conditions (2% FCS) but can be actively displaced by incubation of the cells in medium with AHS. Rat MSCs (rMSCs) can be expanded under similar conditions using supplemented heterologous adult rat serum (ARS(+)). After expansion in FCS, a further 8 days of culture with ARS(+) significantly improves the viability of the rMSCs in vivo after encapsulation in fibrin followed by subcutaneous implantation in rats. Our results have the potential to dramatically improve cellular and genetic therapies using hMSCs.

Equine dendritic cells generated with horse serum have enhanced functionality in comparison to dendritic cells generated with fetal bovine serum.

Science.gov (United States)

Ziegler, Anja; Everett, Helen; Hamza, Eman; Garbani, Mattia; Gerber, Vinzenz; Marti, Eliane; Steinbach, Falko

2016-11-15

Dendritic cells are professional antigen-presenting cells that play an essential role in the initiation and modulation of T cell responses. They have been studied widely for their potential clinical applications, but for clinical use to be successful, alternatives to xenogeneic substances like fetal bovine serum (FBS) in cell culture need to be found. Protocols for the generation of dendritic cells ex vivo from monocytes are well established for several species, including horses. Currently, the gold standard protocol for generating dendritic cells from monocytes across various species relies upon a combination of GM-CSF and IL-4 added to cell culture medium which is supplemented with FBS. The aim of this study was to substitute FBS with heterologous horse serum. For this purpose, equine monocyte-derived dendritic cells (eqMoDC) were generated in the presence of horse serum or FBS and analysed for the effect on morphology, phenotype and immunological properties. Changes in the expression of phenotypic markers (CD14, CD86, CD206) were assessed during dendritic cell maturation by flow cytometry. To obtain a more complete picture of the eqMoDC differentiation and assess possible differences between FBS- and horse serum-driven cultures, a transcriptomic microarray analysis was performed. Lastly, immature eqMoDC were primed with a primary antigen (ovalbumin) or a recall antigen (tetanus toxoid) and, after maturation, were co-cultured with freshly isolated autologous CD5 + T lymphocytes to assess their T cell stimulatory capacity. The microarray analysis demonstrated that eqMoDC generated with horse serum were indistinguishable from those generated with FBS. However, eqMoDC incubated with horse serum-supplemented medium exhibited a more characteristic dendritic cell morphology during differentiation from monocytes. A significant increase in cell viability was also observed in eqMoDC cultured with horse serum. Furthermore, eqMoDC generated in the presence of horse serum

Serum neuron specific enolase - a novel indicator for neuropsychiatric systemic lupus erythematosus?

Science.gov (United States)

Hawro, T; Bogucki, A; Krupińska-Kun, M; Maurer, M; Woźniacka, A

2015-12-01

Neuropsychiatric (NP) lupus, a common manifestation of systemic lupus erythematosus (SLE), is still insufficiently understood, in part, because of the lack of specific biomarkers. Neuron specific enolase (NSE), an important neuronal glycolytic enzyme, shows increased serum levels following acute brain injury, and decreased serum levels in several chronic disorders of the nervous system, including multi infarct dementia, multiple sclerosis and depression. The aim of the study was to evaluate serum NSE levels in SLE patients with and without nervous system involvement, and in healthy controls, and to assess the correlation of NSE serum levels of patients with neuropsychiatric systemic lupus erythematosus (NPSLE) with clinical parameters. The study comprised 47 SLE patients and 28 controls. SLE activity was assessed using the Systemic Lupus Activity Measure (SLAM). A neurologist and a psychiatrist examined all patients. NP involvement was diagnosed according to strict NPSLE criteria proposed by Ainiala and coworkers, as modification to American College of Rheumatology (ACR) nomenclature and case definitions. NSE serum levels were determined by use of an immunoassay. Mean NSE serum concentrations in patients with NPSLE were significantly lower than in non-NPSLE patients (6.3 ± 2.6 µg/L vs. 9.7 ± 3.3 µg/L, p < 0.01) and in controls (8.8 ± 3.3 µg/L, p < 0.05). There were significant negative correlations between NSE serum levels and SLE activity (r = -0.42, p < 0.05) and the number of NPSLE manifestations diagnosed (-0.37; p = 0.001). Decreased serum concentrations of NSE may reflect chronic neuronal damage with declined metabolism of the nervous tissue in patients with NPSLE. © The Author(s) 2015.

Hand-made cloned goat (Capra hircus) embryos—a comparison of different donor cells and culture systems.

Science.gov (United States)

Akshey, Yogesh S; Malakar, Dhruba; De, Arun K; Jena, Manoj K; Garg, Shweta; Dutta, Rahul; Pawar, Sachin Kumar; Mukesh, Manisha

2010-10-01

Nuclear transfer is a very effective method for propagation of valuable, extinct, and endangered animals. Hand-made cloning (HMC) is an efficient alternative to the conventional micromanipulator-based technique in some domestic species. The present study was carried out for the selection of suitable somatic cells as a nuclear donor and development of an optimum culture system for in vitro culture of zona-free goat cloned embryos. Cleavage and blastocyst rates were observed 72.06 ± 2.94% and 0% for fresh cumulus cells, 81.95 ± 3.40% and 12.74 ± 2.12% for cultured cumulus cells, and 92.94 ± 0.91% and 23.78 ± 3.33% for fetal fibroblast cells, respectively. There was a significant (p cloned embryos and donor cells. In conclusion, the present study describes that the fetal fibroblast cell is a suitable candidate as nuclear donor, and the flat surface culture system is suitable for zona-free blastocyst development by the hand-made cloning technique in the goat.

Radioimmunological determination of reverse triiodo thyronine in unextracted serum and serum dialysates

International Nuclear Information System (INIS)

Laurberg, P.; Weeke, J.

1977-01-01

A sensitive radioimmunoassay for measurements of 3,3,5-triiodothyronine (reverse T3,rT3) in small amounts of unextracted serum is described. The interference of rT3 binding proteins in serum was precluded by addition of 8-anilinol-naphthalene sulphonic acid (ANS). The cross reaction of T4 with the rT3 anti-serum varied with the concentration of T4 in the samples. At 50 per cent inhibition of I 125 rT3 binding, the T4 cross reaction was 0.075% (mol/mol). All values were corrected for T4 cross reaction. By the present method total rT3 averaged 0.37 nmol/l in thirty-two normal subjects. Higher values (0.81-1.98 nmol/l) were obtained in seventeen thyrotoxic patients, while the rT3 was very low (<=0.046 nmol/l) in ten patients with severe primary hypothyroidism. A modification of the total rT3 assay was used for measurements of rT3 in serum dialysates (free rT3). The sensitivity was 0.46 pmol/l. This sensitivity did not allow detection of free rT3 in all normal subjects. (Auth.)

In vitro expansion and differentiation of rat pancreatic duct-derived stem cells into insulin secreting cells using a dynamicthree-dimensional cell culture system.

Science.gov (United States)

Chen, X C; Liu, H; Li, H; Cheng, Y; Yang, L; Liu, Y F

2016-06-27

In this study, a dynamic three-dimensional cell culture technology was used to expand and differentiate rat pancreatic duct-derived stem cells (PDSCs) into islet-like cell clusters that can secrete insulin. PDSCs were isolated from rat pancreatic tissues by in situ collagenase digestion and density gradient centrifugation. Using a dynamic three-dimensional culture technique, the cells were expanded and differentiated into functional islet-like cell clusters, which were characterized by morphological and phenotype analyses. After maintaining 1 x 108 isolated rat PDSCs in a dynamic three-dimensional cell culture for 7 days, 1.5 x 109 cells could be harvested. Passaged PDSCs expressed markers of pancreatic endocrine progenitors, including CD29 (86.17%), CD73 (90.73%), CD90 (84.13%), CD105 (78.28%), and Pdx-1. Following 14 additional days of culture in serum-free medium with nicotinamide, keratinocyte growth factor (KGF), and b fibroblast growth factor (FGF), the cells were differentiated into islet-like cell clusters (ICCs). The ICC morphology reflected that of fused cell clusters. During the late stage of differentiation, representative clusters were non-adherent and expressed insulin indicated by dithizone (DTZ)-positive staining. Insulin was detected in the extracellular fluid and cytoplasm of ICCs after 14 days of differentiation. Additionally, insulin levels were significantly higher at this time compared with the levels exhibited by PDSCs before differentiation (P cell culture system, PDSCs can be expanded in vitro and can differentiate into functional islet-like cell clusters.

Utility of free prostate specific antigen serum level and its related parameters in the diagnosis of prostate cancer

Directory of Open Access Journals (Sweden)

Azmi A Haroun

2011-01-01

Full Text Available We evaluated the role of free prostate specific antigen (f-PSA serum level and its related parameters in detecting prostate cancer. This retrospective study was conducted between January 2006 and March 2008. Trans-rectal ultrasound guided prostate biopsy was performed for 107 patients who had total PSA (t-PSA level of either >4 ng/mL with or without palpable nodule or ≤4 ng/mL with palpable nodule on digital rectal examination. The perfor-mance measurements for f-PSA, percent free PSA (%f-PSA and free PSA density (f-PSAD were determined and compared with those for t-PSA and total PSA density (t-PSAD. Descriptive statistics for all variables of interest were calculated, and receiver operating characteristic curves were generated. Nine patients (8.4% had normal histology, 69 patients (64.4% had benign disease and 29 patients (27.1% had prostate cancer. The performance of f-PSA in PCa detection was better than other evaluated parameters. The largest area under the curve for patients in the gray area (t-PSA range 4.1-10 ng/mL was for f-PSA, with a value of 0.64 and a sensitivity and specificity of 44% and 87%, respectively. For %f-PSA, these values were 0.59, 63% and 62%, respectively. For patients with a t-PSA level of 10.1-20 ng/mL, they were 0.68, 67%, and 81%, respectively, for f-PSA, and 0.64, 67%, and 76%, respectively, for %f-PSA. In conclusion, f-PSA serum levels performed better than free to total PSA ratio and t-PSA for prostate cancer screening. It is of clinical value which could affect the biopsy decision avoiding unnecessary interventions.

Diverse Requirements for Microglial Survival, Specification, and Function Revealed by Defined-Medium Cultures.

Science.gov (United States)

Bohlen, Christopher J; Bennett, F Chris; Tucker, Andrew F; Collins, Hannah Y; Mulinyawe, Sara B; Barres, Ben A

2017-05-17

Microglia, the resident macrophages of the CNS, engage in various CNS-specific functions that are critical for development and health. To better study microglia and the properties that distinguish them from other tissue macrophage populations, we have optimized serum-free culture conditions to permit robust survival of highly ramified adult microglia under defined-medium conditions. We find that astrocyte-derived factors prevent microglial death ex vivo and that this activity results from three primary components, CSF-1/IL-34, TGF-β2, and cholesterol. Using microglial cultures that have never been exposed to serum, we demonstrate a dramatic and lasting change in phagocytic capacity after serum exposure. Finally, we find that mature microglia rapidly lose signature gene expression after isolation, and that this loss can be reversed by engrafting cells back into an intact CNS environment. These data indicate that the specialized gene expression profile of mature microglia requires continuous instructive signaling from the intact CNS. Copyright © 2017 Elsevier Inc. All rights reserved.

Serum-dependent effects of tamoxifen and cannabinoids upon C6 glioma cell viability.

Science.gov (United States)

Jacobsson, S O; Rongård, E; Stridh, M; Tiger, G; Fowler, C J

2000-12-15

In the present study, the effects of the combination of tamoxifen ((Z)-2[p-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylamine citrate) and three cannabinoids (Delta(9)-tetrahydrocannabinol [Delta(9)-THC], cannabidiol, and anandamide [AEA]) upon the viability of C6 rat glioma cells was assessed at different incubation times and using different culturing concentrations of foetal bovine serum (FBS). Consistent with previous data for human glioblastoma cells, the tamoxifen sensitivity of the cells was increased as the FBS content of the culture medium was reduced from 10 to 0.4 and 0%. The cells expressed protein kinase C alpha and calmodulin (the concentration of which did not change significantly as the FBS concentration was reduced), but did not express estrogen receptors. Delta(9)-THC and cannabidiol, but not AEA, produced a modest reduction in cell viability after 6 days of incubation in serum-free medium, whereas no effects were seen in 10% FBS-containing medium. There was no observed synergy between the effects of tamoxifen and the cannabinoids upon cell viability.

Isolation of intracellular parasites (Plasmodium falciparum) from culture using free-flow electrophoresis: separation of the free parasites according to stages.

Science.gov (United States)

Heidrich, H G; Mrema, J E; Vander Jagt, D L; Reyes, P; Rieckmann, K H

1982-06-01

Parasitized human erythrocytes were concentrated from continuous cultures of Plasmodium falciparum from 5-7% up to 80-95% using Plasmagel. After aggregation of the cells with phythemagglutinin, the aggregated erythrocytes were fragmented by passing them, with minimal force, through successive nylon filters of decreasing pore size (100 microns-3 microns). The mixture of liberated, free parasites, intact erythrocytes and erythrocyte membrane vesicles was separated using free-flow electrophoresis. Most of the fractions containing free parasites did not show contamination with erythrocyte constituents as determined by light and electron microscopy, polyacrylamide gel electrophoresis, and enzymatic analysis. In addition, the various stages of free parasites of Plasmodium falciparum exhibited different electrical surface charges. Rings and trophozoites were highly negatively charged whereas schizonts and, in particular, merozoites showed low negative charges. Thus, the various stages could be isolated separate from each other.

[Detection of leptospira by culture of vitreous humor and detection of antibodies against leptospira in vitreous humor and serum of 225 horses with equine recurrent uveitis].

Science.gov (United States)

Dorrego-Keiter, Elisa; Tóth, József; Dikker, Lieke; Sielhorst, Jutta; Schusser, Gerald Fritz

2016-01-01

In the ongoing discussion regarding the aetiopathogenesis of equine recurrent uveitis (ERU) it was the aim of the present study to elucidate the relationship of leptospira infection and ERU. In a population of 225 horses leptospira were examined in vitreous humor by culture and leptospira antibody were detected in vitreous humor and serum samples. Preoperative serum samples were collected from 221/225 ERU patients of different age, gender and breed. Undiluted vitreous humor was aseptically taken from 198/225 patients that underwent pars plana vitrectomy at the beginning of surgery and from 27/225 patients' eyeball after enucleation: Serum and vitreous humor were tested for specific leptospiral antibodies by microscopic agglutination test (MAT). Furthermore, vitreous humor was examined by culture. 20 patients which were euthanized due to a live-threatening disease other than ERU served as a control group. A total of 127/221 (57.5%) horses had serum antibodies (≥ 1:100). Most frequently antibodies against L. interrogans serovar Grippotyphosa were detected (79/127), followed by L. interrogans serovar lcterohaemorrhagiae (34/127) and L. interrogans serovar Bratislava (29/127). Only 79/225 horses (35.1%) had leptospiral antibodies in vitreous humor, in which L. interrogans serovar Grippotyphosa (67/79) was identified most frequently followed by L. interrogans serovar Pomona (18/79) and L. interrogans serovar lcterohaemorrhagiae (8/79) which was identified as single or multiple reaction. Isolation of leptospira from vitreous humor was positive in 34/212 horses (16%). 10/20 control horses had a positive antibody titer against leptospira in serum and 2/20 horses in vitreous humor, whereas there was no leptospira detected in culture. The result of 84% negative cultures from vitreous humor of 212 ERU patients is decisive for the diagnosis and therapy of ERU.

