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Adenoviruses are efficient and safe vectors for delivering target antigens and adenovirus-based vaccines have been used against a wide variety of pathogens, including tuberculosis and COVID-19. Cost-effective and scalable biomanufacturing processes are critical for the commercialization of adenovirus-vectored vaccines. Adenoviral vectors are commonly produced through the infection of batch cultures at low cell density cultures, mostly because infections at high cell densities result in reduced cell-specific virus productivity and does not improve volumetric productivity. In this study, we have investigated the feasibility of improving the volumetric productivity by infecting fed-batch cultures at high cell densities. Four commercial and one in-house developed serum-free media were first tested for supporting growth of HEK 293 cells and production of adenovirus type 5 (Ad5) in batch culture. Two best media were then selected for development of fed-batch culture to improve cell growth and virus productivity. A maximum viable cell density up to 16 × 106 cells/mL was achieved in shake flask fed-batch cultures using the selected media and commercial or in-house developed feeds. The volumetric virus productivity was improved by up to six folds, reaching 3.0 × 1010 total viral particles/mL in the fed-batch culture cultivated with the media and feeds developed in house and infected at a cell density of 5 × 106 cells/mL. Additional rounds of optimization of media and feed were required to maintain the improved titer when the fed-batch culture was scaled up in a bench scale (3 L) bioreactor. Overall, the results suggested that fed-batch culture is a simple and feasible process to significantly improve the volumetric productivity of Ad5 through optimization and balance of nutrients in culture media and feeds.
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Oncolytic viruses (OVs) have emerged as a novel cancer treatment modality, and four OVs have been approved for cancer immunotherapy. However, high-yield and cost-effective production processes remain to be developed for most OVs. Here suspension-adapted Vero cell culture processes were developed for high titer production of an OV model, herpes simplex virus type 1 (HSV-1). Our study showed the HSV-1 productivity was significantly affected by multiplicity of infection, cell density, and nutritional supplies. Cell culture conditions were first optimized in shake flask experiments and then scaled up to 3 L bioreactors for virus production under batch and perfusion modes. A titer of 2.7 × 108 TCID50 mL-1 was obtained in 3 L batch culture infected at a cell density of 1.4 × 106 cells mL-1 , and was further improved to 1.1 × 109 TCID50 mL-1 in perfusion culture infected at 4.6 × 106 cells mL-1 . These titers are similar to or better than the previously reported best titer of 8.6 × 107 TCID50 mL-1 and 8.1 × 108 TCID50 mL-1 respectively obtained in labor-intensive adherent Vero batch and perfusion cultures. HSV-1 production in batch culture was successfully scaled up to 60 L pilot-scale bioreactor to demonstrate the scalability. The work reported here is the first study demonstrating high titer production of HSV-1 in suspension Vero cell culture under different bioreactor operating modes.
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Herpesvirus Humano 1 , Virus Oncolíticos , Animales , Chlorocebus aethiops , Herpesvirus Humano 1/genética , Células Vero , Técnicas de Cultivo Celular por Lotes , Reactores Biológicos , Cultivo de VirusRESUMEN
The growing interest in the use of lentiviral vectors (LVs) for various applications has created a strong demand for large quantities of vectors. To meet the increased demand, we developed a high cell density culture process for production of LV using stable producer clones generated from HEK293 cells, and improved volumetric LV productivity by up to fivefold, reaching a high titer of 8.2 × 107 TU/mL. However, culture media selection and feeding strategy development were not straightforward. The stable producer clone either did not grow or grow to lower cell density in majority of six commercial HEK293 media selected from four manufacturers, although its parental cell line, HEK293 cell, grows robustly in these media. In addition, the LV productivity was only improved up to 53% by increasing cell density from 1 × 106 and 3.8 × 106 cells/mL at induction in batch cultures using two identified top performance media, even these two media supported the clone growth to 5.7 × 106 and 8.1 × 106 cells/mL, respectively. A combination of media and feed from different companies was required to provide diverse nutrients and generate synergetic effect, which supported the clone growing to a higher cell density of 11 × 106 cells/mL and also increasing LV productivity by up to fivefold. This study illustrates that culture media selection and feeding strategy development for a new clone or cell line can be a complex process, due to variable nutritional requirements of a new clone. A combination of diversified culture media and feed provides a broader nutrients and could be used as one fast approach to dramatically improve process performance.
