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1.
François LeFloch Bertrand Tessier Sébastien Chenuet Jean-Marc Guillaume Pierre Cans Jean-Louis Goergen Annie Marc 《Cytotechnology》2006,52(1):39-53
The necessity to perform serum-free cultures to produce recombinant glycoproteins generally requires an adaptation procedure
of the cell line to new environmental conditions, which may therefore induce quantitative and qualitative effects on the product,
particularly on its glycosylation. In previous studies, desialylation of EPO produced by CHO cells was shown to be dependent
on the presence of serum in the medium. In this paper, to discriminate between the effects of the adaptation procedure to
serum-free medium and the effects of the absence of serum on EPO production and glycosylation, adapted and non-adapted CHO
cells were grown in serum-free and serum-containing media. The main kinetics of CHO cells were determined over batch processes
as well as the glycosylation patterns of produced EPO by HPCE-LIF. A reversible decrease in EPO production was observed when
cells were adapted to SFX-CHOTM medium, as the same cells partially recovered their production capacity when cultivated in serum-containing medium or in
the enriched SFMTM serum-free medium. More interestingly, EPO desialylation that was not observed in both serum-free media was restored if the
serum-independent cells were recultured in presence of serum. In the same way, while the serum-independent cells did not release
a sialidase activity in both serum-free media, a significant activity was recovered when serum was added. In fact, the cell
adaptation process to serum-free conditions did not specifically affect the sialidase release and the cellular mechanism of
protein desialylation, which appeared to be mainly related to the presence of serum for both adapted and non-adapted cells. 相似文献
2.
Dissociated retinal cells from 8-day-old chick embryos were cultivated in serum-containing and in defined serum-free media. Under the latter conditions, and using polylysine as a substrate, the proliferation of glial cells was almost completely prevented, and pure (>90%) neuronal cultures could be maintained for up to 7 days in vitro. The specific but not the total activities of choline acetyltransferase and of the nicotinic and the muscarinic acetylcholine receptor were increased under serum-free culture conditions. Autoradiographic studies with [125I]α-bungarotoxin, a selective ligand for nicotinic cholinergic receptors, showed that serum-free culture conditions may constitute a useful tool for identifying biochemically different types of retinal neurons in tissue culture. 相似文献
3.
Jianyong Wu Glenn A. King Andrew J. Daugulis Peter Faulkner Mattheus F. A. Goosen 《Cytotechnology》1992,9(1-3):141-147
Spodoptera frugiperda (IPLB-SF-21) insect cells were grown in shake-flasks and infected with a temperature-sensitive baculovirus to express the
gene of chloramphenicol acetyl transferase (CAT) in serum-free medium (SF-900) and two serum-supplemented media (IPL-41 and
Grace's). In temperature-shift experiments (cell growth at 33°C followed by virus replication at 27°C 3–4 days later), virus
and CAT production were much poorer in the serum-free medium than in serum-supplemented media, though cell growth was virtually
the same in the different media tested. In all the three media, highest virus and CAT titers were obtained at the lowest MOI
(multiplicity of infection 0.02). This result is contrary to that obtained in constant-temperature culture (27°C for both
cell growth and virus replication). Virus and CAT production was greatly improved when the entire culture was run at constant
temperature. It appeared that infected cells were severely damaged at 33°C (6°C above the optimal 27°C), resulting in little
or no virus and protein production. As a result of these temperature-shift experiments, a larger-scale (141 air-lift bioreactor)
serum-free culture of Sf-9 insect cells was conducted at constant temperature (27°C) to produce recombinant protein (β-galactosidase).
A cell density as high as 1×107 cells.ml−1, and a β-gal concentration of up to 104,000 unit.ml−1 were achieved. 相似文献
4.