Purification and characterization of enterovirus 71 viral particles produced from vero cells grown in a serum-free microcarrier bioreactor system.

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Chia-Chyi Liu

Full Text Available BACKGROUND: Enterovirus 71 (EV71 infections manifest most commonly as a childhood exanthema known as hand-foot-and-mouth disease (HFMD and can cause neurological disease during acute infection. PRINCIPAL FINDING: In this study, we describe the production, purification and characterization of EV71 virus produced from Vero cells grown in a five-liter serum-free bioreactor system containing 5 g/L Cytodex 1 microcarrier. The viral titer was >10(6 TCID(50/mL by 6 days post infection when a MOI of 10(-5 was used at the initial infection. Two EV71 virus fractions were separated and detected when the harvested EV71 virus concentrate was purified by sucrose gradient zonal ultracentrifugation. The EV71 viral particles detected in the 24-28% sucrose fractions had an icosahedral structure 30-31 nm in diameter and had low viral infectivity and RNA content. Three major viral proteins (VP0, VP1 and VP3 were observed by SDS-PAGE. The EV71 viral particles detected in the fractions containing 35-38% sucrose were 33-35 nm in size, had high viral infectivity and RNA content, and were composed of four viral proteins (VP1, VP2, VP3 and VP4, as shown by SDS-PAGE analyses. The two virus fractions were formalin-inactivated and induced high virus neutralizing antibody responses in mouse immunogenicity studies. Both mouse antisera recognized the immunodominant linear neutralization epitope of VP1 (residues 211-225. CONCLUSION: These results provide important information for cell-based EV71 vaccine development, particularly for the preparation of working standards for viral antigen quantification.

Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications

NARCIS (Netherlands)

Naaijkens, B.A.; Niessen, H.W.M.; Prins, H.J.; Krijnen, P.A.J.; Kokhuis, T.J.A.; de Jong, N.; van Hinsbergh, V.W.M.; Kamp, O.; Helder, M.N.; Musters, R.J.P.; van Dijk, A.; Juffermans, L.J.M.

2012-01-01

Adipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically,

Human serum activates CIDEB-mediated lipid droplet enlargement in hepatoma cells

Energy Technology Data Exchange (ETDEWEB)

Singaravelu, Ragunath [Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Lyn, Rodney K. [Department of Chemistry, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Srinivasan, Prashanth [National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Delcorde, Julie [Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Steenbergen, Rineke H.; Tyrrell, D. Lorne [Department of Medical Microbiology and Immunology, University of Alberta (Canada); Li Ka Shing Institute of Virology, Katz Centre for Pharmacy and Health Research, Edmonton, Alberta T6G 2S2 (Canada); Pezacki, John P., E-mail: John.Pezacki@nrc-cnrc.gc.ca [Department of Chemistry, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada)

2013-11-15

Highlights: •Human serum induced differentiation of hepatoma cells increases cellular lipid droplet (LD) size. •The observed increase in LD size correlates with increased PGC-1α and CIDEB expression. •Induction of CIDEB expression correlates with rescue of VLDL secretion and loss of ADRP. •siRNA knockdown of CIDEB impairs the human serum mediated increase in LD size. •This system represents a cost-efficient model to study CIDEB’s role in lipid biology. -- Abstract: Human hepatocytes constitutively express the lipid droplet (LD) associated protein cell death-inducing DFFA-like effector B (CIDEB). CIDEB mediates LD fusion, as well as very-low-density lipoprotein (VLDL) maturation. However, there are limited cell culture models readily available to study CIDEB’s role in these biological processes, as hepatoma cell lines express negligible levels of CIDEB. Recent work has highlighted the ability of human serum to differentiate hepatoma cells. Herein, we demonstrate that culturing Huh7.5 cells in media supplemented with human serum activates CIDEB expression. This activation occurs through the induced expression of PGC-1α, a positive transcriptional regulator of CIDEB. Coherent anti-Stokes Raman scattering (CARS) microscopy revealed a correlation between CIDEB levels and LD size in human serum treated Huh7.5 cells. Human serum treatment also resulted in a rapid decrease in the levels of adipose differentiation-related protein (ADRP). Furthermore, individual overexpression of CIDEB was sufficient to down-regulate ADRP protein levels. siRNA knockdown of CIDEB revealed that the human serum mediated increase in LD size was CIDEB-dependent. Overall, our work highlights CIDEB’s role in LD fusion, and presents a new model system to study the PGC-1α/CIDEB pathway’s role in LD dynamics and the VLDL pathway.

Human serum activates CIDEB-mediated lipid droplet enlargement in hepatoma cells

International Nuclear Information System (INIS)

Singaravelu, Ragunath; Lyn, Rodney K.; Srinivasan, Prashanth; Delcorde, Julie; Steenbergen, Rineke H.; Tyrrell, D. Lorne; Pezacki, John P.

2013-01-01

Highlights: •Human serum induced differentiation of hepatoma cells increases cellular lipid droplet (LD) size. •The observed increase in LD size correlates with increased PGC-1α and CIDEB expression. •Induction of CIDEB expression correlates with rescue of VLDL secretion and loss of ADRP. •siRNA knockdown of CIDEB impairs the human serum mediated increase in LD size. •This system represents a cost-efficient model to study CIDEB’s role in lipid biology. -- Abstract: Human hepatocytes constitutively express the lipid droplet (LD) associated protein cell death-inducing DFFA-like effector B (CIDEB). CIDEB mediates LD fusion, as well as very-low-density lipoprotein (VLDL) maturation. However, there are limited cell culture models readily available to study CIDEB’s role in these biological processes, as hepatoma cell lines express negligible levels of CIDEB. Recent work has highlighted the ability of human serum to differentiate hepatoma cells. Herein, we demonstrate that culturing Huh7.5 cells in media supplemented with human serum activates CIDEB expression. This activation occurs through the induced expression of PGC-1α, a positive transcriptional regulator of CIDEB. Coherent anti-Stokes Raman scattering (CARS) microscopy revealed a correlation between CIDEB levels and LD size in human serum treated Huh7.5 cells. Human serum treatment also resulted in a rapid decrease in the levels of adipose differentiation-related protein (ADRP). Furthermore, individual overexpression of CIDEB was sufficient to down-regulate ADRP protein levels. siRNA knockdown of CIDEB revealed that the human serum mediated increase in LD size was CIDEB-dependent. Overall, our work highlights CIDEB’s role in LD fusion, and presents a new model system to study the PGC-1α/CIDEB pathway’s role in LD dynamics and the VLDL pathway

Human platelet lysate as a fetal bovine serum substitute improves human adipose-derived stromal cell culture for future cardiac repair applications

NARCIS (Netherlands)

B. Naaijkens (Benno); H.W.M. Niessen (Hans ); H.-J. Prins (H.); P.A.J. Krijnen (Paul); T.J.A. Kokhuis (Tom); N. de Jong (Nico); V.W.M. van Hinsbergh (Victor); O. Kamp (Otto); K. Helder MScN (Onno); R.J.P. Musters (René); A. van Dijk (Annemieke); L.J.M. Juffermans (Lynda)

2012-01-01

textabstractAdipose-derived stromal cells (ASC) are promising candidates for cell therapy, for example to treat myocardial infarction. Commonly, fetal bovine serum (FBS) is used in ASC culturing. However, FBS has several disadvantages. Its effects differ between batches and, when applied clinically,

Human umbilical cord mesenchymal stromal cells exhibit immature nucleus pulposus cell phenotype in a laminin-rich pseudo-three-dimensional culture system.

Science.gov (United States)

Chon, Brian H; Lee, Esther J; Jing, Liufang; Setton, Lori A; Chen, Jun

2013-10-02

Cell supplementation to the herniated or degenerated intervertebral disc (IVD) is a potential strategy to promote tissue regeneration and slow disc pathology. Human umbilical cord mesenchymal stromal cells (HUCMSCs) - originating from the Wharton's jelly - remain an attractive candidate for such endeavors with their ability to differentiate into multiple lineages. Previously, mesenchymal stem cells (MSCs) have been studied as a potential source for disc tissue regeneration. However, no studies have demonstrated that MSCs can regenerate matrix with unique characteristics matching that of immature nucleus pulposus (NP) tissues of the IVD. In our prior work, immature NP cells were found to express specific laminin isoforms and laminin-binding receptors that may serve as phenotypic markers for evaluating MSC differentiation to NP-like cells. The goal of this study is to evaluate these markers and matrix synthesis for HUCMSCs cultured in a laminin-rich pseudo-three-dimensional culture system. HUCMSCs were seeded on top of Transwell inserts pre-coated with Matrigel™, which contained mainly laminin-111. Cells were cultured under hypoxia environment with three differentiation conditions: NP differentiation media (containing 2.5% Matrigel™ solution to provide for a pseudo-three-dimensional laminin culture system) with no serum, or the same media supplemented with either insulin-like growth factor-1 (IGF-1) or transforming growth factor-β1 (TGF-β1). Cell clustering behavior, matrix production and the expression of NP-specific laminin and laminin-receptors were evaluated at days 1, 7, 13 and 21 of culture. Data show that a pseudo-three-dimensional culture condition (laminin-1 rich) promoted HUCMSC differentiation under no serum conditions. Starting at day 1, HUCMSCs demonstrated a cell clustering morphology similar to that of immature NP cells in situ and that observed for primary immature NP cells within the similar laminin-rich culture system (prior study

Studies on Using Cattle and Sheep Hydatid Cyst Fluid Instead of the Fetal Calf Serum in Leishmania Culture

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Hossein Rezvan

2013-12-01

Full Text Available Background: Leishmania is a single cell parasite causing leishmaniasis, which is a common disease between humans and animals. Due to the importance of in-vitro culture of the parasite in leishmania research, developing new methods for in-vitro cultivation of the parasite has always been a goal for leishmania researchers. The main objective of7T 5T7Tthis study was to use sheep and bovine hydatid cyst fluids as alternatives for fetal calf serum (FCS in leishmania in-vitro5T culture5T. Materials and Methods: 7TA total of 5T7T1 million leishmania promastigotes were added to 4 flasks as follow5T7T. A f5T7Tlask containing DMEM medium with 105T7T% 5T7Tfetal bovine serum5T7T, a f5T7Tlask containing DMEM and 10% sheep hydatid cyst fluid5T7T, a f5T7Tlask containing DMEM medium with 105T7T% 5T7Tbovine hydatid cyst fluid and a5T7T f5T7Tlask containing DMEM medium alone. After 2, 45T7T, 5T7T7, 95T7T, 11, 5T7T21 and 24 days, the number of parasites were counted and compared5T7T. Results: The result of this study showed that, DMEM medium enriched with 10% sheep hydatid cyst fluid in 168 hours and medium enriched with 10% bovine hydatid cyst fluid in 96 hours can act as a good alternative for fetal bovine serum in the culture Leishmania major. Conclusion: 5TThe results showed that sheep and bovine hydatid cyst fluid can be used as alternatives to FCS for dense cultivation of leishmania. The results also showed that5T, 5Tthe growth of promastigotes in medium enriched with bovine cyst fluid is more rapid than the medium enriched with sheep5T c5Tyst fluid5T in5T the beginning of cultivation.

Can cultural differences lead to accidents? Team cultural differences and sociotechnical system operations.

Science.gov (United States)

Strauch, Barry

2010-04-01

I discuss cultural factors and how they may influence sociotechnical system operations. Investigations of several major transportation accidents suggest that cultural factors may have played a role in the causes of the accidents. However, research has not fully addressed how cultural factors can influence sociotechnical systems. I review literature on cultural differences in general and cultural factors in sociotechnical systems and discuss how these differences can affect team performance in sociotechnical systems. Cultural differences have been observed in social and interpersonal dimensions and in cognitive and perceptual styles; these differences can affect multioperator team performance. Cultural factors may account for team errors in sociotechnical systems, most likely during high-workload, high-stress operational phases. However, much of the research on cultural factors has methodological and interpretive shortcomings that limit their applicability to sociotechnical systems. Although some research has been conducted on the role of cultural differences on team performance in sociotechnical system operations, considerable work remains to be done before the effects of these differences can be fully understood. I propose a model that illustrates how culture can interact with sociotechnical system operations and suggest avenues of future research. Given methodological challenges in measuring cultural differences and team performance in sociotechnical system operations, research in these systems should use a variety of methodologies to better understand how culture can affect multioperator team performance in these systems.

Label-free LC-MSe in tissue and serum reveals protein networks underlying differences between benign and malignant serous ovarian tumors.

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Wouter Wegdam

Full Text Available PURPOSE: To identify proteins and (molecular/biological pathways associated with differences between benign and malignant epithelial ovarian tumors. EXPERIMENTAL PROCEDURES: Serum of six patients with a serous adenocarcinoma of the ovary was collected before treatment, with a control group consisting of six matched patients with a serous cystadenoma. In addition to the serum, homogeneous regions of cells exhibiting uniform histology were isolated from benign and cancerous tissue by laser microdissection. We subsequently employed label-free liquid chromatography tandem mass spectrometry (LC-MSe to identify proteins in these serum and tissues samples. Analyses of differential expression between samples were performed using Bioconductor packages and in-house scripts in the statistical software package R. Hierarchical clustering and pathway enrichment analyses were performed, as well as network enrichment and interactome analysis using MetaCore. RESULTS: In total, we identified 20 and 71 proteins that were significantly differentially expressed between benign and malignant serum and tissue samples, respectively. The differentially expressed protein sets in serum and tissue largely differed with only 2 proteins in common. MetaCore network analysis, however inferred GCR-alpha and Sp1 as common transcriptional regulators. Interactome analysis highlighted 14-3-3 zeta/delta, 14-3-3 beta/alpha, Alpha-actinin 4, HSP60, and PCBP1 as critical proteins in the tumor proteome signature based on their relative overconnectivity. The data have been deposited to the ProteomeXchange with identifier PXD001084. DISCUSSION: Our analysis identified proteins with both novel and previously known associations to ovarian cancer biology. Despite the small overlap between differentially expressed protein sets in serum and tissue, APOA1 and Serotransferrin were significantly lower expressed in both serum and cancer tissue samples, suggesting a tissue-derived effect in serum

Nanoparticles of Conjugated Methotrexate-Human Serum Albumin: Preparation and Cytotoxicity Evaluations

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Azade Taheri

2011-01-01

Full Text Available Methotrexate-human serum albumin conjugates were developed by a simple carbodiimide reaction. Methotrexate-human serum albumin conjugates were then crosslinked with 1-ethyl-3-(3-dimethylaminopropyl carbodiimide HCl (EDC to form nanoparticles. The size of nanoparticles determined by laser light scattering and TEM was between 90–150 nm. Nanoparticles were very stable at physiologic conditions (PBS pH 7.4, 37∘C and after incubation with serum. The effect of amount of EDC used for crosslinking on the particle size and free amino groups of nanoparticles was examined. The amount of crosslinker showed no significant effect on the size of nanoparticles but free amino groups of nanoparticles were decreased by increasing the crosslinker. The physicochemical interactions between methotrexate and human serum albumin were investigated by differential scanning calorimetry (DSC. Nanoparticles were more cytotoxic on T47D cells compared to free methotrexate. Moreover, methotrexate-human serum albumin nanoparticles decreased the IC50 value of methotrexate on T47D cells in comparison with free methotrexate.