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Técnicas de Cultivo Celular por Lotes , Vectores Genéticos , Recuento de Células , Células Clonales , Medios de Cultivo , Células HEK293 , HumanosRESUMEN
Viral vectors derived from vesicular stomatitis virus (VSV) are important vectors for the development of vaccines and for the treatment of cancer. The efficiency of therapy based on VSV is dependent on the dose of virus used. Therefore it is essential to measure accurately and reproducibly the amount of functional vectors in the samples to be tested. Two common methods used to measure the titer of VSV are TCID50% and plaque assay. In the current study, we compared these two titration methods by using a recombinant VSV expressing the green fluorescent protein (VSV-GFP) as a model virus. Some culture media developed for suspension mammalian cells contain dextran sulfate. We observed that plaque assay, but not TCID50%, can underestimate the virus titer up to 10 fold when VSV-GFP was produced in culture media containing dextran sulfate. Dextran sulfate is commonly used in serum-free culture media to reduce cell aggregation in suspension culture. The inhibitory effect of dextran sulfate on the titration of VSV-GFP was confirmed by supplementing the culture medium with this compound during virus production. Our results also demonstrated that extending the incubation time during plaque assay and TCID50% increases virus titer.
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Medios de Cultivo/química , Sulfato de Dextran/farmacología , Virus de la Estomatitis Vesicular Indiana/crecimiento & desarrollo , Cultivo de Virus , Línea Celular , Compuestos Férricos/farmacología , Proteínas Fluorescentes Verdes/genética , Humanos , Virus de la Estomatitis Vesicular Indiana/genética , Carga Viral , Ensayo de Placa ViralRESUMEN
Vero cells are considered as the most widely accepted continuous cell line by the regulatory authorities (such as WHO) for the manufacture of viral vaccines for human use. The growth of Vero cells is anchorage-dependent. Scale-up and manufacturing in adherent cultures are labor intensive and complicated. Adaptation of Vero cells to grow in suspension will simplify subcultivation and process scale-up significantly, and therefore reduce the production cost. Here we report on a successful adaptation of adherent Vero cells to grow in suspension in a serum-free and animal component-free medium (IHM03) developed in-house. The suspension adapted Vero cell cultures in IHM03 grew to similar or better maximum cell density as what was observed for the adherent Vero cells grown in commercial serum-free media and with a cell doubling time of 40-44â¯h. Much higher cell density (8â¯×â¯106 cells/mL) was achieved in a batch culture when three volume of the culture medium was replaced during the batch culture process. Both adherent and suspension Vero cells from various stages were tested for their authenticity using short tandem repeat analysis. Testing result indicates that all Vero cell samples had 100% concordance with the Vero DNA control sample, indicating the suspension cells maintained their genetic stability. Furthermore, suspension Vero cells at a passage number of 163 were assayed for tumorigenicity, and were not found to be tumorigenic. The viral productivity of suspension Vero cells was evaluated by using vesicular stomatitis virus (VSV) as a model. The suspension cell culture showed a better productivity of VSV than the adherent Vero cell culture. In addition, the suspension culture could be infected at higher cell densities, thus improving the volumetric virus productivity. More than one log of increase in the VSV productivity was achieved in a 3L bioreactor perfusion culture infected at a cell density of 6.8â¯×â¯106 cells/mL.
