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61.
Evaluation of monitoring approaches and effects of culture conditions on recombinant protein production in baculovirus-infected insect cells 总被引:2,自引:0,他引:2
The baculovirus infection process ofSpodoptera frugiperda (Sf9) insect cells in oxygen-controlled bioreactors in serum-free medium was investigated using a recombinantAutographa californica (AcNPV) virus expressing -galactosidase enzyme as a model system. A variety of monitoring techniques including trypan blue exclusion, fluorescent dye staining, oxygen uptake rate (OUR) measurements, and glucose consumption were applied to infected cells to determine the best way of evaluating cell integrity and assessing the course of baculovirus infection. The metabolism of newly-infected cells increased 90% during the first 24 hours, but as infection proceeded, and cells gradually succumbed to the baculovirus infection, the cytopathic effect of the baculovirus on the cells became evident. Oxygen and glucose uptake rate measurements appeared to more accurately assess the condition of infected cells than conventional trypan blue staining, which tended to overestimate cell viability in the mid stages of infection. The optimal harvest time varied, depending on which technique — SDS-PAGE, chromogenic (ONPG) or fluorometric (C12FDG) — was used to monitor -galactosidase production. Specific -galactosidase production was found to be insensitive to a wide range of culture dissolved oxygen tensions, whereas resuspending cells in fresh medium prior to infection increased volumetric productivity approximately two-fold (800,000 units -galactosidase/ml) compared to cultures infected in batch mode and allowed successful infections to occur at higher cell densities.Abbreviations ONPG
ortho-phenyl 2--D-galactopyranoside
- OUR
oxygen uptake rate (-mol O2/liter/hour)
- qglucose
specific glucose uptake rate (mg glucose/106cell/hour)
- qglutamine
specific glutamine uptake rate (mg glutamine/106cell/hour)
- qO2
specific oxygen uptake rate (-mol O2/106cell/hour)
- MOI
virus multiplicity of infection (viral plaque forming units/cell) 相似文献
62.
Suspension cultures of Sf-9 cells at different stages of growth were infected with a recombinant baculovirus expressing -galactosidase, using a range of multiplicities of infection (MOI) of 0.05 to 50. Following infection, the cells were resuspended either in the medium in which they had been grown or in fresh medium. Specific -galactosidase yields were not markedly affected by either MOI or medium change in cultures infected in early exponential phase (3×106 cells mL–1). In cultures infected at later growth stages, -galactosidase yields could only be maintained by medium replacement. The possibility that this requirement for medium replacement is due either to the accumulation of an inhibitory byproduct or nutrient limitation was examined. Alanine, a major byproduct of cultured insect cell metabolism, did not significantly reduce recombinant protein yield when added to infected cultures in concentrations of up to 40 mM. Following a factorial design, various nutrient concentrates were added alone or in combination to cultures infected in late exponential phase. Additions that included both yeastolate ultrafiltrate and an amino acid mixture restored specific -galactosidase yields to levels observed at earlier growth stages or in late stages with medium replacement; the addition of these concentrates, by permitting production at higher cell density, led to increases in the volumetric yield of recombinant protein. Together or separately, the concentrates when added to uninfected late exponential phase cultures, lead to a doubling of the maximum total cell protein level normally supported by unamended medium. 相似文献
63.
Daniel Keppler Patrice Waridel Magnus Abrahamson Daniel Bachmann José Berdoz Bernard Sordat 《生物化学与生物物理学报:疾病的分子基础》1994,1226(2):117-125
The lysosomal cystein proteinase cathepsin B is shown to be secreted by ten human colon carcinoma cell lines and to accumulate in culture media as a latent enzyme. The cell lines also secrete a physiological inhibitor of cathepsin B, cystatin C. A significant correlation was found between secretion of the latent enzyme and the inhibitor (r = 0.755, P < 0.01). The aim of the present study was to modulate the respective secretion of the two antagonists to test whether or not latency of cathepsin B was due to the concomitant secretion of the inhibitor. SW480 colon carcinoma cells were treated with the acidotropic agent ammonium chloride, phorbol 12-myristate 13-acetate, and the inflammatory cytokines TGF-β, TNF-α, and IL-1β. Ammonium chloride significantly increased latent cathepsin B levels without affecting the constitutive secretion of cystatin C. Phorbol 12-myristate 13-acetate induced a 4- to 5-fold increase in secreted latent cathepsin B, but did not alter significantly the accumulation of cystatin C in media. The cytokines, TGF-β, TNF-α, and IL-1β, had no major effect on the expression of these two antagonists. Latent cathepsin B released from human carcinoma cells could be efficiently activated by neutrophil elastate at neutral pH. It is concluded that latent cathepsin B is a true proenzyme rather than an enzyme-inhibitor comples. In addition, our data from neutrophil elastate activation experiments indicate that a proteolytic system for activation of the tumor cell-secreted latent enzyme may exist in vivo. 相似文献
64.