Nanoparticles of Conjugated Methotrexate-Human Serum Albumin: Preparation and Cytotoxicity Evaluations

International Nuclear Information System (INIS)

Taheri, A.; Atyabi, F.; Nouri, F.S.; Ahadi, F.; Derakhshan, M.A.; Dinarvand, R.; Atyabi, F.; Ghahremani, M.H.; Ostad, S.N.; Dinarvand, R.; Amini, M.; Ghahremani, M.H.; Ostad, S.N.; Mansoori, P.

2011-01-01

Methotrexate-human serum albumin conjugates were developed by a simple carbodiimide reaction. Methotrexate-human serum albumin conjugates were then crosslinked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (EDC) to form nanoparticles. The size of nanoparticles determined by laser light scattering and TEM was between 90 150 nm. Nanoparticles were very stable at physiologic conditions (PBS pH 7.4, 37 degree C) and after incubation with serum. The effect of amount of EDC used for crosslinking on the particle size and free amino groups of nanoparticles was examined. The amount of cross linker showed no significant effect on the size of nanoparticles but free amino groups of nanoparticles were decreased by increasing the cross linker. The physicochemical interactions between methotrexate and human serum albumin were investigated by differential scanning calorimetry (DSC). Nanoparticles were more cytotoxic on T 47 D cells compared to free methotrexate. Moreover, methotrexate-human serum albumin nanoparticles decreased the C50 value of methotrexate on T 47 D cells in comparison with free methotrexate.

Evaluation of serum and milk ELISAs for paratuberculosis in Danish dairy cattle

DEFF Research Database (Denmark)

Klausen, Joan; Huda, A.; Ekeroth, Lars

2003-01-01

concurrently from six dairy herds infected with MAP and from two dairy herds without history of infection with MAP. A cut-off value of 7 OD% was used in the ELISAs. At this cut-off value, all six culture-positive herds were positive in the serum ELISA but one was negative in the milk ELISA. All six culture......-positive herds were positive in the CFT. In the two culture-negative herds, the serum and the milk ELISA deemed all serum samples negative at this cut-off value, whereas four serum samples from one of these herds were positive in the CFT. The highest cut-off value enabling the milk ELISA to record all six...... culture-positive herds as positive was 4 OD%. The highest cut-off value enabling the serum ELISA to record all six culture-positive herds as positive was 17 OD%. Individual-sample relative sensitivities of the ELISAs ranged from 49 to 64% and relative specificities were 80-96% at the cut-off values of 4...

Comparison between human serum and Albuminar-20 (TM) supplement for in-vitro fertilization.

Science.gov (United States)

Staessen, C; Van den Abbeel, E; Carlé, M; Khan, I; Devroey, P; Van Steirteghem, A C

1990-04-01

Patient or fetal cord serum is commonly used as a protein supplement to culture media used in in-vitro fertilization (IVF). To eliminate the variability and possible hazards related to the use of human serum, a well-defined protein supplement, Albuminar-20 (Armour Pharmaceutical Cy) was evaluated as a substitute for serum. Prior to its application in the human, Earle's culture media supplemented with 0.5% (w/v) bovine serum albumin, 8% (v/v) decomplemented patient serum or 2.25% (v/v) Albuminar-20 were compared in a mouse bioassay. For the three different conditions, the percentages of blastocysts formed after 120 h in-vitro culture were respectively 91.2, 85.2 and 87.8% (NS). In the human IVF, a controlled comparison was performed from October to December 1988, between Earle's medium supplemented with patients' serum or Albuminar-20. When oocytes and spermatozoa were cultured in these two media, the fertilization rates were similar, 58.9% in human serum versus 59.4% in Albuminar-20. After further culture, the morphological quality of the cleaved embryos was better in the embryos cultured in Albuminar-20. The higher pregnancy rate in Albuminar-20 was correlated with the better morphological appearance of the embryos and their more advanced cleavage stage at the time of transfer. Therefore, Albuminar-20 can be considered as a suitable protein supplement in human IVF.

Measurement of rat serum FSH by radioreceptor assay and comparison with radioimmunoassay

International Nuclear Information System (INIS)

Minegishi, Takashi; Igarashi, Masao; Wakabayashi, Katsumi

1980-01-01

Follicle stimulating hormone (FSH) in rat serum is successfully measured by a radioreceptor assay system employing PMS-treated immature rat ovary. The non-specific inhibitory effect of serum was partially overcome by the addition of merthiolate to every component, while the residual effect was compensated for by using FSH-free serum which was prepared by passing the pooled female diestrous rat sera through an immunoadsorbent column packed with anti-ovine FSH-coupled Sepharose 4B. The assay system consisted of 100 μl of Tris-MgCl 2 -BSA or standard, 100 μl of FSH-free serum or sample, 100 μl of the receptor preparation and 100 μl of 125 I-FSH. The incubation was carried out for 4 hr at 37 0 C and 500 μl of cold Tris-MgCl 2 -BSA was used for the termination. Serum FSH could be measured within a range of 0.125 - 16 ng NIAMDD rat FSH I-3/tube. The mean within-assay coefficient of variation was 10.5%. The mean between-assay coefficient of variation was 11.0%. The assay values obtained by RRA showed a good correlation to those by RIA under the same physiological states of the animals. The ratio of the assay values, RRA/RIA, was found to change according to the sex and the physiological states, e.g. around 1.3 in normal males and 1.7 in orchiectomized animals and 2.21 in female rats. Serum FSH levels in female rats obtained by RRA and RIA changed almost in parallel until 20:00 (hr) of proestrous day, but after the first surge of serum FSH they were not parallel. These facts seem to indicate possible changes in the affinity of FSH with its receptor according to the state of animals and lead to the problem of the heterogeneity of FSH. (author)

Dynamic quantitative analysis of adherent cell cultures by means of lens-free video microscopy

Science.gov (United States)

Allier, C.; Vincent, R.; Navarro, F.; Menneteau, M.; Ghenim, L.; Gidrol, X.; Bordy, T.; Hervé, L.; Cioni, O.; Bardin, S.; Bornens, M.; Usson, Y.; Morales, S.

2018-02-01

We present our implementation of lens-free video microscopy setup for the monitoring of adherent cell cultures. We use a multi-wavelength LED illumination together with a dedicated holographic reconstruction algorithm that allows for an efficient removal of twin images from the reconstructed phase image for densities up to those of confluent cell cultures (>500 cells/mm2). We thereby demonstrate that lens-free video microscopy, with a large field of view ( 30 mm2) can enable us to capture the images of thousands of cells simultaneously and directly inside the incubator. It is then possible to trace and quantify single cells along several cell cycles. We thus prove that lens-free microscopy is a quantitative phase imaging technique enabling estimation of several metrics at the single cell level as a function of time, for example the area, dry mass, maximum thickness, major axis length and aspect ratio of each cell. Combined with cell tracking, it is then possible to extract important parameters such as the initial cell dry mass (just after cell division), the final cell dry mass (just before cell division), the average cell growth rate, and the cell cycle duration. As an example, we discuss the monitoring of a HeLa cell cultures which provided us with a data-set featuring more than 10 000 cell cycle tracks and more than 2x106 cell morphological measurements in a single time-lapse.

Stem cell recovering effect of copper-free GHK in skin.

Science.gov (United States)

Choi, Hye-Ryung; Kang, Youn-A; Ryoo, Sun-Jong; Shin, Jung-Won; Na, Jung-Im; Huh, Chang-Hun; Park, Kyoung-Chan

2012-11-01

The peptide Gly-His-Lys (GHK) is a naturally occurring copper(II)-chelating motifs in human serum and cerebrospinal fluid. In industry, GHK (with or without copper) is used to make hair and skin care products. Copper-GHK plays a physiological role in the process of wound healing and tissue repair by stimulating collagen synthesis in fibroblasts. We also reported that copper-GHK promotes the survival of basal stem cells in the skin. However, the effects of copper-free GHK (GHK) have not been investigated well. In this study, the effects of GHK were studied using cultured normal human keratinocytes and skin equivalent (SE) models. In monolayer cultured keratinocytes, GHK increased the proliferation of keratinocytes. When GHK was added during the culture of SE models, the basal cells became more cuboidal than control model. In addition, there was linear and intense staining of α6 and β1 integrin along the basement membrane. The number of p63 and proliferating cell nuclear antigen positive cells was also significantly increased in GHK-treated SEs than in control SEs. Western blot and slide culture experiment showed that GHK increased the expression of integrin by keratinocytes. All these results showed that GHK increased the stemness and proliferative potential of epidermal basal cells, which is associated with increased expression of integrin. In conclusion, copper-free GHK showed similar effects with copper-GHK. Thus, it can be said that copper-free GHK can be used in industry to obtain the effects of copper-GHK in vivo. Further study is necessary to explore the relationship between copper-free GHK and copper-GHK. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

[Characterization of epithelial primary culture from human conjunctiva].

Science.gov (United States)

Rivas, L; Blázquez, A; Muñoz-Negrete, F J; López, S; Rebolleda, G; Domínguez, F; Pérez-Esteban, A

2014-01-01

To evaluate primary cultures from human conjunctiva supplemented with fetal bovine serum, autologous serum, and platelet-rich autologous serum, over human amniotic membrane and lens anterior capsules. One-hundred and forty-eight human conjunctiva explants were cultured in CnT50(®) supplemented with 1, 2.5, 5 and 10% fetal bovine serum, autologous serum and platelet-rich autologous serum. Conjunctival samples were incubated at 37°C, 5% CO2 and 95% HR, for 3 weeks. The typical phenotype corresponding to conjunctival epithelial cells was present in all primary cultures. Conjunctival cultures had MUC5AC-positive secretory cells, K19-positive conjunctival cells, and MUC4-positive non-secretory conjunctival cells, but were not corneal phenotype (cytokeratin K3-negative) and fibroblasts (CD90-negative). Conjunctiva epithelial progenitor cells were preserved in all cultures; thus, a cell culture in CnT50(®) supplemented with 1 to 5% autologous serum over human amniotic membrane can provide better information of epithelial cell differentiation for the conjunctival surface reconstruction. Copyright © 2013 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

Effect of Culture System on Developmental Competence, Cryosurvival and DNA-Fragmentation of In Vitro Bovine Blastocysts

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Mahdi Hajian

2011-01-01

Full Text Available Background: This study investigated the effect of two in vitro embryo culture systems (co-culturesystem versus cell-free sequential-media on developmental competence, cryosurvival and DNAfragmentationof in vitro developed bovine blastocysts.Materials and Methods: Bovine presumptive zygotes were cultured in Ménézo's B2 (B2 plusvero-cells or sequential synthetic oviductal fluid (SOF for eight days. Subsequently, half of theexpanded blastocysts developed in both groups were vitrified, warmed within 30 minutes and postwarmingembryos along with their corresponding non-vitrified embryos were cultured for twoadditional days in the same medium used before vitrification. Embryo development, cryosurvivaland apoptosis were compared between the groups.Results: For non-vitrified embryos, culture in SOF significantly promoted the potency of embryosto develop into blastocysts compared with the co-culture system. The difference in post vitrificationsurvival rate of SOF blastocysts (83.3% was insignificant compared with co-culture (84.3%.However, while total cell number of warmed blastocysts in the co-culture system was significantlyhigher in the co-culture versus the sequential system (215.4 vs. 170.4, the quality of survived embryosin terms of hatching ability and apoptosis was adversely affected by co-culture compared with SOF(65.0% vs. 74.3%, and 13.5% vs. 10.0%, respectively; p<0.05.Conclusion: Although co-culture system may increase the viability of embryos followingcryopreservation, the potency and dynamics of blastocyst formation significantly increased withsequential media compared to the co-culture system which can compensate for the lower efficiency ofsequential media for vitrification/warming purposes.

Association of PSA, free-PSA and testosteron levels in serum of patients with benign prostate hyperplasia (BPH) and prostate cancer

International Nuclear Information System (INIS)

Wiwin Mailana; Kristina Dwi P; Sri Insani WW; Puji Widayati

2015-01-01

Prostate cancer screening can be done by measuring the concentration levels of PSA, free-PSA and testosterone in serum that examined with radioimmunoassay (RIA). A total of 30 patients of 45-81 years old had enrolled in this study and were taken their venous blood. The aim of research is to know the relationship between PSA and testosterone free-PSA with BPH and prostate cancer. Results showed that there was no correlation between age with BPH and prostate cancer (p = 0.06), but there is a relationship between PSA with BPH and prostate cancer (p = 0.002), the relationship between free-PSA with BPH and prostate cancer (p = 0.001). No correlation was found between PSA ratio with BPH and prostate cancer as well as the absence of a relationship between testosterone with BPH and prostate cancer (p = 0.924). (author)

Konsentrasi Serum Anjing yang Optimum untuk Menumbuhkan dan Memelihara Babesia canis dalam Biakan

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Tutuk Astyawati

2010-12-01

Full Text Available The use of cultivation system in vitro is very important in the future study of Babesia canis. Theaim of this study was to cultivate B. canis in vitro using RPMI media with different concentration of dogsera. B. canis infected erythrocytes were collected from splenectomized infected dog. Parasites werecultivated with RPMI 1640 medium supplemented with normal dog sera at the concentration of 10%, 20%and 40%, the culture were then incubated in 5% CO2 , 37oC temperature for 17 days and subcultured every48 hours. The Percentage of Parasitized Erythrocytes (PPE in culture with 10% dog serum was significantlylower than those 20%, and 40% The used of 20% and 40 % sera were better than 10%. It is recommendedthat 40 % serum can be used for initiation phase of cultivation, whereas 20% concentration were used formaintenance of the culture.

Circulating angiostatin serum level in patients with systemic sclerosis

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Zofia Gerlicz-Kowalczuk

2017-12-01

Full Text Available Introduction : Systemic sclerosis (SSc is achronic connective tissue disease characterized by microangiopathy with inadequate angiogenesis. Angiostatin (AS is a potent antiangiogenic factor specifically inhibiting proliferation and inducing apoptosis of vascular endothelial cells. Aim : To evaluate the level of angiostatin in the serum of patients with SSc. Material and methods : Serum levels of AS were measured in 20 SSc patients and 12 healthy controls. Results : A statistically significant difference in the serum levels of AS in SSc patients was observed compared to the control group (636.51 vs. 869.20 ng/ml; p = 0.012. Significant correlations between limited and disseminated SSc (lSSc/dSSc were not found, however, a difference between lSSc and the control group was demonstrated (620.00 vs. 869.20 ng/ml; p = 0.011. The serum level of AS was not associated positively with organ changes caused by SSc. However, a statistically significant lower serum level of AS was observed in patients with SSc and no esophageal (p = 0.008 or pulmonary changes (p = 0.007 compared to the control group. Conclusions : Our results reveal significant differences in AS level in SSc patients compared to the healthy controls, and suggest that a low level of AS may occur as a result of impaired angiogenesis.