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Células Vero/virología , Vacunas Virales/inmunología , Cultivo de Virus/métodos , Animales , Técnicas de Cultivo Celular por Lotes/métodos , Reactores Biológicos/virología , Recuento de Células/métodos , Línea Celular , Chlorocebus aethiops , Medios de Cultivo/metabolismo , Medio de Cultivo Libre de Suero/metabolismo , Virus de la Estomatitis Vesicular Indiana/inmunología , Vesiculovirus/inmunologíaRESUMEN
During the last decade, oncolytic viruses such as vesicular stomatitis virus (VSV) have gained tremendous popularity as efficient vaccines for infectious diseases as well as for the treatment of cancer. Our laboratory has developed two stable cell lines, 293SF-3F6 (derived from HEK293A cells) and SF-BMAdR cells (a variant of A549 that expresses the E1 region of human adenovirus). These two cell lines were adapted to grow efficiently in suspension culture and in serum-free medium. In this report we evaluated the production of a recombinant VSV expressing the green fluorescent protein (VSV-GFP) in these two stable cell lines. At a relatively low cell density of 500,000 cells per ml, 293SF-3F6 produced 4.6 times more infectious particles than SF-BMAdR cells. There was a positive correlation between volumetric virus titer and cell density up to 2.E + 07 cells/ml. A fed-batch process using an in-house medium and feed was developed to support the growth of 293SF-3F6 cells up to a concentration of 1.E + 07 cells/ml for infection at higher cell density and VSV production at high titer. Shifting the temperature from 37 °C to 34 °C at infection time improved VSV titer up to 3.3 fold. After scaling up the optimal condition from small scale (3 ml) to larger volumes (50 & 200 ml), the maximal volumetric titer obtained using the 293SF-3F6 cells was in average 2.9E + 10 extracellular infectious particles/ml. In conclusion, our data demonstrated that 293SF-3F6 cells, for which a cGMP master cell bank is available, is a performant cell line to scale up VSV production in suspension culture using serum-free medium.
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Vesiculovirus/fisiología , Animales , Recuento de Células , Línea Celular , Chlorocebus aethiops , Medio de Cultivo Libre de Suero , Proteínas Fluorescentes Verdes , Humanos , Replicación ViralRESUMEN
Tuberculosis (TB) is the second leading cause of death by infectious disease worldwide. The only available TB vaccine is the Bacille Calmette-Guerin (BCG). However, parenterally administered Mycobacterium bovis BCG vaccine confers only limited immune protection from pulmonary tuberculosis in humans. There is a need for developing effective boosting vaccination strategies. AdAg85A, an adenoviral vector expressing the mycobacterial protein Ag85A, is a new tuberculosis vaccine candidate, and has shown promising results in pre-clinical studies and phase I trial. This adenovirus vectored vaccine is produced using HEK 293 cell culture. Here we report on the optimization of cell culture conditions, scale-up of production and purification of the AdAg85A at different scales. Four commercial serum-free media were evaluated under various conditions for supporting the growth of HEK293 cell and production of AdAg85A. A culturing strategy was employed to take advantages of two culture media with respective strengths in supporting the cell growth and virus production, which enabled to maintain virus productivity at higher cell densities and resulted in more than two folds of increases in culture titer. The production of AdAg85A was successfully scaled up and validated at 60L bioreactor under the optimal conditions. The AdAg85A generated from the 3L and 60L bioreactor runs was purified through several purification steps. More than 98% of total cellular proteins was removed, over 60% of viral particles was recovered after the purification process, and purity of AdAg85A was similar to that of the ATCC VR-1516 Ad5 standard. Vaccination of mice with the purified AdAg85A demonstrated a very good level of Ag85A-specific antibody responses. The optimized production and purification conditions were transferred to a GMP facility for manufacturing of AdAg85A for generation of clinical grade material to support clinical trials.
