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1.
M Hammami A Oudot G Maume M Chessebeuf-Padieu P Padieu B F Maume 《Cell biology international reports》1988,12(7):527-536
The development of a method for the serum-free culture of the Y-1 mouse adrenocortical tumor cell line has permitted a detailed search for factors regulating cellular growth and steroidogenesis. The serum-free medium (SFM) was made of Ham's F10 basal medium supplemented with free fatty acids adsorbed on albumin. The SFM complemented with calcium, arachidonic acid and cholesterol, i.e. SFM-S, induced cell proliferation to a density at confluency higher than that obtained with 1% serum-supplemented medium (1%-SSM) and allowed to sustain cell growth for more than six passages. When albumin was replaced by a dextran polymer (Mr = 2 x 10(6)) used as a carrier of lipids instead of albumin (which resulted in a serum-free and protein-free medium, SPFM), the cell number was 75% of that observed with the SFM-S. The addition to SPFM of beta-globulin alone or combined with insulin caused a 2- or 3-fold increase in the final cell density, respectively. The ability of the Y-1 cell line to produce steroids in response to ACTH was found to be higher in SFM-S or SPFM than in 1%-SSM. Furthermore, the addition of beta-globulin to SPFM stimulated steroid hormone biosynthesis with a marked increase in 11 beta-hydroxylated steroid production. These studies demonstrate that the use of a defined mixture of nutriments and of few growth factors permits to sustain not only the cellular proliferation of the Y-1 cell line but also its differentiated function of ACTH-induced steroidogenesis. 相似文献
2.
Alfred R Radford J Fan J Boon K Krawetz R Rancourt D Kallos MS 《Biotechnology progress》2011,27(3):811-823
Large numbers of cells will be required for successful embryonic stem cell (ESC)-based cellular therapies or drug discovery, thus raising the need to develop scaled-up bioprocesses for production of ESCs and their derived progeny. Traditionally, ESCs have been propagated in adherent cultures in static flasks on fibroblasts layers in serum-containing medium. Direct translation of two-dimensional flatbed cultures to large-scale production of the quantities of cells required for therapy simply by increasing the number of dishes or flasks is not practical or economical. Here, we describe successful scaled-up production of ESCs on microcarriers in a stirred culture system in a serum-free medium. Cells expanded on CultiSpher S, Cytodex 3, and Collagen microcarriers showed superior cell-fold expansions of 439, 193, and 68, respectively, without excessive agglomeration, compared with 27 in static culture. In addition, the ESCs maintained their pluripotency after long-term culture (28 days) in serum-free medium. This is the first time mESCs have been cultured on microcarriers without prior exposure to serum and/or fibroblasts, while also eliminating the excessive agglomeration plaguing earlier studies. These protocols provide an economical, practical, serum-free means for expanding ESCs in a stirred suspension bioprocess. 相似文献
3.
Cell multiplication and type II collagen production by rabbit articular chondrocytes cultivated in a defined medium 总被引:2,自引:0,他引:2
M. Adolphe B. Froger X. Ronot M. T. Corvol N. Forest 《Experimental cell research》1984,155(2):527-536
The complexity and the variations in the efficiency of different batches of serum stimulated the preparation of a serum-free medium which could promote not only growth, but also the differentiation properties of rabbit articular chondrocytes in culture. The serum-free medium (SFM) developed in this study contained insulin, transferrin, Na-selenite, human fibronectin bovine serum albumin (BSA), brain growth factor (BGF) or fibroblast growth factor (FGF), hydrocortisone and multiplication stimulating activity (MSA). Primary or secondary cultures of chondrocytes in such a medium attained a proliferation rate equal to 70-80% of that obtained with chondrocytes grown in a serum control medium. The deletion of various factors from SFM indicates that BGF or FGF are the most stimulating of growth factors. Insulin was beneficial when used individually; when combined with BGF or FGF, they had a synergistic effect on cell proliferation. MSA seemed not to play any role in chondrocyte growth in culture. The SFM medium did not modify either the morphology or the progression of cells into the cell cycle. It moreover allowed the maintenance of the specific function of chondrocytes to synthesize type II collagen. 相似文献
4.
Summary The cell culture on serum-coated microcarriers yielded higher efficiency of cell attachment to microcarriers and more favorable
initial cell distribution on microcarriers than on the conventional microcarriers. By employing serum-coated microcarriers,
the maximum cell density was increased by 46% in low serum medium and by 30% in 10% (v/v) serum-supplemented medium. Serum
coating of microcarriers could provide cell attachment factors and may replace costly attachment factors supplemented in low
serum medium and serum-free medium. 相似文献
5.