Cultured Cortical Neurons Can Perform Blind Source Separation According to the Free-Energy Principle

Science.gov (United States)

Isomura, Takuya; Kotani, Kiyoshi; Jimbo, Yasuhiko

2015-01-01

Blind source separation is the computation underlying the cocktail party effect––a partygoer can distinguish a particular talker’s voice from the ambient noise. Early studies indicated that the brain might use blind source separation as a signal processing strategy for sensory perception and numerous mathematical models have been proposed; however, it remains unclear how the neural networks extract particular sources from a complex mixture of inputs. We discovered that neurons in cultures of dissociated rat cortical cells could learn to represent particular sources while filtering out other signals. Specifically, the distinct classes of neurons in the culture learned to respond to the distinct sources after repeating training stimulation. Moreover, the neural network structures changed to reduce free energy, as predicted by the free-energy principle, a candidate unified theory of learning and memory, and by Jaynes’ principle of maximum entropy. This implicit learning can only be explained by some form of Hebbian plasticity. These results are the first in vitro (as opposed to in silico) demonstration of neural networks performing blind source separation, and the first formal demonstration of neuronal self-organization under the free energy principle. PMID:26690814

Cultured Cortical Neurons Can Perform Blind Source Separation According to the Free-Energy Principle.

Directory of Open Access Journals (Sweden)

Takuya Isomura

2015-12-01

Full Text Available Blind source separation is the computation underlying the cocktail party effect--a partygoer can distinguish a particular talker's voice from the ambient noise. Early studies indicated that the brain might use blind source separation as a signal processing strategy for sensory perception and numerous mathematical models have been proposed; however, it remains unclear how the neural networks extract particular sources from a complex mixture of inputs. We discovered that neurons in cultures of dissociated rat cortical cells could learn to represent particular sources while filtering out other signals. Specifically, the distinct classes of neurons in the culture learned to respond to the distinct sources after repeating training stimulation. Moreover, the neural network structures changed to reduce free energy, as predicted by the free-energy principle, a candidate unified theory of learning and memory, and by Jaynes' principle of maximum entropy. This implicit learning can only be explained by some form of Hebbian plasticity. These results are the first in vitro (as opposed to in silico demonstration of neural networks performing blind source separation, and the first formal demonstration of neuronal self-organization under the free energy principle.

A novel liquid medium for the efficient growth of the salmonid pathogen Piscirickettsia salmonis and optimization of culture conditions.

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Mirtha Henríquez

Full Text Available Piscirickettsia salmonis is the bacterium that causes Piscirickettsiosis, a systemic disease of salmonid fish responsible for significant economic losses within the aquaculture industry worldwide. The growth of the bacterium for vaccine formulation has been traditionally accomplished by infecting eukaryotic cell lines, a process that involves high production costs and is time-consuming. Recent research has demonstrated that it is possible to culture pure P. salmonis in a blood containing (cell-free medium. In the present work we demonstrate the growth of P. salmonis in a liquid medium free from blood and serum components, thus establishing a novel and simplified bacteriological medium. Additionally, the new media reported provides improved growth conditions for P. salmonis, where biomass concentrations of approximately 800 mg cell dry weight L(-1 were obtained, about eight times higher than those reported for the blood containing medium. A 2- level full factorial design was employed to evaluate the significance of the main medium components on cell growth and an optimal temperature range of 23-27°C was determined for the microorganism to grow in the novel liquid media. Therefore, these results represent a breakthrough regarding P. salmonis research in order to optimize pure P. salmonis growth in liquid blood and serum free medium.

Differentiation of monkey embryonic stem cells to hepatocytes by feeder-free dispersion culture and expression analyses of cytochrome p450 enzymes responsible for drug metabolism.

Science.gov (United States)

Maruyama, Junya; Matsunaga, Tamihide; Yamaori, Satoshi; Sakamoto, Sakae; Kamada, Noboru; Nakamura, Katsunori; Kikuchi, Shinji; Ohmori, Shigeru

2013-01-01

We reported previously that monkey embryonic stem cells (ESCs) were differentiated into hepatocytes by formation of embryoid bodies (EBs). However, this EB formation method is not always efficient for assays using a large number of samples simultaneously. A dispersion culture system, one of the differentiation methods without EB formation, is able to more efficiently provide a large number of feeder-free undifferentiated cells. A previous study demonstrated the effectiveness of the Rho-associated kinase inhibitor Y-27632 for feeder-free dispersion culture and induction of differentiation of monkey ESCs into neural cells. In the present study, the induction of differentiation of cynomolgus monkey ESCs (cmESCs) into hepatocytes was performed by the dispersion culture method, and the expression and drug inducibility of cytochrome P450 (CYP) enzymes in these hepatocytes were examined. The cmESCs were successfully differentiated into hepatocytes under feeder-free dispersion culture conditions supplemented with Y-27632. The hepatocytes differentiated from cmESCs expressed the mRNAs for three hepatocyte marker genes (α-fetoprotein, albumin, CYP7A1) and several CYP enzymes, as measured by real-time polymerase chain reaction. In particular, the basal expression of cmCYP3A4 (3A8) in these hepatocytes was detected at mRNA and enzyme activity (testosterone 6β-hydroxylation) levels. Furthermore, the expression and activity of cmCYP3A4 (3A8) were significantly upregulated by rifampicin. These results indicated the effectiveness of Y-27632 supplementation for feeder-free dispersed culture and induction of differentiation into hepatocytes, and the expression of functional CYP enzyme(s) in cmESC-derived hepatic cells.

Characterization of cryopreserved primary human corneal endothelial cells cultured in human serum-supplemented media

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Lucas Monferrari Monteiro Vianna

2016-02-01

Full Text Available ABSTRACT Purpose: To compare cryopreserved human corneal endothelial cells (HCECs grown in human serum-supplemented media (HS-SM with cryopreserved HCECs grown in fetal bovine serum-supplemented media (FBS-SM. Methods: Three pairs of human corneas from donors aged 8, 28, and 31 years were obtained from the eye bank. From each pair, one cornea was used to start a HCEC culture using HS-SM; the other cornea was grown in FBS-SM. On reaching confluence, the six cell populations were frozen using 10% dimethyl sulfoxidecontaining medium. Thawed cells grown in HS-SM were compared with those grown in FBS-SM with respect to morphology, growth curves, immunohistochemistry, real time-reverse transcriptase polymerase chain reaction (RT-PCR for endothelial cell markers, and detachment time. Results: No difference in morphology was observed for cells grown in the two media before or after cryopreservation. By growth curves, cell counts after thawing were similar in both media, with a slight trend toward higher cell counts in FBS-SM. Cells grown in both the media demonstrated a similar expression of endothelial cell markers when assessed by immunohistochemistry, although HCEC marker gene expression was higher in cells grown in HS-SM than in those grown in FBS-SM as assessed by RT-PCR. With FBS-SM, there was a tendency of longer detachment time and lower cell passages. Conclusions: HS-SM was similar to FBS-SM for cryopreservation of cultured HCECs as assessed by analysis of cell morphology, proliferation, and protein expression, although marker gene expression was higher in cells grown in HS-SM than in those grown in FBS-SM. Detachment time was longer with FBS-SM and in lower passages.

Effects of egg white protein supplementation on muscle strength and serum free amino acid concentrations.

Science.gov (United States)

Hida, Azumi; Hasegawa, Yuko; Mekata, Yuko; Usuda, Mika; Masuda, Yasunobu; Kawano, Hitoshi; Kawano, Yukari

2012-10-19

The aim of this study was to evaluate the effects of egg white protein compared to carbohydrate intake prior to exercise on fat free mass (FFM), one repetition maximum (1RM) muscle strength and blood biochemistry in female athletes. Thirty healthy female collegiate athletes were recruited for this study and matched by sport type, body fat percentage and 1RM leg curl muscle strength. Participants were randomly divided into two groups: protein group (15.0 g egg white protein; 75 kcal) and carbohydrate group (17.5 g maltodextrin, 78 kcal). Supplements were administered daily at the same time in a double-blind manner prior to training during an 8-week period. Measurements were performed before and after the 8-week regimen. The mean dietary energy intake did not change throughout the study period. FFM and 1RM assessments (i.e., leg curl, leg extension, squat, and bench press) increased in both groups. Furthermore, serum urea and serum citrulline levels after the 8-week regimen increased significantly only in the protein group. Our findings indicated that compared to the carbohydrate supplement, the protein supplement was associated with some changes in protein metabolites but not with changes in body composition or muscle strength.

Effects of Egg White Protein Supplementation on Muscle Strength and Serum Free Amino Acid Concentrations

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Yukari Kawano

2012-10-01

Full Text Available The aim of this study was to evaluate the effects of egg white protein compared to carbohydrate intake prior to exercise on fat free mass (FFM, one repetition maximum (1RM muscle strength and blood biochemistry in female athletes. Thirty healthy female collegiate athletes were recruited for this study and matched by sport type, body fat percentage and 1RM leg curl muscle strength. Participants were randomly divided into two groups: protein group (15.0 g egg white protein; 75 kcal and carbohydrate group (17.5 g maltodextrin, 78 kcal. Supplements were administered daily at the same time in a double-blind manner prior to training during an 8-week period. Measurements were performed before and after the 8-week regimen. The mean dietary energy intake did not change throughout the study period. FFM and 1RM assessments (i.e., leg curl, leg extension, squat, and bench press increased in both groups. Furthermore, serum urea and serum citrulline levels after the 8-week regimen increased significantly only in the protein group. Our findings indicated that compared to the carbohydrate supplement, the protein supplement was associated with some changes in protein metabolites but not with changes in body composition or muscle strength.

Effects of Egg White Protein Supplementation on Muscle Strength and Serum Free Amino Acid Concentrations

Science.gov (United States)

Hida, Azumi; Hasegawa, Yuko; Mekata, Yuko; Usuda, Mika; Masuda, Yasunobu; Kawano, Hitoshi; Kawano, Yukari

2012-01-01

The aim of this study was to evaluate the effects of egg white protein compared to carbohydrate intake prior to exercise on fat free mass (FFM), one repetition maximum (1RM) muscle strength and blood biochemistry in female athletes. Thirty healthy female collegiate athletes were recruited for this study and matched by sport type, body fat percentage and 1RM leg curl muscle strength. Participants were randomly divided into two groups: protein group (15.0 g egg white protein; 75 kcal) and carbohydrate group (17.5 g maltodextrin, 78 kcal). Supplements were administered daily at the same time in a double-blind manner prior to training during an 8-week period. Measurements were performed before and after the 8-week regimen. The mean dietary energy intake did not change throughout the study period. FFM and 1RM assessments (i.e., leg curl, leg extension, squat, and bench press) increased in both groups. Furthermore, serum urea and serum citrulline levels after the 8-week regimen increased significantly only in the protein group. Our findings indicated that compared to the carbohydrate supplement, the protein supplement was associated with some changes in protein metabolites but not with changes in body composition or muscle strength. PMID:23201768

A defined, feeder-free, serum-free system to generate in vitro hematopoietic progenitors and differentiated blood cells from hESCs and hiPSCs.

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Giorgia Salvagiotto

2011-03-01

Full Text Available Human ESC and iPSC are an attractive source of cells of high quantity and purity to be used to elucidate early human development processes, for drug discovery, and in clinical cell therapy applications. To efficiently differentiate pluripotent cells into a pure population of hematopoietic progenitors we have developed a new 2-dimensional, defined and highly efficient protocol that avoids the use of feeder cells, serum or embryoid body formation. Here we showed that a single matrix protein in combination with growth factors and a hypoxic environment is sufficient to generate from pluripotent cells hematopoietic progenitors capable of differentiating further in mature cell types of different lineages of the blood system. We tested the differentiation method using hESCs and 9 iPSC lines generated from different tissues. These data indicate the robustness of the protocol providing a valuable tool for the generation of clinical-grade hematopoietic cells from pluripotent cells.

Modular Approach to Designing Computer Cultural Systems: Culture as a Thermodynamic Machine

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Leland Gilsen

2015-01-01

Full Text Available Culture is a complex non-linear system. In order to design computer simulations of cultural systems, it is necessary to break the system down into sub-systems. Human culture is modular. It consists of sets of people that belong to economic units. Access to, and control over matter, energy and information is postulated as the key to development of cultural simulations. Because resources in the real world are patchy, access to and control over resources is expressed in two related arenas: economics (direct control and politics (non-direct control. The best way to create models for cultural ecology/economics lies in an energy-information-economic paradigm based on general systems theory and an understanding of the "thermodynamics" of ecology, or culture as a thermodynamic machine.

Free leptin index and PAPP-A: a first trimester maternal serum screening test for pre-eclampsia

DEFF Research Database (Denmark)

Hedley, Paula L; Placing, Sophie; Wøjdemann, Karen

2010-01-01

BACKGROUND: Prophylaxis with low-dose aspirin may reduce the risk of pre-eclampsia (PE) if introduced in first trimester. The performance of first trimester maternal serum screening for PE using free leptin index (fLI) and PAPP-A, where fLI = leptin/leptin soluble receptor was studied. METHODS: F......: First trimester serum samples from 126 PE pregnancies and 289 control pregnancies were studied. fLI and PAPP-A were converted into gestational age and maternal weight independent log MoM values of PAPP-A and fLI. The screening performance of markers was studied by receiver......-operator-characteristics curves. The performance of population screening was estimated by Monte Carlo simulation. RESULTS: fLI was significantly (p controls [mean log MoM -0.0368 (SD: 0.3132)] and PAPP-A was significantly (p ....0133 (SD: 0.2661)] compared to controls [mean log MoM 0.0474 (SD: 0.2521)] in PE pregnancies. There was no correlation between fLI and PAPP-A in control or PE pregnancies. Combined fLI and PAPP-A screening for PE had estimated population detection rates of 22% and 35% for false positives rates of 6% and 12...

Detection of dengue virus from mosquito cell cultures inoculated with human serum in the presence of actinomycin D.

Science.gov (United States)

Ramos, C; Villaseca, J M; García, H; Hernández, D G; Ramos-Castañeda, J; Imbert, J L

1995-01-01

We report the use of cultures of mosquito cells (TRA-284) to detect dengue virus in serum from cases of dengue fever in the state of Puebla, México. Using the conventional procedure 56 of 171 samples (32.7%) were positive. The negative sera (67.3%) were passaged 'blind' in mosquito cell cultures but no virus was detected. However, when these sera were incubated in the presence of actinomycin D (an inhibitor of deoxyribonucleic acid transcription) 20 of the 115 samples (17.4%) became positive. This procedure increased the virus detection rate from 32.7% to 44.4%. Serotypes 1 and 4 were identified for the first time in the state of Puebla, where the transmission of dengue virus is increasing. The addition of actinomycin D to mosquito cell cultures may improve the detection of dengue virus and could be a useful tool for virological surveillance in endemic countries.

A simple and efficient feeder-free culture system to up-scale iPSCs on polymeric material surface for use in 3D bioprinting.