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Adenoviridae , Técnicas de Cultivo de Célula , Vacunas contra la Tuberculosis/biosíntesis , Aciltransferasas/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Formación de Anticuerpos , Antígenos Bacterianos/inmunología , Reactores Biológicos , Medio de Cultivo Libre de Suero , Femenino , Células HEK293 , Humanos , Inmunogenicidad Vacunal , Ratones , Ratones Endogámicos BALB C , Vacunas contra la Tuberculosis/inmunología , Tuberculosis Pulmonar/prevención & controlRESUMEN
Mammalian cell cultures are increasingly used for the production of complex biopharmaceuticals including viral vectors and vaccines. HEK293 is the predominant cell line used for the transient expression of recombinant proteins and a well-established system for the production of viral vectors. Understanding metabolic requirements for high productivity in HEK293 cells remains an important area of investigation. Many authors have presented approaches for increased productivity through optimization of cellular metabolism from two distinct perspectives. One is a non-targeted approach, which is directed to improving feeding strategies by addition of exhausted or critical substrates and eventually removal of toxic metabolites. Alternatively, a targeted approach has attempted to identify specific targets for optimization through better understanding of the cellular metabolism under different operating conditions. This review will present both approaches and their successes with regards to improvement of viral production in HEK293 cells outlining the key relations between HEK293 cell metabolism and viral vector productivity. Also, we will summarize the current knowledge on HEK293 metabolism indicating remaining issues to address and problems to resolve to maximize the productivity of viral vectors in HEK293 cells.
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Portadores de Fármacos/aislamiento & purificación , Portadores de Fármacos/metabolismo , Células Epiteliales/metabolismo , Vacunas/aislamiento & purificación , Vacunas/metabolismo , Técnicas de Cultivo de Célula , Línea Celular , Humanos , Tecnología FarmacéuticaRESUMEN
Hyperosmotic stress has been widely explored as a means of improving specific antibody productivity in mammalian cell cultures. In contrast, a decrease in cell-specific productivity of adenovirus production has been reported in several studies in which virus production in HEK 293 cell cultures was conducted under hyperosmotic conditions. However, production of viral vectors and, in particular, adenoviral vectors is the result of two consecutive phases: the growth phase and the virus production phase. In this study, the singular and combined effects of osmolality on the phases of cell growth and virus production were evaluated in culture media with osmolalities ranging from 250 to 410mOsm. A two-factor, five-level full factorial design was used to investigate the effect of osmotic stress on cell physiology, as determined through the characterization of cell growth, cell metabolism, cell viability, cell cycle, cell RNA and total protein content, and total virus yield/cell-specific virus productivity. Overall, the results show that the growth of cells under hyperosmotic conditions induced favorable physiological states for viral production, and the specific virus productivity was improved by more than 11-fold when the medium's osmolality was increased from 250 to 410mOsm during the cell growth phase. Both hypo- and hyperosmotic stresses in the virus production phase reduced virus productivity by as much as a factor of six. Optimal virus productivity was achieved by growing cells in media with an osmolality of 370mOsm or greater, followed by a virus production phase at an osmolality of 290mOsm. Compared to standard culture and production conditions in isotonic media, the shift from high to low osmolality between the two phases resulted in a two- to three-fold increase in virus yields. This hyperosmotic pressure effect on virus productivity was reproduced in five different commercial serum-free media.
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Adenoviridae/metabolismo , Medios de Cultivo , Presión Osmótica/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos , Cultivo de Virus/métodos , Adenoviridae/crecimiento & desarrollo , Ciclo Celular , Proliferación Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Vectores Genéticos/metabolismo , Células HEK293 , Humanos , ARN/metabolismoRESUMEN
Rabies virus is an important causative agent of disease resulting in an acute infection of the nervous system and death. Although curable if treated in a timely manner, rabies remains a serious public health issue in developing countries, and the indigenous threat of rabies continues in developed countries because of wildlife reservoirs. Control of rabies in wildlife is still an important challenge for governmental authorities. There are a number of rabies vaccines commercially available for control of wildlife rabies infection. However, the vaccines currently distributed to wildlife do not effectively immunize all at-risk species, particularly skunks. A replication competent recombinant adenovirus expressing rabies glycoprotein (AdRG1.3) has shown the most promising results in laboratory trials. The adenovirus vectored vaccine is manufactured using HEK 293 cells. This study describes the successful scale-up of AdRG1.3 adenovirus production from 1 to 500 L and the manufacturing of large quantities of bulk material required for field trials to demonstrate efficacy of this new candidate vaccine. The production process was streamlined by eliminating a medium replacement step prior to infection and the culture titer was increased by over 2 fold through optimization of cell culture medium. These improvements produced a more robust and cost-effective process that facilitates industrialization and commercialization. Over 17,000 L of AdRG1.3 adenovirus cultures were manufactured to support extensive field trials. AdRG1.3 adenovirus is formulated and packaged into baits by Artemis Technologies Inc. using proprietary technology. Field trials of AdRG1.3 rabies vaccine baits have been conducted in several Canadian provinces including Ontario, Quebec and New Brunswick. The results from field trials over the period 2006-2009 demonstrated superiority of the new vaccine over other licensed vaccines in immunizing wild animals that were previously difficult to vaccinate.