Catherine Jumarie Christiane Malo 《In vitro cellular & developmental biology. Animal》1994,30(11):753-760
Summary Caco-2 cell human colon adenocarcinoma cell line was used to study the hormonal regulation of small intestinal epithelial
cell differentiation. We had previously shown that insulin-transferrin-selenium and triiodothyronine (5 × 10−8
M)-supplemented medium can best replace serum after 2 days of culture for both the maintenance and differentiation of Caco-2
cells. The present study demonstrates that precoating petri dishes with complete serum allows the growth and differentiation
of Caco-2 cells seeded directly in serum-free medium. On the other hand, precoating with dialyzed serum inhibits alkaline
phosphatase and dipeptidyl-dipeptidase IV activities by more than 50%. The results obtained with complete serum-precoated
culture plates indicate that there is no synergy between insulin and triiodothyronine because cells maintained in transferrin-selenium
and triiodothyronine-supplemented medium, with or without insulin, express comparable enzyme activities. Moreover, large increases
in alkaline phosphatase and dipeptidyl-dipeptidase IV activities were observed when triiodothyronine was added to the culture
medium by the time confluency was reached. In contrast, γ-glutamyltransferase was lowered to a greater extent when triiodothyronine
was present from the beginning of culture. These findings show that triiodothyronine preferentially stimulates alkaline phosphatase
and dipeptidyl-dipeptidase IV activities during the differentiation period whereas it selectively inhibits γ-glutamyltransferase
during the proliferation phase. Triiodothyronine acts in a dose-dependent manner. 相似文献
65.
The influence of temperature, shoot age, and medium on gall induction by Subanguina picridis on Russian knapweed (Acroptilon repens) was examined in vitro. The optimal temperature for gall formation was 20 C. Gall induction was delayed as the temperature decreased, and decreased as shoot age increased. Bud primordia (0-day-old shoots and 5-day-old shoots) with an average length of 4.2 mm and 7.9 mm were the most suitable tissues for nematode development and gall formation. Gall formation was more effective on B5G medium than on MSG. Young shoots under slow growth were most suitable for mass rearing of S. picridis. 相似文献
66.
An optimized, defined minimal medium was developed to support balanced growth of Escherichia coli X90 harboring a recombinant plasmid. Foreign protein expression was repressed in these studies. A pulse injection technique was used to identify the growth responses to nutrients in a chemostat. Once the nutrients essential for growth had been identified, the yield coefficients for individual medium components. These yield coefficients were used to develop an optimized, glucose-limited defined minimal medium that supports balanced cell growth in chemostat culture. The biomass and substrate concentrations follow the Monod chemostat model. The maximum specific growth rate determined in a washout experiment is 0.87 h(-1) for this strain in the optimized medium. the glucose yield factor is 0.42 g DCW/g glucose and the maintenance coefficient is zero in the glucose-limited chemostat culture. (c) 1993 John Wiley & Sons, Inc. 相似文献
67.
【目的】南极洲具备独特的环境和相对的生物地理隔离,南极洲各类生境中蕴藏了大量尚未培养和难培养的微生物,也是新颖微生物物种的重要来源之一。本研究以南极冰锥洞这类特殊生境为研究对象,通过培养条件的多样化提升南极微生物的培养率和多样性,揭示南极冰锥洞可培养微生物类群多样性,为该环境可培养微生物功能研究奠定基础,也为南极极端环境未培养微生物的培养方法提供借鉴。【方法】通过采用不同培养基添加复苏促进因子(resuscitation promoting factor, Rpf)的方式,提高南极柯林斯冰盖冰锥洞生境中微生物的可培养率,探究该生境中微生物的多样性。采用4种不同营养水平的培养基,平行添加Rpf进行菌株培养,经分离纯化与16S rRNA基因鉴定,分析冰锥洞可培养微生物的多样性及培养条件对多样性的影响。【结果】本研究共分离培养细菌407株,涵盖5个门、18个科、29个属,其中:放线菌门(Actinomycetota)为优势门,占72.73%;微杆菌科(Microbacteriaceae)为优势科,占69.78%;Lacisediminihabitans属为优势属,占45.70%。从培养基效果... 相似文献
68.