Jack Litwin 《Cytotechnology》1992,10(2):169-174
Vero cell lines, usually considered anchorage-dependent, could be grown as cell-aggregates in suspension culture with serum-free media. Several different combinations of base media gave growth results above 106 cells/ml (NCTC 135:SFRE 199-1; NCTC 135:Waymouth MB 752/1; NCTC 135:RPMI 1640). Insulin was not essential for growth and Bovine Serum Albumin could be diluted out of the media if linoleic acid was present. The size and density of the aggregates formed varied depending on the media used. 相似文献
5.
Baculovirus vectors constitute important tools for therapeutic protein production and mammalian cell transduction for gene therapy applications. A prerequisite for such applications is that the cell lines in which baculoviruses are propagated be maintained in serum-free media that are devoid of potential human pathogens. However, in serum-free media, the performance of baculovirus-based systems can be significantly reduced. In this report, we show that silkmoth-derived host cell lines for the Bombyx mori-nuclear polyhedrosis virus (BmNPV) that are transformed with the gene for the promoting protein (PP), a silkmoth-derived secreted factor containing a lipid-binding domain, display enhanced susceptibility to BmNPV infection and enhanced budded virus productivity in serum-free media. For transformed silkmoth cells maintained in serum-free media, the rate of BmNPV entry is enhanced by two orders of magnitude relative to the untransformed cells, while the rate of budded virus production is increased five-fold. The infectivity-enhancing effect can be also conferred to normal cells grown in serum-free media by addition of conditioned media from the transformed cells, which contain the secreted recombinant PP. Thus, PP substitutes for serum factors whose presence facilitates baculovirus entry into the cells. However, the effects of silkmoth-derived PP may be specific to the BmNPV-silkmoth system since little or no changes in viral infectivity are obtained by PP expression in Trichoplusia ni-derived High-Fivetrade mark cells grown in serum-free media and infected with a different baculovirus (AcNPV). 相似文献
6.
Falkensammer C Jöhrer K Gander H Ramoner R Putz T Rahm A Greil R Bartsch G Thurnher M 《Cancer immunology, immunotherapy : CII》2006,55(10):1228-1237
Objective: While previous reports clearly demonstrated antiproliferative effects of IL-4 on renal cell carcinoma (RCC) in vitro, the administration of IL-4 to patients with metastatic RCC in clinical trials could not recapitulate the promising preclinical results. In the present study we wanted to examine the context of IL-4 action and to establish conditions of enhanced IL-4 efficacy. Methods: Primary and permanent human RCC cells were cultured in either serum-supplemented or chemically defined, serum-free culture medium in the presence or absence of cytokines. Cell proliferation was assessed as [3H]-thymidine incorporation. Cell apoptosis was measured using the fluorescent DNA intercalator 7-aminoactinomycin D and flow cytometry. In addition, culture media conditioned by RCC were subjected to cytokine antibody array and cytokine multiplex analysis. Results: Our results indicate that the previously reported antiproliferative effects of IL-4 are serum-dependent. Under serum-free conditions, IL-4 failed to exhibit growth-inhibitory effects or was even growth-stimulatory. In a chemically defined, serum-free medium (AIM-V), however, IL-4 inhibited the TNF-α induced proliferation of RCC. IL-4 and TNF-α synergistically induced apoptosis of RCC as well as a complex cytokine response by RCC, which included the synergistic upregulation of RANTES and MCP-1. Conclusions: IL-4 alone has little effect on the spontaneous proliferation of RCC but can prevent the enhancement of proliferation induced by growth promoters like FBS and TNF-α. The concomitant growth inhibitory, apoptosis-inducing, and cytokine-enhancing effects of IL-4 in combination with TNF-α on RCC support the view that Th2 cytokines may be required for productive immune responses against RCC. 相似文献
7.