Science.gov (United States)

Wong, Chui-Wei; Chen, You-Tzung; Chien, Chung-Liang; Yu, Tien-Yu; Rwei, Syang-Peng; Hsu, Shan-Hui

2018-01-01

The 3D bioprinting and cell/tissue printing techniques open new possibilities for future applications. To facilitate the 3D bioprinting process, a large amount of living cells are required. Induced pluripotent stem cells (iPSCs) represent a promising cell source for bioprinting. However, the maintenance and expansion of undifferentiated iPSCs are expensive and time consuming. Therefore, in this study a culture method to obtain a sufficient amount of healthy and undifferentiated iPSCs in a short-term period was established. The iPSCs could be passaged for twice on tissue culture polystyrene (TCPS) dish with the conditional medium and could adapt to the feeder-free environment. Feeder-free dishes were further prepared from chitosan, chitosan-hyaluronan, silk fibroin, and polyurethane (PU1 and PU2) two-dimensional substrates. The iPSCs cultured on the chitosan substrates showed a higher proliferation rate without losing the stemness feature. Among the different materials, PU2 could be prepared as a thermoresponsive hydrogel, which was a potential ink for 3D bioprinting. The iPSCs cultured on PU2 substrates well survived when further embedded in PU2 hydrogel. Moreover, PU2 hydrogel printed with iPSCs remained structural integrity. The use of PU2 hydrogel to embed iPSCs reduced the injury to iPSCs by shear stress. These results indicate that iPSCs could be expanded on chitosan or PU2 membranes without the feeder layer and then printed in PU2 hydrogel. The combination of these steps could offer a new possibility for future applications of iPSC-based 3D bioprinting in tissue engineering. Copyright © 2017 Elsevier B.V. All rights reserved.

Serum testosterone concentration in chloroquine- treated rats ...

African Journals Online (AJOL)

ONOS

2010-07-05

Jul 5, 2010 ... The effects of ascorbic acid (vitamin C) and alpha-tocopherol (vitamin E) were studied on serum testosterone ... chloroquine are probably mediated via the generation of free radicals. ... Effects of ascorbic acid and alpha-tocopherol on serum testosterone concentration in chloroquine-treated rats. Groups.

Anaerobic thermophilic culture-system

Energy Technology Data Exchange (ETDEWEB)

Ljungdahl, L G; Wiegel, J K.W.

1981-04-14

A mixed culture system of Thermoanaerobacter ethanolicus and Clostridium thermocellum is employed for anaerobic, thermophilic ethanol fermentation of cellulose. By cellulase action, monosaccharides are formed which inhibit the growth of C. thermocellum, but are fermented by T. ethanolicus. Thus, at a regulated pH-value of 7.5, this mixed culture system of micro organisms results in a cellulose fermentation with a considerably higher ethanol yield.

Ratio of serum free triiodothyronine to free thyroxine in Graves' hyperthyroidism and thyrotoxicosis caused by painless thyroiditis.

Science.gov (United States)

Yoshimura Noh, Jaeduk; Momotani, Naoko; Fukada, Shuji; Ito, Koichi; Miyauchi, Akira; Amino, Nobuyuki

2005-10-01

The serum T3 to T4 ratio is a useful indicator for differentiating destruction-induced thyrotoxicosis from Graves' thyrotoxicosis. However, the usefulness of the serum free T3 (FT3) to free T4 (FT4) ratio is controversial. We therefore systematically evaluated the usefulness of this ratio, based on measurements made using two widely available commercial kits in two hospitals. Eighty-two untreated patients with thyrotoxicosis (48 patients with Graves' disease and 34 patients with painless thyroiditis) were examined in Kuma Hospital, and 218 patients (126 with Graves' disease and 92 with painless thyroiditis) and 66 normal controls were examined in Ito Hospital. The FT3 and FT4 values, as well as the FT3/FT4 ratios, were significantly higher in the patients with Graves' disease than in those with painless thyroiditis in both hospitals, but considerable overlap between the two disorders was observed. Receiver operating characteristic (ROC) curves for the FT3 and FT4 values and the FT3/FT4 ratios of patients with Graves' disease and those with painless thyroiditis seen in both hospitals were prepared, and the area under the curves (AUC), the cut-off points for discriminating Graves' disease from painless thyroiditis, the sensitivity, and the specificity were calculated. AUC and sensitivity of the FT(3)/FT(4) ratio were smaller than those of FT(3) and FT(4) in both hospitals. The patients treated at Ito hospital were then divided into 4 groups according to their FT4 levels (A: 2.3 approximately 5.4 ng/dl), and the AUC, cut-off points, sensitivity, and specificity of the FT(3)/FT(4) ratios were calculated. The AUC and sensitivity of each group increased with the FT4 levels (AUC: 57.8%, 72.1%, 91.1%, and 93.4%, respectively; sensitivity: 62.6%, 50.0%, 77.8%, and 97.0%, respectively). The means +/- SE of the FT3/FT4 ratio in the Graves' disease groups were 3.1 +/- 0.22, 3.1 +/- 0.09, 3.2 +/- 0.06, and 3.1 +/- 0.07, respectively, versus 2.9 +/- 0.1, 2.6 +/- 0.07, 2.5 +/- 0

Development of a quick serum IL-6 measuring system in rheumatoid arthritis.

Science.gov (United States)

Koyama, Kensuke; Ohba, Tetsuro; Ishii, Kentaro; Jung, Giman; Haro, Hirotaka; Matsuda, Kenichi

2017-07-01

Interleukin-6 (IL-6) plays a crucial role in the pathogenesis of rheumatoid arthritis (RA). Both fulfillment of remission criteria and assessment of other methods of evaluation of RA are important for preventing joint damage progression. Measurement of serum IL-6 concentrations has been reported to be useful for monitoring RA disease activity. However, it takes at least 4-5h to measure serum IL-6 concentrations using traditional methods, which limits its utility during routine assessment in daily clinical practice settings. We established a novel method that enables measurement of serum IL-6 within 24min and requires a very small blood volume. We investigated the accuracy and efficacy of this system in RA patients. One hundred fifty blood samples collected from 76 patients were measured using the two systems. We first developed the prototype of the Human IL-6 RAYFAST. Then, we examined the correlation between the prototype RAYFAST and chemiluminescent enzyme immunoassay (CLEIA) methods. Finally, we compared IL-6 concentrations and clinical parameters using both systems. The correlation between RAYFAST (x) and CLEIA (y) for IL-6 was y=0.895x-5.94, r=0.941 (p<0.0001). Serum IL-6 concentrations in RAYFAST correlated with DAS28-CRP (r=0.372, p<0.05) and DAS28-ESR (r=0.397, p<0.01). Serum IL-6 concentrations in CLEIA correlated with DAS28-CRP (r=0.313, p<0.001) and DAS28-ESR (r=0.353, p<0.001). This new cytokine quick measure system is as accurate as CLEIA methods. Serum IL-6 concentrations can be measured in 24min using the prototype RAYFAST. It might be usable in the daily clinical practice setting, thereby contributing to improved RA management. Copyright © 2017 Elsevier Ltd. All rights reserved.

Copyright for Interactive Systems: Stratagems for Tourism and Cultural Heritage Promotion

Science.gov (United States)

Cipolla-Ficarra, Francisco V.; Cipolla-Ficarra, Miguel; Ficarra, Valeria M.

We present a series of strategies followed from the interactive design for the realization of a hypermedia system aimed at promoting in an original, simple and universal way the cultural and tourism heritage of a wide rural area in two Italian regions: Emilia Romagna and Lombardy. Besides, the main stratagems followed are disclosed to overcome the existing hurdles when it comes to copyright for the free diffusion of the tourism view of the area, such as can be photography or video, for instance. Finally, we present the first vademecum to be considered before making on-line and off-line interactive systems in Italy.

Effects of all-trans retinol and cigarette smoke condensate on hamster tracheal epithelium in organ culture. I. A cell proliferation study

NARCIS (Netherlands)

Rutten, A.A.J.J.L.; Wilmer, J.W.G.M.; Beems, R.B.

1988-01-01

The effects of cigarette smoke condensate (CSC) and all-trans retinol on the cell proliferative activity of vitamin A-deprived hamster tracheal epithelium have been studied in vitamin A-deficient, serum-free, hormone-supplemented medium in organ culture. In the absence of retinol, CSC induced a

Serum protein electrophoresis values for free-ranging and zoo-based koalas (Phascolarctos cinereus).

Science.gov (United States)

Pye, Geoffrey W; Ellis, William; Fitzgibbon, Sean; Opitz, Brian; Keener, Laura; Arheart, Kristopher L; Cray, Carolyn

2012-03-01

In a clinical setting, especially with species of special interest, it is important to use all clinical pathology testing options for general health monitoring and diagnosis. Protein electrophoresis (EPH) has previously been shown to be an important adjunct tool in veterinary medicine. Serum samples from 18 free-ranging and 12 zoo-based koalas (Phascolarctos cinereus) were subject to EPH analysis. Significant differences were found between the two groups for the following values: total protein, albumin, beta globulins, and albumin-globulin ratio (P < 0.05). By using the combined data, the minimum-maximum values for the EPH fractions were as follows: total protein 5.0-7.8 g/dl, albumin 2.8-4.7 g/dl, alpha-1 globulins 0.5-1.1 g/dl, alpha-2 globulins 0.3-0.7 g/dl, beta globulins 0.4-1.0 g/dl, gamma globulins 0.2-1.0 g/dl, and albumin-globulin ratio 1.0-2.1.

Application of a nitrocellulose immunoassay for quantitation of proteins secreted in cultured media

International Nuclear Information System (INIS)

LaDuca, F.M.; Dang, C.V.; Bell, W.R.

1986-01-01

A macro immunoassay was developed to quantitate proteins (antigens) secreted in the culture media of primary rat hepatocytes. Dilutions of protein standards and undiluted spent culture media were applied to numbered sheets of nitrocellulose (NC) paper by vacuum filtration (in volumes up to 1 ml) through a specially designed macrofiltration apparatus constructed of plexiglas. Sequential incubation of the NC with bovine serum albumin blocking buffer, monospecific antibody, and 125 I Protein A enabled quantitation of protein concentration by determination of NC bound radioactivity. Linear and reproducible standard curves were obtained with fibrinogen, albumin, transferrin, and haptoglobin. A high degree of coefficient of correlation between radioactivity (cmp) and protein concentration was found. Intra- and inter-test reproducibility was excellent. By using monospecific antibodies, single proteins (i.e., fibrinogen), as low as 32 ng/ml, could be quantified in heterogeneous protein mixtures and in spent culture media. The assay was sensitive to the difference of fibrinogen secretion under nonstimulatory (serum-free hormonally define medium, SFHD) and stimulatory (SFHD plus hydrocortisone) culture conditions. The procedure and techniques described are applicable to the quantitation of any protein in a suitable buffer

Serum complexed and free prostate specific antigen levels are lower in female elite athletes in comparison to control women [version 1; referees: 2 approved

Directory of Open Access Journals (Sweden)

Emma Eklund

2017-07-01

Full Text Available Background: We hypothesize that prostate specific antigen (PSA, a protein that it is under regulation by androgens, may be differentially expressed in female elite athletes in comparison to control women. Methods: We conducted a cross-sectional study of 106 female athletes and 114 sedentary age-matched controls.  Serum from these women was analyzed for complexed prostate specific antigen (cPSA and free prostate specific antigen (fPSA, by fifth generation assays with limits of detection of around 6 and 140 fg/mL, respectively.  A panel of estrogens, androgens and progesterone in the same serum was also quantified by tandem mass spectrometry.  Results: Both components of serum PSA (cPSA and fPSA were lower in the elite athletes vs the control group (P=0.033 and 0.013, respectively.  Furthermore, estrone (p=0.003 and estradiol (p=0.004 were significantly lower, and dehydroepiandrosterone  (p=0.095 and 5-androstene-3β, 17β-diol (p=0.084 tended to be higher in the athletes vs controls. Oral contraceptive use was similar between groups and significantly associated with increased cPSA and fPSA in athletes (p= 0.046 and 0.009, respectively.  PSA fractions were not significantly associated with progesterone changes. The Spearman correlation between cPSA and fPSA in both athletes and controls was 0.75 (P < 0.0001 and 0.64 (P < 0.0001, respectively.  Conclusions: Elite athletes have lower complexed and free PSA, higher levels of androgen precursors and lower levels of estrogen in their serum than sedentary control women. Abbreviations: cPSA, complexed PSA; fPSA, free PSA; PCOS, polycystic ovarian syndrome; E1, estrone; E2, estradiol; DHEA, dehydroepiandrosterone, Testo, testosterone; DHT, dihydrotestosterone; PROG, progesterone; Delta 4, androstenedione; Delta 5, androst-5-ene-3β, 17β-diol; BMD, body mineral density; LLOQ, lower limit of quantification; ULOQ, upper limit of quantification; LOD, limit of detection; ACT, α1-antichymotrypsin

The Cultural-Rhetorical Role of Free Jazz: Forging an Identity in the Sixties.

Science.gov (United States)

Francesconi, Robert

The free jazz movement of the 1960s provided a rhetorical parallel in music to the verbal messages of black power and black nationalism. The use of Third World musical patterns represented an attempt to reinforce the revolutions in perceptions that black Americans held of themselves, their cultural heritage, and relationships to the rest of the…

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

Science.gov (United States)

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. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Human platelet lysate: Replacing fetal bovine serum as a gold standard for human cell propagation?

Science.gov (United States)

Burnouf, Thierry; Strunk, Dirk; Koh, Mickey B C; Schallmoser, Katharina

2016-01-01

The essential physiological role of platelets in wound healing and tissue repair builds the rationale for the use of human platelet derivatives in regenerative medicine. Abundant growth factors and cytokines stored in platelet granules can be naturally released by thrombin activation and clotting or artificially by freeze/thaw-mediated platelet lysis, sonication or chemical treatment. Human platelet lysate prepared by the various release strategies has been established as a suitable alternative to fetal bovine serum as culture medium supplement, enabling efficient propagation of human cells under animal serum-free conditions for a multiplicity of applications in advanced somatic cell therapy and tissue engineering. The rapidly increasing number of studies using platelet derived products for inducing human cell proliferation and differentiation has also uncovered a considerable variability of human platelet lysate preparations which limits comparability of results. The main variations discussed herein encompass aspects of donor selection, preparation of the starting material, the possibility for pooling in plasma or additive solution, the implementation of pathogen inactivation and consideration of ABO blood groups, all of which can influence applicability. This review outlines the current knowledge about human platelet lysate as a powerful additive for human cell propagation and highlights its role as a prevailing supplement for human cell culture capable to replace animal serum in a growing spectrum of applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Serum nitric oxide metabolites and disease activity in patients with systemic sclerosis.

Science.gov (United States)

Mok, Mo Yin; Fung, Peter Chin Wah; Ooi, Clara; Tse, Hung Fat; Wong, Yik; Lam, Yui Ming; Wong, Woon Sing; Lau, Chak Sing

2008-03-01

There is no surrogate marker in serum for defining disease activity in scleroderma (SSc). Nitric oxide (NO), which regulates vasodilation and possesses pro-inflammatory actions, has been implicated in the pathogenesis of SSc. We compared serum NO(x) (total nitrate and nitrite) level in SSc patients to healthy controls and evaluated its correlation with detailed symptomatology and scoring systems for various organ involvement. Symptoms and physical findings that suggested disease activity in regard to various organs were documented. Lung function test, high-resolution computed tomographic (HRCT) scan of thorax and echocardiography were performed. Serum NO(x) was measured by chemiluminescence. Serum NO(x) levels in SSc (n = 43) were significantly higher (72.4 +/- 47.8 microM) than age- and sex-matched controls (n = 41; 37.1 +/- 13.5 microM; p n = 9; OR 145.3, p = 0.01) were predictive factors for elevated serum NO(x). Prednisolone use was associated with lower serum NO(x) level (OR 0.06, p = 0.04). Elevated PAP of increasing severity was found to be associated with higher level of serum NO(x) (p = 0.004 by trend). Serum NO(x) in SSc patients were elevated compared to healthy controls. Serum NO(x) level was determined by multiple factors including age, prednisolone use, and elevated PAP.