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Adenoviridae/aislamiento & purificación , Portadores de Fármacos/aislamiento & purificación , Vectores Genéticos/aislamiento & purificación , Vacunas Antirrábicas/aislamiento & purificación , Virus de la Rabia/genética , Tecnología Farmacéutica/métodos , Adenoviridae/genética , Adenoviridae/crecimiento & desarrollo , Animales , Técnicas de Cultivo de Célula/métodos , Línea Celular , Humanos , Vacunas Antirrábicas/genética , Vacunas Sintéticas/genética , Vacunas Sintéticas/aislamiento & purificaciónRESUMEN
Adenovirus production is currently operated at low cell density because infection at high cell densities still results in reduced cell-specific productivity. To better understand nutrient limitation and inhibitory metabolites causing the reduction of specific yields at high cell densities, adenovirus production in HEK 293 cultures using NSFM 13 and CD 293 media were evaluated. For cultures using NSFM 13 medium, the cell-specific productivity decreased from 3,400 to 150 vp/cell (or 96% reduction) when the cell density at infection was increased from 1 to 3 x 10(6) cells/mL. In comparison, only 50% of reduction in the cell-specific productivity was observed under the same conditions for cultures using CD 293 medium. The effect of medium osmolality was found critical on viral production. Media were adjusted to an optimal osmolality of 290 mOsm/kg to facilitate comparison. Amino acids were not critical limiting factors. Potential limiting nutrients including vitamins, energy metabolites, bases and nucleotides, or inhibitory metabolites (lactate and ammonia) were supplemented to infected cultures to further investigate their effect on the adenovirus production. Accumulation of lactate and ammonia in a culture infected at 3 x 10(6) cells/mL contributed to about 20% reduction of the adenovirus production yield, whereas nutrient limitation appeared primarily responsible for the decline in the viral production when NSFM 13 medium was used. Overall, the results indicate that multiple factors contribute to limiting the specific production yield at cell densities beyond 1 x 10(6) cells/mL and underline the need to further investigate and develop media for better adenoviral vector productions.
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Adenoviridae/crecimiento & desarrollo , Adenoviridae/metabolismo , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Adenoviridae/efectos de los fármacos , Técnicas de Cultivo de Célula , Células Cultivadas , Medios de Cultivo/farmacología , HumanosRESUMEN
Prader-Willi syndrome (PWS) is a complex, genetic, multisystem disorder. Its major clinical features include neonatal hypotonia and failure to thrive, mental retardation, hypogonadism, short hands and feet, hyperphagia-caused obesity, and characteristic appearance. The genetic basis of PWS is also complex. It is caused by the absence of expression of the active paternal genes such as the SNRPN, NDN, and possibly others in the PWS critical region on 15q11-13. PWS is in effect a contiguous gene syndrome resulting from deletion of the paternal copies of the imprinted. Consensus in clinical diagnostic criteria was established in 1993. However, identifying relevant patients for tests remains a challenge for most practitioners, as many features of the disorder are nonspecific, and others can be subtle or evolved over time. Consequently, molecular genetic tests can be used to diagnose PWS accurately, allowing early diagnosis of the syndrome. High resolution G-banding, high resolution cytogenetic methylation-specific PCR (MS-PCR), and fluorescence in situ hybridization (FISH) are routinely used to diagnose PWS. In this study, four Chinese patients, with typical PWS features, were detected by MS-PCR and FISH. Three were cytogenetically normal, but lacked paternal expression of proximal chromosome 15q because of maternal uniparental disomy (UPD). The other one, however, demonstrated an unbalanced de novo translocation 46, XX, t (7; 15).