AIM: To compare commercially available serum-free media with common, standard, growth medium for their ability to support growth of Cryptosporidium parvum in HCT-8 cell cultures. METHODS AND RESULTS: Twelve serum-free media formulations with or without additional supplements were tested against a standard growth medium containing 2% FBS in HCT-8 cell cultures. After a 48-h incubation period, the level of parasite development was determined by ELISA. The extent of development in the serum-free media was determined as a percentage of infections compared with those obtained using a standard growth medium. CONCLUSIONS: Several of the serum-free media formulations, which included MDCK, UltraMDCK, PC-1, UltraCHO and UltraCulture, compared favourably with a traditional, standard growth medium. Moreover, increasing FBS concentrations to 10% actually resulted in an overall decrease in development in many cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Several serum-free medium formulations are available which allow development of C. parvumin vitro at levels comparable with standard media employing FBS. These serum-free media are particularly useful for applications, which may require a more defined medium without the presence of FBS. Moreover, the elimination of FBS as a variable allows investigators the ability to more closely regulate their experimental systems when growing C. parvum in cell cultures. 相似文献
8.
David G. Chilton Betty H. Johnson Laurence Danel-Moore Simon Kawa E. Brad Thompson 《In vitro cellular & developmental biology. Plant》1990,26(6):561-570
Summary CEM-C7, a human leukemic CD4+ T-lymphocyte cell line and three of its subclones, CEM-4R4, CEM-3R43, and ICR-27, previously
cultured in a medium supplemented with 5 to 10% fetal bovine serum, have been adapted to serum-free media. The best medium
of those tested was RPMI 1640 supplemented with 5 μg/ml each transferrin and insulin + 5 ng/ml sodium selinite ± 0.1% bovine
serum albumin. While growing either with or without albumin, the several clonal lines of CEM cells displayed growth similar
to serum-supplemented cultures. Cell proliferation of CEM-C7 cells cultured in both serum-free media has been sustained for
3 mo, with culture doubling times of about 25 h for both serum-supplemented and serum-free cultures (viability ≥ 90%). Cell
morphology remained essentially the same in serum-free or serum containing media. The expression of CD4, a marker for T-derived
lymphoid cells, was not significantly different in serum-free medium. When grown in serum-free medium, CEM-C7 cells exhibited
increased steroid responsiveness as evidenced by increased glucocorticoid receptor binding sites, increased induction of glutamine
synthetase, and cell lysis at lower concentrations of steroid. Receptor mutant subclones of CEM-C7, which are proven to be
completely unresponsive to micromolar concentrations of dexamethasone when grown in serum-supplemented medium, become partially
sensitive to the hormone after growth in defined medium. The increased sensitivity of CEM-C7 cells and its subclones to dexamethasone
in serum-free medium returned to previous levels when these cells were recultured in serum-containing medium. Our results
suggest that substances in serum influence steroid effects on these cells and that the molecular details of glucocorticoid
hormone action may be pursued more precisely in a clearly defined culture medium.
This work was conducted in conjunction with the Walls Medical Research Foundation. 相似文献
9.
The replacement of serum in hybridoma cultures is considered. The focus is on the effects of serum-free media on hybridoma growth and monoclonal antibody secretion. Comparative literature data with serum supplemented cultures are discussed with an analysis of serum-free formulations and selection rules for the serum-free ingredients. In general, serum-free media which are "lipid rich" can sustain cell growth rates approaching that of serum supplemented cultures. Specific antibody secretion rate, however, is usually higher in serum-free media, irrespective of the lipid content. 相似文献
10.
Yuhsi Matuo Nozomu Nishi Hideyuki Tanaka Ikuharu Sasaki John T. Issacs Fumio Wada 《In vitro cellular & developmental biology. Plant》1988,24(10):1053-1056
Summary AT-3 cells, one of anaplastic cell lines established from the Dunning prostatic carcinoma of rats, were able to grow under
serum-free conditions in a state of suspension detached from a substratum. Radioimmunoassays using monoclonal antibody against
rat insulin-like growth factor II (IGF-II) revealed the presence of IGF-II-related peptide in acid-ethanol extracts extracsts
of lyophilized serum-free media conditioned by AT-3 cell. The peptide contents in the culture media increased with increase
in cell number; 71 ng at 3.0 × 106 cells and 449 ng at 4.6 × 107 cells. IGF-II-related peptide was hardly detectable in acid-ethanol extracts of AT-3 cells harvested after 13-days culture.