Homogeneity in the relationship of serum cholesterol to coronary deaths across different cultures: 40-year follow-up of the Seven Countries Study

NARCIS (Netherlands)

Menotti, A.; Lanti, M.; Kromhout, D.; Blackburn, H.; Jacobs, D.; Nissinen, A.; Dontas, A.; Kafatos, A.; Nedeljkovic, S.; Adachi, H.

2008-01-01

Background: The aim was to investigate whether multivariate coefficients of serum cholesterol in the prediction of coronary heart disease (CHD) deaths were similar across different cultures in a long-term follow-up. Design: Thirteen cohorts for a total of 10 157 men aged 40¿59 years at entry,

Effects of CO 2 concentration and moisture content of sugar-free media on the tissue-cultured plantlets in a large growth chamber

Science.gov (United States)

Qu, Y. H.; Lin, C.; Zhou, W.; Li, Y.; Chen, B.; Chen, G. Q.

2009-01-01

The dynamic fluctuations of CO 2 concentration in the tissue culture growth chamber after transplantation of petunia, chrysanthemum and tomato plantlets were recorded with a real-time control system to determine the critical CO 2 concentration levels of 35 μl l -1 at which CO 2 enrichment is needed. The experimental data showed that the tissue-cultured plantlets of petunia, chrysanthemum and tomato had the same CO 2 concentration dynamics. The results indicated that CO 2 enrichment was proper on the second day after transplantation. Petunia plantlets were used to conduct experiments under PPFD of 80 μmol m -2 s -1, and CO 2 concentrations of 350 ± 50 μl l -1, 650 ± 50 μl l -1 and 950 ± 50 μl l -1 as well as medium moisture contents of 60%, 70% and 80%, with the result that plantlets grew better under CO 2 concentration of 650 ± 50 μl l -1 than under the other two concentrations with all the different media water contents. Three media water contents under the same CO 2 concentration produced plantlets with the same quality. The impacts of CO 2 concentrations on plantlets are more important than those of the media water contents. Sugar-free tissue culture, as compared with the conventional culture, showed that CO 2 enrichment to 350 ± 50 μl l -1 can promote the growth of the cultured plantlets. Sugar-free tissue culture produced healthy plantlets with thick roots, almost equivalent to the common plantlets.

Hydroponic Culture

Science.gov (United States)

Steucek, G. L.; Yurkiewicz, W. J.

1973-01-01

Describes a hydroponic culture technique suitable for student exercises in biology. This technique of growing plants in nutrient solutions enhances plant growth, and is an excellent way to obtain intact plants with root systems free of soil or other particulate matter. (JR)

How Universal Are Free Will Beliefs? Cultural Differences in Chinese and U.S. 4- and 6-Year-Olds.

Science.gov (United States)

Wente, Adrienne O; Bridgers, Sophie; Zhao, Xin; Seiver, Elizabeth; Zhu, Liqi; Gopnik, Alison

2016-05-01

This study explores the development of free will beliefs across cultures. Sixty-seven Chinese 4- and 6-year-olds were asked questions to gauge whether they believed that people could freely choose to inhibit or act against their desires. Responses were compared to those given by the U.S. children in Kushnir, Gopnik, Chernyak, Seiver, and Wellman (). Results indicate that children from both cultures increased the amount of choice they ascribed with age. For inhibition questions, Chinese children ascribed less choice than the U.S. children. Qualitative explanations revealed that the U.S. children were also more likely to endorse notions of autonomous choice. These findings suggest both cultural differences and similarities in free will beliefs. © 2016 The Authors. Child Development © 2016 Society for Research in Child Development, Inc.

Three-dimensional spheroid culture of human umbilical cord mesenchymal stem cells promotes cell yield and stemness maintenance.

Science.gov (United States)

Li, Yi; Guo, Gang; Li, Li; Chen, Fei; Bao, Ji; Shi, Yu-Jun; Bu, Hong

2015-05-01

Mesenchymal stem cell (MSC) transplantation is a promising treatment of many diseases. However, conventional techniques with cells being cultured as a monolayer result in slow cell proliferation and insufficient yield to meet clinical demands. Three-dimensional (3D) culture systems are gaining attention with regard to recreating a complex microenvironment and to understanding the conditions experienced by cells. Our aim is to establish a novel 3D system for the culture of human umbilical cord MSCs (hUC-MSCs) within a real 3D microenvironment but with no digestion or passaging. Primary hUC-MSCs were isolated and grown in serum-free medium (SFM) on a suspension Rocker system. Cell characteristics including proliferation, phenotype and multipotency were recorded. The therapeutic effects of 3D-cultured hUC-MSCs on carbon tetrachloride (CCl4)-induced acute liver failure in mouse models were examined. In the 3D Rocker system, hUC-MSCs formed spheroids in SFM and maintained high viability and active proliferation. Compared with monolayer culture, the 3D-culture system yielded more hUC-MSCs cells within the same volume. The spheroids expressed higher levels of stem cell markers and displayed stronger multipotency. After transplantation into mouse, 3D hUC-MSCs significantly promoted the secretion of interferon-γ and interleukin-6 but inhibited that of tumor necrosis factor-α, thereby alleviating liver necrosis and promoting regeneration following CCl4 injury. The 3D culture of hUC-MSCs thus promotes cell yield and stemness maintenance and represents a promising strategy for hUC-MSCs expansion on an industrial scale with great potential for cell therapy and biotechnology.

Upregulation of endothelin ETB receptor-mediated vasoconstriction in rat coronary artery after organ culture

DEFF Research Database (Denmark)

Eskesen, Karen; Edvinsson, Lars

2006-01-01

The aim of this study was to examine if endothelin ET(B) receptor-mediated contraction occurred in isolated segments of rat coronary arteries during organ culture. Presence of contractile endothelin ET(B) receptors was studied by measuring the change in isometric tension in rings of left anterior......(+)-solution was not modified after 1 day in culture medium. The experiments indicate that organ culture of rat coronary arteries upregulate endothelin ET(B) receptor-mediated contraction by inducing synthesis of new protein....... descending coronary arteries isolated from hearts of rats as response to application of the selective endothelin ET(B) receptor agonist, Sarafotoxin 6c and endothelin-1. In segments cultured 1 day in serum free Dulbecco's Modified Eagle's Medium, Sarafotoxin 6c induced a concentration dependent contraction...

Modification and standardization of the culture of early postimplantation embryos for toxicological studies

Energy Technology Data Exchange (ETDEWEB)

Klug, S.; Lewandowski, C.; Neubert, D.

1985-12-01

The method of culturing ''whole'' rat embryos (days 9.5-11.5 of gestation, i.e. at the early stage of organogenesis) as modified and standardized in our laboratory is presented. We have succeeded in using bovine serum as culture medium instead of rat serum as recommended in the original procedure. Experimental conditions are described for obtaining reproducible results. An improved scoring system was developed which, in connection with a computerized documentation, greatly facilitates the evaluation of the data.

Elimination of remaining undifferentiated induced pluripotent stem cells in the process of human cardiac cell sheet fabrication using a methionine-free culture condition.

Science.gov (United States)

Matsuura, Katsuhisa; Kodama, Fumiko; Sugiyama, Kasumi; Shimizu, Tatsuya; Hagiwara, Nobuhisa; Okano, Teruo

2015-03-01

Cardiac tissue engineering is a promising method for regenerative medicine. Although we have developed human cardiac cell sheets by integration of cell sheet-based tissue engineering and scalable bioreactor culture, the risk of contamination by induced pluripotent stem (iPS) cells in cardiac cell sheets remains unresolved. In the present study, we established a novel culture method to fabricate human cardiac cell sheets with a decreased risk of iPS cell contamination while maintaining viabilities of iPS cell-derived cells, including cardiomyocytes and fibroblasts, using a methionine-free culture condition. When cultured in the methionine-free condition, human iPS cells did not survive without feeder cells and could not proliferate or form colonies on feeder cells or in coculture with cells for cardiac cell sheet fabrication. When iPS cell-derived cells after the cardiac differentiation were transiently cultured in the methionine-free condition, gene expression of OCT3/4 and NANOG was downregulated significantly compared with that in the standard culture condition. Furthermore, in fabricated cardiac cell sheets, spontaneous and synchronous beating was observed in the whole area while maintaining or upregulating the expression of various cardiac and extracellular matrix genes. These findings suggest that human iPS cells are methionine dependent and a methionine-free culture condition for cardiac cell sheet fabrication might reduce the risk of iPS cell contamination.

Differential solubility of curcuminoids in serum and albumin solutions: implications for analytical and therapeutic applications

Directory of Open Access Journals (Sweden)

Quitschke Wolfgang W

2008-11-01

Full Text Available Abstract Background Commercially available curcumin preparations contain a mixture of related polyphenols, collectively referred to as curcuminoids. These encompass the primary component curcumin along with its co-purified derivatives demethoxycurcumin and bisdemethoxycurcumin. Curcuminoids have numerous biological activities, including inhibition of cancer related cell proliferation and reduction of amyloid plaque formation associated with Alzheimer disease. Unfortunately, the solubility of curcuminoids in aqueous solutions is exceedingly low. This restricts their systemic availability in orally administered formulations and limits their therapeutic potential. Results Methods are described that achieve high concentrations of soluble curcuminoids in serum. Solid curcuminoids were either mixed directly with serum, or they were predissolved in dimethyl sulfoxide and added as aliquots to serum. Both methods resulted in high levels of curcuminoid-solubility in mammalian sera from different species. However, adding aliquots of dimethyl sulfoxide-dissolved curcuminoids to serum proved to be more efficient, producing soluble curcuminoid concentrations of at least 3 mM in human serum. The methods also resulted in the differential solubility of individual curcuminoids in serum. The addition of dimethyl sulfoxide-dissolved curcuminoids to serum preferentially solubilized curcumin, whereas adding solid curcuminoids predominantly solubilized bisdemethoxycurcumin. Either method of solubilization was equally effective in inhibiting dose-dependent HeLa cell proliferation in culture. The maximum concentration of curcuminoids achieved in serum was at least 100-fold higher than that required for inhibiting cell proliferation in culture and 1000-fold higher than the concentration that has been reported to prevent amyloid plaque formation associated with Alzheimer disease. Curcuminoids were also highly soluble in solutions of purified albumin, a major component of

Cutoff Values of Serum Carcinoembryonic Antigen (CEA) in Normal Korean Adults and Factors Influencing Serum CEA Level

International Nuclear Information System (INIS)

Kim, Jong Soon; Kim, Sun Wook; Chung, June Key; Lee, Dong Soo

1994-01-01

Carcinoembryonic Antigen is one of most frequently checked tumor markers in cancer management. We performed statistical analysis with serum CEA data of 2626 persons who received regular health examination and were thought to be free of active disease to determine the cutoff values of serum CEA level in normal Korean adults and to study the factors influencing serum CEA levels in normal subjects. 1) The cutoff values of serum CEA in normal Korean adults in general were 9.28 ng/ml for men, 5.90 ng/ml for women. 2) Serum CEA level was influenced by age, present smoking history, sex, and abnormal findings in chest X ray. 3) Serum CEA level had no correlation with the history of amount of alcohol consumption or obesity. 4) Cutoff values of serum CEA in normal Korean adults were tabulated according to age, sex, and smoking history. Serum CEA level was influenced by age, sex, present smoking history and abnormal findings in chest X ray and cutoff values of serum CEA were tabulated according to age, sex, and smoking history.

Correlation between serum E-selectin levels and panoramic nailfold capillaroscopy in systemic sclerosis.

Science.gov (United States)

Valim, V; Assis, L S S; Simões, M F J; Trevisani, V F M; Pucinelli, M L C; Andrade, L E C

2004-09-01

E-selectin is expressed by the activated endothelium and its plasma levels are increased in patients with systemic sclerosis. Eighteen patients fulfilling the American Rheumatism Association criteria for systemic sclerosis, 15 females and 3 males, 42-70 years old, 9 with diffuse and 9 with limited forms, were sequentially recruited for this study. Serum E-selectin levels were determined by commercially available ELISA and their association with nailfold capillaroscopic abnormalities was investigated. Nailfold capillaries were analyzed by 16X magnification wide-field capillaroscopy. Two parameters on capillaroscopy were used to correlate to serum E-selectin: deletion and ectasia. Data were analyzed statistically by the Student t-test and Spearman correlation. Two-tailed P values below 0.05 were considered significant. E-selectin range was 38 to 200 ng/ml (80 +/- 39.94). There was a correlation between serum E-selectin levels and the deletion capillaroscopic score (r = 0.50, P capillaroscopy. The stronger correlation of deletion score in capillaroscopy in early disease suggests that serum E-selectin levels might be a useful biochemical marker of disease activity in systemic sclerosis.

Inositol and hepatic lipidosis. II. Effect of inositol supplementation and time from parturition on serum insulin, thyroxine and triiodothyronine and their relationship to serum and liver lipids in dairy cows.

Science.gov (United States)

Gerloff, B J; Herdt, T H; Wells, W W; Nachreiner, R F; Emery, R S

1986-06-01

Percutaneous liver biopsies and blood samples were obtained from 80 dairy cows in nine Michigan herds over the peripartum period. Thirty-nine cows were fed 17 g of supplemental inositol and 41 were fed a placebo. Liver biopsies were assayed for total myoinositol and triglyceride (TG) concentrations. Blood samples were assayed for serum dextran precipitable cholesterol, nonesterified fatty acids (NEFA), insulin, thyroxine (T4), free (FT4), triiodothyronine (T3) and free T3 (FT3) concentrations. Serum concentrations of insulin and the thyroid hormones decreased near parturition, with lowest concentrations occurring in the immediate postpartum period. Concentrations of T3 correlated well with T4, and the concentrations of free thyroid hormones reflected concentrations of total thyroid hormones. The percentage of hormone in the free fraction remained constant over time. Serum insulin, T3 and T4 were negatively correlated with serum NEFA and liver TG concentrations. Thyroid hormone concentrations were positively correlated with serum dextran precipitable cholesterol concentrations. Inositol supplementation was associated with reduced circulating T3 and FT3 concentrations, but not T4 and FT4 concentrations. Changes in hormone concentrations at parturition and their relationship to liver TG and serum NEFA concentrations were consistent with a metabolic adaptation by the dairy cow to the negative energy balance of early lactation.