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Pueblo Asiatico/genética , Cromosomas Humanos Par 15 , Síndrome de Prader-Willi/genética , Translocación Genética , Adolescente , Niño , Metilación de ADN , Salud de la Familia , Femenino , Humanos , Hibridación Fluorescente in Situ , Masculino , Obesidad Mórbida/genética , Obesidad Mórbida/patología , Reacción en Cadena de la Polimerasa , Síndrome de Prader-Willi/patología , Adulto JovenRESUMEN
Type 2 diabetes mellitus (T2DM) is a heterogeneous disorder characterized by defects in insulin secretion and action and obesity plays an important role in the deterioration of glucose metabolism. In the present study we evaluated the degree of insulin resistance and first-phase insulin secretion of beta-cell in obese subjects with normal glucose tolerance (NGT), impaired glucose tolerance (IGT), and T2DM in Chinese. A total of 220 subjects underwent standard 75 g oral glucose tolerance test (OGTT) and insulin-modified frequently sampled intravenous glucose tolerance test (FSIGT). Insulin sensitivity index (S I) was assessed by the reduced sample number (n = 12) of Bergman's minimal model method with FSIGT. Insulin secretion capacities were determined by the insulinogenic index (I 30 min - I 0 min)/(G 30 min - G 0 min) in OGTT and the acute insulin response to glucose (AIR) in FSIGT. The disposition index (DI), the product of AIR and S I was used to determine whether AIR was adequate to compensate for insulin resistance. The S I in healthy lean control group was significantly higher than that in NGT, IGT, and T2DM group, but there was no significant difference among NGT, IGT, and T2DM group. The AIR in NGT group was significantly greater than that in control group, but then it was progressively decreased in IGT and T2DM group. The value of DI in control group was significantly higher than that in those three abnormal groups, and was decreased from NGT to IGT and T2DM group with significant difference. It indicates that obese subjects with different glucose tolerances have a similar degree of insulin resistance but differ in insulin secretion in Chinese Han population.
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Diabetes Mellitus Tipo 2/etiología , Resistencia a la Insulina , Células Secretoras de Insulina/patología , Obesidad/complicaciones , Adulto , Anciano , Glucemia/análisis , Estudios de Casos y Controles , China , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/patología , Femenino , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/sangre , Insulina/metabolismo , Secreción de Insulina , Masculino , Persona de Mediana Edad , Obesidad/sangre , Obesidad/patologíaRESUMEN
Yeastolate is effective in promoting growth of insect cell and enhancing production of recombinant protein, thus it is a key component in formulating serum-free medium for insect cell culture. However, yeastolate is a complex mixture and identification of the constituents responsible for cell growth promotion has not yet been achieved. This study used sequential ethanol precipitation to fractionate yeastolate ultrafiltrate (YUF) into six fractions (F1-F6). Fractions were characterized and evaluated for their growth promoting activities. Fraction F1 was obtained by 65% ethanol precipitation. When supplemented to IPL-41 medium at a concentration of 1 g L(-1), fraction F1 showed 71% Sf-9 cell growth improvement and 22% beta-galactosidase production enhancement over YUF (at 1 g L(-1 )in IPL-41 medium). However, the superiority of F1 over YUF on promoting cell growth gradually diminished as its concentration in IPL-41 medium increased. At 4 g L(-1), the relative activity of F1 was 93% whereas YUF was 100% at the same concentration. At 1 g L(-1), four other fractions (F2-F5) precipitated with higher ethanol concentrations and F6, the final supernatant, showed growth promoting activities ranging from 32 to 80% as compared to YUF (100%). Interestingly, a synergistic effect on promoting cell growth was observed when F6 was supplemented in IPL-41 medium in presence of high concentrations of F1 (>3 g L(-1)). The results suggest that ethanol precipitation was a practical method to fractionate growth-promoting components from YUF, but more than one components contributed to the optimum growth of Sf-9 cells. Further fractionation, isolation and identification of individual active components would be needed to better understand the role of these components on the cell metabolism.