The growth behavior of the two human colon tumor cell lines (SW 480, primary and SW 620, metastatic), originating from the same patient, was studied in six different serum-free media (SFM) [GF3, Chee's essential medium plus insulin, transferrin and selenium; GF3F, GF3 plus fetuin; GF4, GF3 plus linoleic acid-BSA; GF5, GF4 plus fetuin; GF5E, GF5 plus EGF; GF5T, GF5 plus triiodothyronine]. SW 480 grew in all of the SFM. In contrast, SW 620 grew only in four SFM. The cells did not grow in GF3 and GF4. When grown in SFM, SW 480 attached much more firmly to the dishes than SW 620 as determined by the time required to detach the cells with trypsin-EDTA (SW 480, greater than 20 min and SW 620, less than 5 min). It was speculated that SW 480 cells excrete proteins in SFM which influence attachment and growth of the cells. Growth behavior of SW 480 cells which did not grow in GF3, was studied using GF3 medium and SW 480 substratum dishes. SW 620 cells readily attached to the SW 480 substratum dishes and grew. Furthermore, when SW 620 cells were grown on substratum prepared from serum-supplemented medium incubated in the absence of cells (serum substratum), the cell growth was comparable to the cell growth on SW 480 substratum in GF3. Substratum from SW 480 cells and the serum substratum were compared for their components using SDS-PAGE system. The SW 480 substratum contains many more components than serum substratum. A protein band at 60 kD appears to be common in both SW 480 and serum substrata. 相似文献
6.
Kinetics and metabolic specificities of Vero cells in bioreactor cultures with serum-free medium 总被引:1,自引:0,他引:1
Sébastien Quesney Annie Marc Catherine Gerdil Cyrille Gimenez Jacqueline Marvel Yves Richard Bernard Meignier 《Cytotechnology》2003,42(1):1-11
The aim of this study was to understand the metabolism kinetics of Vero cells grown on microcarriers in bioreactors in serum-free
medium (SFM). We sought to determine what nutrients are essential for Vero cells and how they are consumed. Contrary to glucose
and to most of the amino acids, glutamine and serine were very quickly depleted in this medium and can be supposed to be responsible
for cell apoptosis. Lactate and ammonium ions did not reach toxic levels for Vero cells. We payed more attention to the lactate
metabolism. Usually we observed that after about 2 days lactate was consumed in serum-containing media, but its concentration
plateaud in SFM. Moreover, the addition of serum in SFM provoked lactate consumption and the rate of glucose and glutamine
consumption was twice as high as in the SFM not supplemented with serum. The depletion of glutamine and serine and the metabolic
deviations leading to a shortage of intermediate products required for other metabolic pathways probably contribute to the
lower cell yield and higher cell death rate in SFM.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
7.
Serum-free culture of AtT 20 pituitary cells: A system for neuroendocrine studies under defined conditions 总被引:2,自引:0,他引:2
Helmut Vedder 《In vitro cellular & developmental biology. Plant》1990,26(11):1068-1072
Summary The growth of the mouse pituitary cell line AtT 20 was studied under different in vitro conditions. A completely defined,
serum-free culture medium supported the survival of cells for a period of more than 2 mo. The medium, designed SFI, consisted
of basal medium supplemented with transferrin, insulin, putrescine, and selenium. For maintenance of cells during long-term
culture, no additional compounds were necessary. The time-dependent increases in cell number during culture with fetal bovine
serum (FBS) and under serum-free conditions showed similar properties. Analysis of the effects of different substrata on cell
growth demonstrated that polylysine supported adhesion and initial growth of cells to a greater extent than untreated plastic
or FBS adsorbed to culture dishes. Synthesis and regulation of proopiomelanocortin (POMC)-mRNA, the precursor-mRNA of adrenocorticotropin
(ACTH), could be detected by Northern blot analysis under basal conditions and after incubation with steroids and corticotropin-releasing
hormone (CRH), indicating the serum-independent expression of important cellular properties. 相似文献
8.