Clinical evaluation of free to total prostate specific antigen ratio in serum

International Nuclear Information System (INIS)

Cheng Wei; Deng Shouzhen; Lin Xiangtong

1999-01-01

Free and total prostate specific antigen (F-PSA and T-PSA) in serum were measured with immunoradiometric assay and the F/T-PSA ratio was calculated in 175 patients with T-PSA levels in the range of 4-20 μg/L. Among them 141 patients were benign prostatic hyperplasia (BPH), 23 were untreated prostate cancer (Pca untreated) and 11 were treated prostate cancer (Pca treated). The results showed that difference in F-PSA and F/T-PSA ratio for BPH group and Pca untreated group were statistically significant (P<0.01). The effectiveness of F/T-PSA ratio for Pca (89.9%) was higher than F-PSA (54.8%). The receiver-operating characteristic (ROC) curve showed an improved diagnostic efficacy of F/T-PSA ratio compared with T-PSA for discrimination between BPH and Pca. If mean F/T-PSA ratio value + 1 SE (13.2%) was used in BPH group as discrimination limits of Pca patients, the diagnostic accuracy of BPH group and Pca untreated group were 90.8% and 82.6% respectively. Thereby F/T-PSA ratio may be useful for the differentiation between BPH and prostate cancer

Association of thyroid function with human serum ghrelin and leptin levels

International Nuclear Information System (INIS)

Wang Jinping; Xu Hao; Wu Qiulian

2008-01-01

Objective: To investigate the effect of different status of thyroid function (hypothyroidism and hyperthyroidism as well as euthyroid status) on serum ghrelin and leptin levels. Methods: The levels of serum ghrelin and leptin were determined by radio immunoassay in 46 untreated subjects with hyperthyroidism, 15 hyperthyroid patients achieved a euthyroid status after radioiodine 131 I therapy, 21 cases of hypothyroidism and 18 cases of normal controls, respectively. Meanwhile, the serum levels of free triiodothyronine (FT 3 ), free thyroxine (FT 4 ) and thyroid-stimulating hormone (TSH) were measured by chemiluminescence immune assay. Results: (1) The levels of serum ghrelin in untreated hyperthyroidism were significantly lower than those in hyperthyroid patients achieved a euthyroid status (t=3.21, P 3 (r=-0.29, P 4 (r=-0.26, P< 0.05), positively correlated with serum TSH (r=0.36, P<0.05); serum leptin levels did not correlate with thyroid hormone. Conclusion: The levels of serum ghrelin were differently under different thyroid functional status and correlated with thyroid hormone, while serum leptin were not. (authors)

Sex differences in the aging pattern of renin-angiotensin system serum peptidases.

Science.gov (United States)

Fernández-Atucha, A; Izagirre, A; Fraile-Bermúdez, A B; Kortajarena, M; Larrinaga, G; Martinez-Lage, P; Echevarría, E; Gil, J

2017-01-01

Serum peptidases, such as angiotensin-converting enzyme (ACE), angiotensin-converting enzyme-2 (ACE2), neutral endopeptidase (NEP), aminopeptidase N (APN), and aminopeptidase A (APA), are important elements of the renin-angiotensin system (RAS). Dysregulation of these enzymes has been associated with hypertension and cardiovascular risk. In the present study, serum activities of RAS peptidases were analyzed to evaluate the existence of sexual differences, with a possible different pattern in pre- and post-andropausal/post-menopausal participants. One hundred and eighteen healthy men and women between 41 and 70 years of age (58 women and 60 men) were recruited to participate in the study. Serum RAS-regulating enzymes were measured by spectrofluorimetry. Enzymatic activity was recorded as units of enzyme per milliliter of serum (U/mL). Significantly lower serum APA activity was observed in men with respect to women; no sex differences were detected for ACE, ACE2, NEP, or APN. Significantly lower APA and ACE serum activity were observed in older men compared to older women. In contrast, younger (menopausia, on the critical serum enzymatic activities of the RAS, which could correlate with sexual differences in cardiovascular risk.

Culture of human cell lines by a pathogen-inactivated human platelet lysate.

Science.gov (United States)

Fazzina, R; Iudicone, P; Mariotti, A; Fioravanti, D; Procoli, A; Cicchetti, E; Scambia, G; Bonanno, G; Pierelli, L

2016-08-01

Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety.

The impact of hypoxemia on serum total and free prostate-specific antigen levels in patients with chronic obstructive pulmonary disease.

Science.gov (United States)

Ozge, Cengiz; Bozlu, Murat; Ozgur, Eylem Sercan; Tek, Mesut; Tunckiran, Ahmet; Muslu, Necati; Ilvan, Ahmet

2015-05-01

Prostate-specific antigen (PSA) is the most important biochemical marker in the diagnosis and follow-up of patients with prostate cancer. In recent years, a relationship between PSA levels and hypoxic conditions has been described. However, no study has investigated the PSA levels in patients with chronic obstructive pulmonary disease (COPD). The aim of the present study was to investigate the impact of hypoxemia on serum total (tPSA) and free PSA (fPSA) levels in patients with COPD. Between January 2010 and January 2014, 95 male patients who hospitalized for acute exacerbations of COPD and 80 control subjects were enrolled in the study. Serum tPSA and fPSA levels and f/tPSA ratios were determined in all patients on the first day of hospitalization (exacerbation) and 7 days after the treatment (stable state). Statistical analysis included paired t test and Mann-Whitney U test. No statistically significant differences were found between COPD and control groups with regard to the baseline characteristics, except for smoking status. The levels of serum tPSA and fPSA during exacerbation of COPD were significantly higher than the levels of the stable period (p 0.05). Hypoxemia during acute exacerbation of COPD can cause a rise in serum tPSA and fPSA levels, but f/tPSA ratio is not affected. Acute exacerbation of COPD may be added to list of the events in which PSA measurements must be interpreted with caution.

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

Science.gov (United States)

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.

Serum levels of ficolin-3 (Hakata antigen) in patients with systemic lupus erythematosus

DEFF Research Database (Denmark)

Andersen, T.; Munthe-Fog, L.; Garred, P.

2009-01-01

OBJECTIVE: Ficolin-3 is a serum protein of putative importance in autoimmunity. Our objective was to investigate any differential expression of ficolin-3 in patients with systemic lupus erythematosus (SLE) or its clinical subsets. METHODS: Serum levels of ficolin-3 (S-ficolin-3) were determined...

Determination of free cisplatin in medium by differential pulse polarography after ultrasound and cisplatin treatment of a cancer cell culture

International Nuclear Information System (INIS)

Bernard, Vladan; Skorpikova, Jirina; Mornstein, Vojtech; Fojt, Lukas

2011-01-01

The in vitro study was carried out for detection of the cisplatin in free form and in culture medium, depending on various conditions of sonodynamic human ovarian cancer cells A2780 treatment by differential pulse polarography (DPP). For sonodynamic treatment, we used cisplatin alone and combined cisplatin/ultrasound treatments. The ultrasound exposure intensity of 1.0 and 2.0 Wcm 2 in far field for incubation periods 1, 24 and 48 h was used. The parameters of DPP measurements were - 1 s drop time, 5 mV.s -1 voltage scan rate, 50 mV modulation amplitude and negative scanning direction; platinum wire served as counter electrode and Ag|AgCl|3 M KCI as reference electrode. The results showed the dependence of free platinum quantities in culture medium on incubation time and treatment protocol. We found difference in concentration of free cisplatin between conventional application of cisplatin and sonodynamic treatment. The sonodynamic combined treatment of cisplatin and ultrasound field showed a higher cisplatin content in the culture medium than cisplatin treatment alone; a difference of 20% was observed for incubation time 48 h. The results also showed the influence of a time sequence of ultrasound and cytostatics in the sonodynamic treatment. The highest amount of free cisplatin in the solution was found for primary application of cisplatin and the subsequent ultrasound exposure. The quantity of free cisplatin increased with time, namely for time intervals 1-24 h. There was no difference between the DPP signal of cisplatin in reaction mixture containing cells in small quantities and micro-filtered mixture without cells. Thus, the DPP method is suitable for the detection and quantification of free cisplatin in the culture medium of cell suspension. Ultrasound field can be important factor during cytostatic therapy. (author)

Temporal effects of maternal and pregnancy characteristics on serum pregnancy-associated plasma protein-A and free β-human chorionic gonadotropin at 7-14 weeks' gestation

DEFF Research Database (Denmark)

Ball, S; Ekelund, C; Wright, D

2013-01-01

OBJECTIVE: The aim of this study was to investigate gestational age-dependent effects of racial origin, smoking status and mode of conception on maternal serum levels of free β-human chorionic gonadotropin (β-hCG) and pregnancy-associated plasma protein-A (PAPP-A) at 7-14 weeks' gestation. METHODS...

A Comparison of Free Time Activity Choices of Third Culture Kids in Albania and Children in the UK

Science.gov (United States)

Purdon, Anne

2018-01-01

This study aims to consider children's perspectives about free time activity choices. Through the use of drawings, favourite free time activities of third culture kids in Albania are compared with those of children in the UK. The sample comprises four boys and three girls from four to eight years from each country. Further conversations reveal…

Use of a novel serum ELISA method and the tonsil-carrier state for evaluation of Mycoplasma hyosynoviae distributions in pig herds with or without clinical arthritis

DEFF Research Database (Denmark)

Nielsen, Elisabeth Okholm; Lauritsen, Klara Tølbøll; Friis, Niels Filskov

2005-01-01

on porcine serum following experimental Mycoplasma infections as well as by analysis of samples from one Danish herd known to be free of M. hyosynoviae and samples from two Norwegian herds without clinical suspicion of M. hyosynoviae infections since their establishment. The epidemiology of M. hyosynoviae...... determined by culture of cross-sectional samples of whole-blood (n = 238) and tonsil scrapings (n = 322), respectively. Levels of serum antibodies (n = 396) were measured by the novel indirect ELISA test. There was no significant difference in the ELISA results between the MhA and the MhC herds. Pigs...

The bone mass density in men aged over 50 and its relation to the concentration of free and total testosterone in the blood serum

International Nuclear Information System (INIS)

Purzycka-Jazdon, A.; Lasek, W.; Serafin, Z.; Manysiak, S.

2003-01-01

As the mean length of life increases, osteoporosis affects a growing number of men and women, thus becoming an important medical and socioeconomic problem in many countries. Pathogenesis and the prevalence of the osteoporosis in women are well established, however, in men, they are still controversial. In this study, the bone mass density (BMD) of the lumbar spine was determined in 100 healthy men age 50-83, using quantitative computed tomography (QCT). Also, the total serum and free testosterone was measured. The mean BMD was 123.1I39.3 mg/cm 3 , and the values below a fracture threshold were noted in 39% of subjects. The mean concentration of total and free serum testosterone was 4.3I1.7 ng/ml and 6.2I3.7 pg/ml, respectively. There was a significant (p 3 , respectively). There was no correlation found between total testosterone and BMD. Results indicate that reduced bone mass density in males over 50 is as frequent as recently reported in females. Moreover, sex hormones seem to be related to osteoporosis development in men as well. (author)

Silver nanoparticle protein corona composition in cell culture media.

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Shannahan, Jonathan H; Lai, Xianyin; Ke, Pu Chun; Podila, Ramakrishna; Brown, Jared M; Witzmann, Frank A

2013-01-01

The potential applications of nanomaterials as drug delivery systems and in other products continue to expand. Upon introduction into physiological environments and driven by energetics, nanomaterials readily associate proteins forming a protein corona (PC) on their surface. This PC influences the nanomaterial's surface characteristics and may impact their interaction with cells. To determine the biological impact of nanomaterial exposure as well as nanotherapeutic applications, it is necessary to understand PC formation. Utilizing a label-free mass spectrometry-based proteomics approach, we examined the composition of the PC for a set of four silver nanoparticles (AgNPs) including citrate-stabilized and polyvinlypyrrolidone-stabilized (PVP) colloidal silver (20 or 110 nm diameter). To simulate cell culture conditions, AgNPs were incubated for 1 h in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, washed, coronal proteins solubilized, and proteins identified and quantified by label-free LC-MS/MS. To determine which attributes influence PC formation, the AgNPs were characterized in both water and cell culture media with 10% FBS. All AgNPs associated a common subset of 11 proteins including albumin, apolipoproteins, keratins, and other serum proteins. 110 nm citrate- and PVP-stabilized AgNPs were found to bind the greatest number of proteins (79 and 85 respectively) compared to 20 nm citrate- and PVP-stabilized AgNPs (45 and 48 respectively), suggesting a difference in PC formation based on surface curvature. While no relationships were found for other protein parameters (isoelectric point or aliphatic index), the PC on 20 nm AgNPs (PVP and citrate) consisted of more hydrophobic proteins compared to 110 nm AgNPs implying that this class of proteins are more receptive to curvature-induced folding and crowding in exchange for an increased hydration in the aqueous environment. These observations demonstrate the significance of electrostatic

Silver nanoparticle protein corona composition in cell culture media.

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Jonathan H Shannahan

Full Text Available The potential applications of nanomaterials as drug delivery systems and in other products continue to expand. Upon introduction into physiological environments and driven by energetics, nanomaterials readily associate proteins forming a protein corona (PC on their surface. This PC influences the nanomaterial's surface characteristics and may impact their interaction with cells. To determine the biological impact of nanomaterial exposure as well as nanotherapeutic applications, it is necessary to understand PC formation. Utilizing a label-free mass spectrometry-based proteomics approach, we examined the composition of the PC for a set of four silver nanoparticles (AgNPs including citrate-stabilized and polyvinlypyrrolidone-stabilized (PVP colloidal silver (20 or 110 nm diameter. To simulate cell culture conditions, AgNPs were incubated for 1 h in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, washed, coronal proteins solubilized, and proteins identified and quantified by label-free LC-MS/MS. To determine which attributes influence PC formation, the AgNPs were characterized in both water and cell culture media with 10% FBS. All AgNPs associated a common subset of 11 proteins including albumin, apolipoproteins, keratins, and other serum proteins. 110 nm citrate- and PVP-stabilized AgNPs were found to bind the greatest number of proteins (79 and 85 respectively compared to 20 nm citrate- and PVP-stabilized AgNPs (45 and 48 respectively, suggesting a difference in PC formation based on surface curvature. While no relationships were found for other protein parameters (isoelectric point or aliphatic index, the PC on 20 nm AgNPs (PVP and citrate consisted of more hydrophobic proteins compared to 110 nm AgNPs implying that this class of proteins are more receptive to curvature-induced folding and crowding in exchange for an increased hydration in the aqueous environment. These observations demonstrate the significance of

Correlation of Serum Androgens and Pituitary Hormone Levels with Serum PSA Less Than 2.5 NG/ML

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Mustafa Sofikerim

2007-01-01

Full Text Available The aim of this clinical study was to determine whether there is a relationship between total serum testosterone, free testosterone, FSH (Follicle-Stimulating Hormone, LH (Luteinizing Hormone and serum prostate specific antigen (PSA levels. We postulated that such a correlation existed then the use of hormone specific reference ranges might enhance the usefullness of PSA concentrations <2.5 ng/mL as a marker for prostate cancer.

The elevated serum urea:creatinine ratio in canine babesiosis in South Africa is not of renal origin.