These results indicate that AT-3 cells produce IGF-II-related peptide ana may release it into the culture media.
Editor's statement One or more members of the insulin-like growth factor family have been established previously as mitogen
for isolated prostate cells. This report suggests that IGF-II member of the family may be involved in autocrine support of
cells from highly malignant prostate tumors. 相似文献
11.
Joanne Keenan Dermot Pearson Lorraine O’Driscoll Patrick Gammell Martin Clynes 《Cytotechnology》2006,51(1):29-37
DeltaFerrinTM, a yeast-derived recombinant human transferrin produced by Delta Biotechnology Ltd. (Nottingham UK), was found to be a suitable replacement for holo human transferrin in serum-free culture media of the MDCK cell line (chosen because of its transferrin dependence) in short-term screening assays. Long-term subculture was achieved with DeltaFerrinTM supporting growth equivalent to that of holo human transferrin. DeltaFerrinTM and a selection of chemical iron chelators were found in short-term assays to be equivalent to holo human transferrin in supporting growth of MDCK, BHK-21-PPI-C16 and Vero-PPI. In long-term subcultures, however, only DeltaFerrinTM was found to support cell growth in a manner essentially equivalent to holo human transferrin in all three cell lines. For both BHK and Vero variants tested, recombinant preproinsulin production was unaltered by replacing holo human transferrin with DeltaFerrinTM. As such, this is the first report of a recombinant human transferrin produced under animal-free conditions that can act as a universal iron chelator for cells grown in serum-free media (SFM). 相似文献
12.
Several new cell culture media designed specifically for the expression of recombinant antibodies in Chinese hamster ovary (CHO) cells were investigated for the presence of bovine IgG. Three serum-free media, three protein-free (animal component free) media, as well as one chemically defined medium were included in the study. Employing a combination of affinity chromatography (Protein G or A columns), SDS-PAGE analysis, and peptide mass fingerprinting, two of the serum-free media were found to contain bovine IgG in the range of approximately 0.5 mg/L. The other five media did not contain detectable levels of contaminating Protein A or G-binding proteins such as bovine IgG. 相似文献
13.
Primary astroglial cultures (14 days of age) from cerebral cortex, striatum, and hippocampus of newborn rat brain contained similar amounts of soluble proteins. Uptake and incorporation of [3H]valine into soluble protein measured after 30 and 60 min of incubation, respectively, was on a similar level in the various cultures. [3H]valine labeling of protein bands from the cell soluble fractions and incubation media of hemisphere cultures, and which were separated by isoelectric focusing (IEF) or sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), indicated that proteins are released to the extracellular medium after being synthesized within cultivated cells. Acidic and high molecular weight proteins were more heavily labelled in the incubation media than in the cell soluble fractions. Two-dimensional electrophoresis (IEF×SDS-PAGE) of soluble proteins from the different cultures showed similar patterns, which were quite different from the serum-free extracellular protein patterns. Both fractions were different from the pattern obtained from fetal calf-serum. In striatum and hippocapus culture media a spot was seen with Ip 6.0–6.8 and m.w. 105,000, and in the media from cerebellar cultures another spot was observed with Ip 5.2–5.6 and m.w. 55,000. The results show that the different cultures are similar in their protein synthetic capacity and protein composition. The specific differences observed in proteins obtained from the serum-free incubation media might indicate specific properties among astroglial cells from various brain regions. 相似文献
14.