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Overproduction of proinflammatory factors is associated with obesity and diabetes. Interleukin (IL)-18 as a member of IL-1 cytokine family is increased in obese, in diabetic, and even in polycystic ovary syndrome (PCOS) patients. In the present study we evaluated the association of serum IL-18 levels with insulin resistance in PCOS women. Forty-two PCOS women and 38 control subjects were enrolled in this study and matched with respect to age and body mass index (BMI). Serum IL-18 levels and hormones were measured for all subjects. Furthermore, euglycemic hyperinsulinemic clamp test was performed in selected 30 PCOS women and 11 control subjects. Serum IL-18 levels were elevated in PCOS women compared with the control (p = 0.033). IL-18 levels were positively correlated with homeostasis model assessment index (HOMA) beta index, which assesses beta cell function (p = 0.035), but were inversely correlated with clamp indices, which best represent insulin resistance status: M, Clamp ISI*100, and MCRg values (p = 0.006, 0.010, and 0.009 respectively). No correlation was found between IL-18 and age, BMI, waist-to-hip ratio (WHR), lipid profile, dehydroepiandrosterone-sulfate (DHEAS), sex hormone- binding globulin (SHBG), or fasting insulin levels. In conclusion, in the present study, serum IL-18 levels were significantly increased in PCOS women and firmly associated with insulin resistance displayed by euglycemic hyperinsulinemic clamp test. It indicates that IL-18 may be a contributing factor linking inflammation and insulin resistance in PCOS women.
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Técnica de Clampeo de la Glucosa/métodos , Resistencia a la Insulina , Interleucina-18/sangre , Síndrome del Ovario Poliquístico/sangre , Síndrome del Ovario Poliquístico/epidemiología , Glucemia/análisis , Índice de Masa Corporal , HDL-Colesterol/sangre , LDL-Colesterol/sangre , Deshidroepiandrosterona/sangre , Diabetes Mellitus Tipo 2/epidemiología , Femenino , Hormona Folículo Estimulante/sangre , Humanos , Insulina/sangre , Hormona Luteinizante/sangre , Obesidad/epidemiología , Síndrome del Ovario Poliquístico/complicaciones , Globulina de Unión a Hormona Sexual/análisis , Triglicéridos/sangreRESUMEN
<p><b>OBJECTIVE</b>To compare the characteristics of over weight and obesity subjects defined by three criteria: World Health Organization (WHO) in 1999, the National Cholesterol Education Program Adult Treatment Panel Nll (NCEP-ATP II ) in 2001, and the International Diabetes Federation (IDF) in 2005.</p><p><b>METHODS</b>Medical history collection, physical examination, oral glucose tolerance test ( OGTT) , and frequently sampled intravenous glucose tolerance test (FSIGTT) were performed in 371 subjects with body mass index (BMI)> or =23 kg/m(2). Fasting blood samples were taken to test lipid profiles, urea acid level and so on. Data was analyzed including comparison and relationship statistics.</p><p><b>RESULTS</b>Data from FSIGTT showed that insulin resistance existed in all over weight and obesity subjects. The relationship between waist and sensitivity index (SI) was superior than that between BMI and SI ( r = - 0. 198 and r = - 0. 194, P < 0. 001). Totally 107 subjects (28. 8%) did not meet any definitions, 187 (50. 4%) met ATP definition, 98 (26. 4%) met WHO definition, and 234 (63. 1% ) met IDF definition. ATP and IDF definitions had the highest coincidence rate (70. 3% ) , and subjects in these two groups almost had basically the same manifestations. The age of onset in WHO group was higher than those in other two groups. Meanwhile, subjects in the WHO group was characterized by lower BMI, higher hyperlipidemia, and significantly increased fasting and postprandial blood glucose level. The MS component scores were positively correlated with age, BMI, waist, and waist-to-hip ratio (WHR) , and were negatively correlated with SI. However, no correlation was found between MS component scores and gender, fat% , or acute insulin response to glucose index.</p><p><b>CONCLUSIONS</b>Some of over weight and obesity subjects only have insulin resistance and have no metabolism syndrome. The group defined by IDF criteria of the metabolic syndrome (MS) has the highest incidence rate. Limited by the requirements of insulin resistance evaluation, the definition proposed by WHO is somewhat unfeasible for practices. ATP definition may be replaced by IDF definition, because the populations defined by ATP definition is covered by the latter. Meanwhile, the abnormalities of fat mass distribution in the body ( i. e. , central obesity) , rather than its contents, may exacerbate the occurrence of MS, as well as the development of insulin resistance.</p>