Noritsugu Yabe Kouji Komiya Tetsuya Takezono Hisao Matsui 《In vitro cellular & developmental biology. Animal》1993,29(10):795-806
Summary Lysozyme at 1 to 100μg/ml of exposure levels augmented or inhibited proliferative response of human peripheral blood lymphocytes stimulated with
interleukin-2 (IL-2). This contradictory effect of lysozyme depended on IL-2 concentration, activating state of lymphocytes,
addition time of lysozyme, and serum existence. Lymphocytes increased their IL-2-mediated proliferating ability in response
to lysozyme when stimulated with less than suboptimal concentration of IL-2. Lymphocyte activation with anti-CD3 antibody
changed the augmented proliferative response into the inhibited response by lysozyme addition whereas elimination of MHC class
II molecule-expressing cells augmented the response. Addition of lysozyme within 1 h after IL-2 exposure was most effective
in promoting the proliferation whereas additions after 16 to 24 h were ineffective or inhibitory. Addition after longer than
24 h inversely restored the proliferative response. Serum seemed to retard lysozyme action because either sequential serum
addition 1 h after exposure of IL-2 and lysozyme to cells or exposure of IL-2 and serum after pretreatment of cells with lysozyme
changed the proliferative responsiveness from inhibition into augmentation. Thus lysozyme may regulate lymphocyte proliferation
responding to a magnitude of antigenic stimuli and to the progression of cellular events that periodically occur. 相似文献
69.
M. Kondo W. E. Finkbeiner J. H. Widdicombe 《In vitro cellular & developmental biology. Animal》1993,29(1):19-24
Summary Tracheal epithelial cells were grown on Nuclepore filters coated with human placental collagen. When grown immersed in medium
containing fetal bovine serum, cells displayed an undifferentiated ultrastructure (no cilia and a cell height of ∼ 10 μm).
Short-circuit current (Isc) was approximately 1/10 that of the native epithelium. By contrast, when grown in hormonally defined, serum-free medium with
an air interface, cells showed Isc equal to or greater than the original tissue, possessed cilia, and had a cell height of ∼ 50 μm. Responses in Isc to mediators were similar to those of the original tissue, but differed from those of dog or human tracheal epithelium. Given
the ready availability and low cost of the native tissues, bovine tracheal cultures grown in serum-free medium with an air
interface should prove useful in studies of airway epithelial physiology. 相似文献
70.
Growth of cells in a new defined protein-free medium 总被引:1,自引:0,他引:1
Kjell Bertheussen 《Cytotechnology》1993,11(3):219-231
The development of a new stable synthetic serum replacement (SSR) is described, which allows the cultivation of mammalian cells in a defined, protein-free medium containing only dialyzable components. With a low concentration of insulin (RPMI-SR2 medium), growth rates of the transformed cell lines L929, HELA S3, and the hybridoma 1E6 were comparable to growth rates obtained with a serum-containing medium. The same medium also supported long-term cultivation of non-dividing mouse macrophages. The main principle of SSR is a metal ion buffer containing a balanced mixture of iron and trace metals. Stability against precipitation of important metals is achieved by the combined use of EDTA and citric acid as chelating agents. Efficient iron supply is mediated through the inclusion of the compound Aurintricarboxylic acid as a synthetic replacement for transferrin. SSR also contains a growth-promoting surfactant, Pluronic F68. Thus SSR provides a general foundation for growth and differentiation normally provided by serum.Limitations of other serum-free medium designs are discussed here: 1) the inability of transferrin to chelate all metals in the medium; and 2) the use of inorganic iron salts or iron citrate as an iron supplement leads to rapid precipitation of iron hydroxide in the medium. Both these problems are solved in the design of SSR. 相似文献