Stem cell-based therapies depend on the reliable expansion of patient-derived mesenchymal stem cells (MSCs) in vitro. The supplementation of cell culture media with serum is associated with several risks; accordingly, serum-free media are commercially available for cell culture. Furthermore, hypoxia is known to accelerate the expansion of MSCs. The present study aimed to characterize the properties of periodontal ligament-derived MSCs (PDLSCs) cultivated in serum-free and serum-containing media, under hypoxic and normoxic conditions. Cell growth, gene and protein expression, cytodifferentiation potential, genomic stability, cytotoxic response, and in vivo hard tissue generation of PDLSCs were examined. Our findings indicated that cultivation in serum-free medium does not affect the MSC phenotype or chromosomal stability of PDLSCs. PDLSCs expanded in serum-free medium exhibited more active growth than in fetal bovine serum-containing medium. We found that hypoxia does not alter the cell growth of PDLSCs under serum-free conditions, but inhibits their osteogenic and adipogenic cytodifferentiation while enabling maintenance of their multidifferentiation potential regardless of the presence of serum. PDLSCs expanded in serum-free medium were found to retain common MSC characteristics, including the capacity for hard tissue formation in vivo. However, PDLSCs cultured in serum-free culture conditions were more susceptible to damage following exposure to extrinsic cytotoxic stimuli than those cultured in medium supplemented with serum, suggesting that serum-free culture conditions do not exert protective effects against cytotoxicity on PDLSC cultures. The present work provides a comparative evaluation of cell culture in serum-free and serum-containing media, under hypoxic and normoxic conditions, for applications in regenerative medicine. 相似文献
15.
Martin Lubin 《In vitro cellular & developmental biology. Animal》1993,29(7):597-600
Summary Human fibroblast, bladder carcinoma, and breast carcinoma cells in commercial serum-free media or balanced salt solutions
rapidly lose K+ and gain Na+. This rapid K+ loss is caused by one or more impurities in phenol red. Adding serum or albumin to media or to balanced salts prevents K+ loss. Quinine also prevents part of this loss in fibroblasts and breast carcinoma cells, suggesting that the impurity acts
on an ion channel. 相似文献
16.
Insulin is often included in serum-free media for animal cell cultivation. However, the necessity of insulin for a specific cell line is rather uncertain. In this article we report the effects of insulin on the cultivation of a hybridoma cell line in a serum-free medium. It was found that insulin affected neither the cell growth nor the antibody production. The specific growth rate and specific antibody production rate were very similar in the cultures with or without insulin. However, the presence of insulin affected the nutrient consumption rate and cell metabolism. Including insulin in the medium resulted in a higher specific glucose consumption rate, a shorter exponential growth stage, and a lower final antibody concentration. The elimination of insulin from the medium allowed antibody to accumulate to a concentration substantially higher than that in the insulin-containing cultuvre. (c) 1995 John Wiley & Sons, Inc. 相似文献
17.
Insulin-like growth factor-II (IGF-II) is highly expressed in fetal tissues and may act as an autocrine growth factor during early embryogenesis. The SH-SY5Y human neuroblastoma cell line also expresses IGF-II and its receptors and responds to exogenous IGF-II with increased DNA synthesis, cell division, and neuritic outgrowth. For this study, we tested the hypothesis that IGF-II mediates autocrine growth of SH-SY5Y cells in serum-free media. SH-SY5Y cells plated at high densities proliferated in serum-free media, whereas sparsely plated cells did not. IGF-II mRNA levels increased within 24 hours of serum deprivation and were associated with increased immunoreactive IGF-II protein. Exogenous addition of IGF-II increased 3H-TdR incorporation and cell number in a dose- and time-dependent fashion. By nuclear labelling experiments using 5-Bromo-2′ deoxyuridine (BrdU), we detected a twofold higher percentage of S phase nuclei after a 24-hour incubation in IGF-II. Treatment of SH-SY5Y cells with anti-IGF-II antibodies in serum-free media inhibited cell proliferation, and this inhibition was partially overcome by the addition of increasing concentrations of IGF-II. Collectively, our results indicate that IGF-II mediates an autocrine growth mechanism in SH-SY5Y cells that is associated with increased IGF-II expression. © 1993 Wiley-Liss, Inc. 相似文献
18.