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Adolescente , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Índice de Masa Corporal , China , Epidemiología , Prueba de Tolerancia a la Glucosa , Resistencia a la Insulina , Síndrome Metabólico , Diagnóstico , Epidemiología , Obesidad , Relación Cintura-CaderaRESUMEN
Lipids (fatty acids) at a concentration range of 10-100 microg/L are essential components included in most serum-free cell culture medium formulations. A gas chromatography/mass spectrometry (GC/MS) method for the micro-quantitation of lipids, determined as fatty acid methyl esters (FAMEs), in complex serum-free cell culture media was developed. The interference of derivatizing reagents, extraction solvents and medium additives in the micro-quantitation of lipids was also examined. The results show that the concentration of fatty acids such as palmitic and stearic acids detected in derivatizing reagents or extraction solvents was in the range of 10-230 microg/L. Tween-80, a surfactant and medium additive, produced nearly 20 FAMEs alone when methylated using a derivatizing agent. Moreover, the surfactant Pluronic F-68, a medium additive, interfered in the FAME recovery. Procedures, which include use of low volumetric ratio of reagent to medium and precipitation of the above surfactants, were developed to minimize background FAMEs to levels which do not significantly affect the quantitation of medium lipids and to diminish the interference caused by Pluronic F-68. Fatty acid concentrations in several complex serum-free culture media were quantitated by this method and were very close to the values indicated in their formulations.
Asunto(s)
Medio de Cultivo Libre de Suero/análisis , Lípidos/análisis , Animales , Células Cultivadas , Ésteres/análisis , Ésteres/síntesis química , Ácidos Grasos/análisis , Cromatografía de Gases y Espectrometría de Masas , Indicadores y Reactivos , Insectos , Mamíferos , Polisorbatos , Estándares de Referencia , Soluciones , SolventesRESUMEN
A method using flow cytometry (FCM) analysis was developed to quantitate baculovirus total particles produced in insect cell cultures. The method is a direct count of particles and involves staining of the baculovirus DNA with SYBR Green I, a highly fluorescent nucleic acid specific dye. Sample preparation of cell-free supernatant containing budded viral particles involves fixation with paraformaldehyde, freeze-thaw treatment, viral membrane permeabilization with Triton X-100, and sample heating to improve staining efficiency and enhance baculovirus particle green fluorescence intensities. In this study, the effects of the different treatment steps and medium composition on viral particle counts were examined in order to identify optimal preparation conditions. FCM analysis linearity was established over a viral concentration range of two logs with a lower detection limit at 10(5) viral particles per ml. Robustness and reproducibility of the method were assessed using samples from large-scale bioreactor cultures. The events (or virus particle counts) obtained by FCM analysis were usually higher than the titres obtained by end-point dilution assay (EPDA). Results from 16 different viral stocks showed an average ratio of 3.7 total particles (FCM) to infectious particles (EPDA). Essentially, the FCM analysis reported below shortens baculovirus quantitation time to 2 h and provides a good estimation of virus titers. It is believed that these findings will contribute to acceleration of process development in the area of baculovirus expression technology in general and specifically in process where stoichiometric multi-viral infections of cells are critical to the expression of complex products.