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de Scally, M P; Leisewitz, A L; Lobetti, R G; Thompson, P N

2006-12-01

Pigmented serum, usually due to free haemoglobin and/or bilirubin, is a common finding in dogs with babesiosis, resulting in interference with all biochemical tests that rely on photochemistry. This is particularly true of urea and creatinine determinations, complicating the diagnosis of acute renal failure, which is a serious complication of babesiosis. A disproportionately raised serum urea concentration of unknown origin occurs in severely anaemic canine babesiosis patients and gives rise to an increased serum urea:creatinine ratio. The assay for cystatin-C, an excellent measure of glomerular filtration rate, is unaffected by free serum haemoglobin, and due to its different intrinsic origins, is free of influence by the metabolic derangements and organ pathology, other than renal disease, encountered in canine babesiosis. Serum cystatin-C was used to compare the concentrations of serum urea and serum creatinine in dogs with the severely anaemic form of canine babesiosis as well as a canine babesiosis-free reference group. Mean serum urea and mean serum urea:creatinine ratio were significantly elevated in the babesia-infected group relative to the reference population in this study. Mean serum creatinine and mean serum cystatin-C were within the reference ranges. Therefore an elevated urea:creatinine ratio in canine babesiosis in the presence of a normal serum creatinine concentration is considered to be caused by an elevated serum urea concentration and is most likely of non-renal origin. Serum creatinine was therefore as specific a measure of renal function as serum cystatin-C in canine babesiosis in this study. The sensitivity of serum creatinine as a measure of renal function was not established by this study. Serum urea, however, proved to be of little use compared to serum cystatin-C and serum creatinine. Serum urea should therefore not be used to diagnose renal failure in canine babesiosis.

Proteins in growth regulation during early development. Comprehensive three year report, 1974--1977

Energy Technology Data Exchange (ETDEWEB)

Klein, N.W.

1977-08-01

Progress is reported on the following research projects: response of embryo regions to nutrition; synthesis of serum proteins by the yolk-sac; serum protein synthesis in relation to protein nutrition, protease secretion, teratogenic agents, genetic abnormalities, yolk-sac cell cultures, and cell free systems; and effects of serum proteins on rat embryos, chick embryos without yolk-sacs, and isolated brains. (HLW)

CULTURAL FEATURES SHARED BY INFORMATION SYSTEMS USERS

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Marilena Maldonado

2006-11-01

Full Text Available Problems may arise when organizational culture is not considered in the development of information systems, such as difficulties in system implementation, since users do not accept changes in their work cultures. However, current methodology designs do not contemplate cultural factors. The objective of this investigation was to identify the main cultural features shared by the users of information systems in an Argentinean university. As result of this work it was possible to identify the memes shared by the members of the community selected, and to categorize such memes according to their incidence grade. This work seeks to be an initial step towards the construction of systems that evolve along with the organizational culture they are an integral part of.

Ubiquinone modified printed carbon electrodes for cell culture pH monitoring.

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McBeth, Craig; Dughaishi, Rajaa Al; Paterson, Andrew; Sharp, Duncan

2018-08-15

The measurement of pH is important throughout many biological systems, but there are limited available technologies to enable its periodical monitoring in the complex, small volume, media often used in cell culture experiments across a range of disciplines. Herein, pad printed electrodes are developed and characterised through modification with: a commercially available fullerene multiwall carbon nanotube composite applied in Nafion, casting of hydrophobic ubiquinone as a pH probe to provide the electrochemical signal, and coated in Polyethylene glycol to reduce fouling and potentially enhance biocompatibility, which together are proven to enable the determination of pH in cell culture media containing serum. The ubiquinone oxidation peak position (E pa ) provided an indirect marker of pH across the applicable range of pH 6-9 (R 2 = 0.9985, n = 15) in complete DMEM. The electrochemical behaviour of these sensors was also proven to be robust; retaining their ability to measure pH in cell culture media supplemented with serum up to 20% (v/v) [encompassing the range commonly employed in cell culture], cycled > 100 times in 10% serum containing media and maintain > 60% functionality after 5 day incubation in a 10% serum containing medium. Overall, this proof of concept research highlights the potential applicability of this, or similar, electrochemical approaches to enable to detection or monitoring of pH in complex cell culture media. Copyright © 2018 Elsevier B.V. All rights reserved.

ASSESSMENT OF THE RESIDUAL TUMOR IN PATIENTS WITH MULTIPLE MYELOMA BASED ON THE ANALYSIS OF THE FREE LIGHT CHAINS OF IMMUNOGLOBULINS IN BLOOD SERUM

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T. A. Мitina

2013-01-01

Full Text Available Efficiency of the multiple myeloma treatment with chemotherapy including bortezomib was assessed based on determination of the level of immunoglobulin free light chains in blood serum. The method enables estimation of changes in kinetic parameters of the residual tumor, detection of the disease course prognosis, and the choice of the optimal approach to the disease therapy.

Label-Free Raman Hyperspectral Imaging of Single Cells Cultured on Polymer Substrates.

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Sinjab, Faris; Sicilia, Giovanna; Shipp, Dustin W; Marlow, Maria; Notingher, Ioan

2017-12-01

While Raman hyperspectral imaging has been widely used for label-free mapping of biomolecules in cells, these measurements require the cells to be cultured on weakly Raman scattering substrates. However, many applications in biological sciences and engineering require the cells to be cultured on polymer substrates that often generate large Raman scattering signals. Here, we discuss the theoretical limits of the signal-to-noise ratio in the Raman spectra of cells in the presence of polymer signals and how optical aberrations may affect these measurements. We show that Raman spectra of cells cultured on polymer substrates can be obtained using automatic subtraction of the polymer signals and demonstrate the capabilities of these methods in two important applications: tissue engineering and in vitro toxicology screening of drugs. Apart from their scientific and technological importance, these applications are examples of the two most common measurement configurations: (1) cells cultured on an optically thick polymer substrate measured using an immersion/dipping objective; and (2) cells cultured on a transparent polymer substrate and measured using an inverted optical microscope. In these examples, we show that Raman hyperspectral data sets with sufficient quality can be successfully acquired to map the distribution of common biomolecules in cells, such as nucleic acids, proteins, and lipids, as well as detecting the early stages of apoptosis. We also discuss strategies for further improvements that could expand the application of Raman hyperspectral imaging on polymer substrates even further in biomedical sciences and engineering.

Correlation between serum E-selectin levels and panoramic nailfold capillaroscopy in systemic sclerosis

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Valim V.

2004-01-01

Full Text Available E-selectin is expressed by the activated endothelium and its plasma levels are increased in patients with systemic sclerosis. Eighteen patients fulfilling the American Rheumatism Association criteria for systemic sclerosis, 15 females and 3 males, 42-70 years old, 9 with diffuse and 9 with limited forms, were sequentially recruited for this study. Serum E-selectin levels were determined by commercially available ELISA and their association with nailfold capillaroscopic abnormalities was investigated. Nailfold capillaries were analyzed by 16X magnification wide-field capillaroscopy. Two parameters on capillaroscopy were used to correlate to serum E-selectin: deletion and ectasia. Data were analyzed statistically by the Student t-test and Spearman correlation. Two-tailed P values below 0.05 were considered significant. E-selectin range was 38 to 200 ng/ml (80 ± 39.94. There was a correlation between serum E-selectin levels and the deletion capillaroscopic score (r = 0.50, P < 0.035. This correlation was even stronger within the first 48 months of diagnosis (r = 0.63, P < 0.048. On the other hand, no association was observed between selectin and ectasia. Patients with diffuse disease presented higher serum E-selectin levels than patients with limited disease, although the difference was not statistically significant (96.44 ± 48.04 vs 63.56 ± 21.77 ng/dl; P = 0.08. The present study is the first showing a correlation between soluble serum E-selectin levels and alterations in capillaroscopy. The stronger correlation of deletion score in capillaroscopy in early disease suggests that serum E-selectin levels might be a useful biochemical marker of disease activity in systemic sclerosis.

Correlation between serum E-selectin levels and panoramic nailfold capillaroscopy in systemic sclerosis

Directory of Open Access Journals (Sweden)

V. Valim

2004-09-01

Full Text Available E-selectin is expressed by the activated endothelium and its plasma levels are increased in patients with systemic sclerosis. Eighteen patients fulfilling the American Rheumatism Association criteria for systemic sclerosis, 15 females and 3 males, 42-70 years old, 9 with diffuse and 9 with limited forms, were sequentially recruited for this study. Serum E-selectin levels were determined by commercially available ELISA and their association with nailfold capillaroscopic abnormalities was investigated. Nailfold capillaries were analyzed by 16X magnification wide-field capillaroscopy. Two parameters on capillaroscopy were used to correlate to serum E-selectin: deletion and ectasia. Data were analyzed statistically by the Student t-test and Spearman correlation. Two-tailed P values below 0.05 were considered significant. E-selectin range was 38 to 200 ng/ml (80 ± 39.94. There was a correlation between serum E-selectin levels and the deletion capillaroscopic score (r = 0.50, P < 0.035. This correlation was even stronger within the first 48 months of diagnosis (r = 0.63, P < 0.048. On the other hand, no association was observed between selectin and ectasia. Patients with diffuse disease presented higher serum E-selectin levels than patients with limited disease, although the difference was not statistically significant (96.44 ± 48.04 vs 63.56 ± 21.77 ng/dl; P = 0.08. The present study is the first showing a correlation between soluble serum E-selectin levels and alterations in capillaroscopy. The stronger correlation of deletion score in capillaroscopy in early disease suggests that serum E-selectin levels might be a useful biochemical marker of disease activity in systemic sclerosis.

SYSTEMIC AND CULTURAL DIFFERENCES

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Ranka Jeknić

2007-01-01

Full Text Available This paper analyses one by one the neo-liberalism, social-democracy, radicalism and political-islamism, as four typical socio-political and economic attitudes toward individualism and collectivism as cultural dimensions in the contemporary socio-political and economic contex of globalization. The paper points out principal differences between these four standpoints, and after that, makes conclusions and points out some problematic questions in the conection with the cultural and systemic differences. Their comparative analysis is in the connection with new sociological theories of culture: functionalistic orientation, marxistic and postmodernistic.

Lung Cancer Serum Biomarker Discovery Using Label Free LC-MS/MS

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Zeng, Xuemei; Hood, Brian L.; Zhao, Ting; Conrads, Thomas P.; Sun, Mai; Gopalakrishnan, Vanathi; Grover, Himanshu; Day, Roger S.; Weissfeld, Joel L.; Wilson, David O.; Siegfried, Jill M.; Bigbee, William L.

2011-01-01

Introduction Lung cancer remains the leading cause of cancer-related death with poor survival due to the late stage at which lung cancer is typically diagnosed. Given the clinical burden from lung cancer, and the relatively favorable survival associated with early stage lung cancer, biomarkers for early detection of lung cancer are of important potential clinical benefit. Methods We performed a global lung cancer serum biomarker discovery study using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a set of pooled non-small cell lung cancer (NSCLC) case sera and matched controls. Immunoaffinity subtraction was used to deplete the top most abundant serum proteins; the remaining serum proteins were subjected to trypsin digestion and analyzed in triplicate by LC-MS/MS. The tandem mass spectrum data were searched against the human proteome database and the resultant spectral counting data were used to estimate the relative abundance of proteins across the case/control serum pools. The spectral counting derived abundances of some candidate biomarker proteins were confirmed with multiple reaction monitoring MS assays. Results A list of 49 differentially abundant candidate proteins was compiled by applying a negative binomial regression model to the spectral counting data (pbiomarkers with statistically significant differential abundance across the lung cancer case/control pools which, when validated, could improve lung cancer early detection. PMID:21304412

125I Radioimmunoassay of serum ursodeoxycholyl conjugates

International Nuclear Information System (INIS)

Hill, A.; Ross, P.E.; Bouchier, I.A.D.

1983-01-01

A radioimmunoassay for serum ursodeoxycholic conjugates using an iodine-125 ligand has been developed. The bile acid was present in normal fasting serum (0.19 +- SD 0.19 μmol/l, n=24) and 2-hour post-prandial serum (0.8 +- SD 0.8 μmol/l, n=16). Gallstone patients undergoing oral ursodeoxycholic acid therapy had significantly higher post-prandial serum levels (21.5 +- SD 14.0 μmol/l, n=15) by radioimmunoassay. Gas liquid chromatography analysis indicated that in normal serum ursodeoxycholic acid was totally conjugated, whereas sera from gallstone patients contained a proportion as the free bile acid (10.2 +- SD 8.1 μmol/l, n=15). Following an oral dose of ursodeoxycholic acid, both unconjugated and conjugated forms of the bile acid appeared in the serum of healthy individuals. (Auth.)

Device-Free Indoor Activity Recognition System

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Mohammed Abdulaziz Aide Al-qaness

2016-11-01

Full Text Available In this paper, we explore the properties of the Channel State Information (CSI of WiFi signals and present a device-free indoor activity recognition system. Our proposed system uses only one ubiquitous router access point and a laptop as a detection point, while the user is free and neither needs to wear sensors nor carry devices. The proposed system recognizes six daily activities, such as walk, crawl, fall, stand, sit, and lie. We have built the prototype with an effective feature extraction method and a fast classification algorithm. The proposed system has been evaluated in a real and complex environment in both line-of-sight (LOS and none-line-of-sight (NLOS scenarios, and the results validate the performance of the proposed system.

Serum biochemical reference intervals for free-living Sand Tiger sharks (Carcharias taurus) from east Australian waters.

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Otway, Nicholas M

2015-06-01

Sharks are top-order predators in ocean food chains and the star attractions in aquaria worldwide. Unfortunately, blood biochemistry reference intervals (RI) have been determined for few species. The study aims to establish serum biochemical RI for free-living Sand Tiger sharks (Carcharias taurus) off eastern Australia. Thirty-seven sharks were captured and their sex, length, weight, reproductive maturity, and health status were recorded. After blood collection, serum analytes were quantified using standard analytical and statistical methods. Reference intervals, means, medians, and 90% confidence intervals were generated. Physiologic data from live and necropsied sharks were used to enhance the study results. Thirty healthy sharks were included in the study. Albumin could not be detected. With the exception of ALP activity, values were unaffected by sex, length, weight, age, and life-history stage. The means (RI) were: sodium 258 (249-267) mmol/L, potassium 5.0 (4.3-5.7) mmol/L, chloride 242 (227-257) mmol/L, inorganic phosphate 1.8 (1.7-2.0) mmol/L, total calcium 3.9 (3.3-4.4) mmol/L, magnesium 1.9 (1.6-2.2) mmol/L, glucose 2.7 (2.2-3.2) mmol/L, urea 377 (360-394) mmol/L, ALP 20 (8-31) U/L, ALT 3 U/L (no RI), AST 29 (13-45) U/L, CK 42 (5-79) U/L, total protein 30 (24-36) g/L, triglyceride 0.3 (0.1-0.6) mmol/L, cholesterol 1.4 (0.9-2.1) mmol/L, creatinine 32 μmol/L (no RI), total bilirubin 1.5 μmol/L (no RI), and osmolarity 1082 (1027-1136) mmol/L. These preliminary RI will assist with the clinical evaluation and treatment of captive and free-living Sand Tiger sharks worldwide. Studies with more animals will increase the precision of upper and lower reference limits. © 2015 American Society for Veterinary Clinical Pathology.

Replacement of serum with sericin in in vitro maturation and culture media: Effects on embryonic developmental competence of Sanjabi sheep embryo during breeding season.

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