Stepwise screening of media supplements using factorial design and analysis was employed in the development of serum-free medium for a recombinant Chinese hamster ovary cell line. The effects (growth and target protein production) of different combinations were measured at two time points to ensure adequate response. The results were analysed by a computer program specialized in factorial analysis. The formulation deduced from the previous experiment was used as the new basal medium for the next screening. Certain significant nutrients were studied again in a more advanced formulation in order to analyse the potential synergistic effects with new media components. Compared to cells grown in serum-containing medium, cells adapted to the final formulation of the serum-free medium had a comparable growth rate but a four fold increase in the active protein production.Abbreviations ANOVA
Analysis of variance
- BSA
bovine serum albumin
- CHO
Chinese hamster ovary
- FBS
fetal bovine serum
- MTT
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
- PBS
phosphate buffered saline
- SFM
serum-free medium 相似文献
19.
Cheryl A. Conover Ron G. Rosenfeld Raymond L. Hintz 《In vitro cellular & developmental biology. Plant》1989,25(6):521-527
Summary The biological actions of insulinlike growth factor I (IGF-I) measured under serum-free assay conditions were found to be
significantly influenced by prior subculture conditions for adult human fibroblasts. Glucocorticoids seemed to be the major
medium variable affecting IGF-I action. IGF-I added to serum-free cultures had little or no effect on [14C]aminoisobutyric acid uptake or [3H]thymidine incorporation in human fibroblasts previously maintained in media containing serum with low glucocorticoid levels
or in serum stripped of endogenous steroids. However, a 48-h preincubation with dexamethasone resulted in a marked synergistic
increase in IGF-I stimulation of [14C]aminoisobutyric acid uptake and [3Hthymidine incorporation in these cultures. In contrast, IGF-I in serum-free medium seemed to be a potent mitogenic and metabolic
stimulus for human fibroblasts which had been subcultured in media with a high glucocorticoid content, either endogenous,
or supplemented. After these culture conditions, a 48-h preexposure to dexamethasone had no further enhancing effect on IGF-I
action. Dexamethasone also potentiated IGF-I, insulin, and epidermal growth factor stimulation of fibroblast replication depending
on the earlier subcultivation conditions. Thus, glucocorticoids are important modulators of IGF-I bioactivity in cultured
human fibroblasts. Serum glucocorticoids can exert a profound influence on the biological phenomena measured in cell culture,
even when the serum has been removed before the actual experiment, and must be carefully taken into account for accurate evaluation
of the biological function of IGF-I and other growth factors.
This work was supported in part by grants DK28229, DK36054, CA42106, RCDA01275, and DK24085 from the National Institutes of
Health, Bethesda, MD. 相似文献
20.
The effects of epidermal growth factor (EGF) were studied in rat pituitary tumor cells, GH3, grown in serum-supplemented and serum-free chemically defined media. EGF (1 nM) increased the cell number to 132% of the control cultured in the defined medium during a 6-day incubation period, while it decreased the cell number to 60% of the control in the serum-supplemented medium. EGF altered the morphology of the cells grown in the defined medium more markedly to an elongated conformation than that of cells grown in the serum-supplemented medium. EGF also stimulated prolactin (PRL) production by culture in the presence or absence of serum. The effects of the cell density of GH3 on the action of EGF were shown to appear in two ways. The mitogenic influence of EGF was more effective on, and more responsive to, high-density cells, whereas the stimulatory action on PRL production was less effective on high-density cells. However, the inhibitory effects on cellular growth appeared independently of cell densities. The results obtained with 125I-EGF binding experiments indicated that the number of binding sites, affinity, and internalization of EGF receptors were similar in either serum-supplemented or serum-free culture. At low cell density, the number of available 125I-EGF binding sites per cell was larger than at high cell density. These results suggested that there was no apparent correlation between EGF binding and its differing effects on the growth of GH3 cultured in the serum-supplemented and the defined medium. 相似文献