Hybridoma cell growth and anti-neuroblastoma monoclonal antibody production in spinner flasks using a protein-free medium with microcarriers 总被引:1,自引:0,他引:1
The main disadvantages of foetal calf serum as the world-wide common serum supplement for cell growth are its content of various proteins of variable concentrations between batches as well as its high cost. The use of serum-free and protein-free media is gradually becoming one of the goals of cell culture especially for standardizing culture conditions or for simple purification of cell products like monoclonal antibodies. The mouse hybridoma cells 14/2/1 were cultivated either in protein-free UltraDOMA medium or in serum-containing RPMI medium with and without microcarriers to generate high quantities of monoclonal antibodies against neuroblastoma tumour cells. Cell growth rate, IgG production, viability, glucose and lactate concentrations, attachment rate and doubling time have been used as investigation criteria. Modifications of culture procedures (static or stirred), inoculum density, and microcarrier concentration caused an improvement of monoclonal antibody production. The kinetics of antibody synthesis was best in spinner culture with 2 ml of microcarriers in protein-free medium. These results of short-term microcarrier culture in stirred spinner flasks indicate that IgG yields in protein-free medium 2.5-fold higher to those in serum-supplemented medium can be achieved. 相似文献
9.
Comparison of manufacturing techniques for adenovirus production 总被引:2,自引:0,他引:2
We have compared three different production methods, which may be suitable for the large scale production of adenovirus vectors
for human clinical trials. The procedures compared 293 cells adapted to suspension growth in serum-free medium in a stirred
tank bioreactor, 293 cells on microcarriers in serum-containing medium in a stirred tank bioreactor, and 293 cells grown in
standard tissue culture plasticware. With a given virus, yields varied between 2000 and 10,000 infectious units/cell. The
stirred tank bioreactor routinely produced between 4000 and 7000 infectious units/cell when 293 cells were grown on microcarriers.
The 293 cells adapted to suspension growth in serum-free medium in the same stirred tank bioreactor yielded between 2000 and
7000 infectious units/cell. Yields obtained from standard tissue culture plasticware were up to 10,000 infectious units/cell.
Cell culture conditions were monitored for glucose consumption, lactate production, and ammonia accumulation. Glucose consumption
and lactate accumulation correlated well with the cell growth parameters. Ammonia production does not appear to be significant.
Based on virus yields, ease of operation and linear scalability, large-scale adenovirus production seems feasible using 293
cells (adapted to suspension/serum free medium or on microcarriers in serum containing medium) in a stirred tank bioreactor.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
10.
Masaaki Ogata Ken-ichi Wakita Kazuo Kimura Yasumasa Marumoto Kiyomoto Oh-i Shin Shimizu 《Applied microbiology and biotechnology》1993,38(4):520-525
Cultivation of gene-engineered Chinese hamster ovary (CHO-K1) cells that produce recombinant human soluble thrombomodulin (rsTM) was investigated to optimize conditions for high-level expression of the protein in a serum-free medium. For economic protein production, oxygenation of cultures with pure O2 permitted sufficient cell growth for high rsTM production with only 1 g/l of microcarriers and a low foetal bovine serum concentration. A longer growth phase (over 5 days) with serum was important to establish sufficient growth of this cell line on the microcarriers for subsequent serum-free culture, and to support a long-term production phase (about 2 months). In the production phase, a high glucose concentration (6.15 g/l) in the serum-free medium was very effective for prolonging the harvest cycle interval. Under these conditions, up to 100 mg/l rsTM was expressed in the conditioned medium. The rates of glucose consumption (G) and lactae production (L) were measured periodically and their ratio (L/G ratio) correlated with rsTM productivity. When the average L/G ratio was lower, reflecting a lower lactate production rate due to appropriate oxygenation of the culture, the specific rsTM production rate increased. Thus it may be possible to estimate protein productivity from L/G ratios calculated from the glucose and lactate measurements.
Correspondence to: M. Ogata 相似文献
11.
J. Fantini J. P. Galons B. Abadie P. Canioni P. J. Cozzone J. Marvaldi A. Tirard 《In vitro cellular & developmental biology. Plant》1987,23(9):641-646
Summary Human colonic adenocarcinoma cells have been successfully grown on polystyrene microcarriers by modifying the culture conditions
used in monolayer culture. The method can be divided into two culture phases: a) a phase of spreading, wherein cells were
seeded in presence of serum-supplemented medium; b) a phase of active growth wherein spread cells on the beads were allowed
to grow in a serum-free medium. Under these conditions, optimal spreading and growth of HT 29 and HRT 18 cells on the microcarriers
were obtained. A differential propagation was observed between HT 29-D4 and HT 29-D9 cells (both clonal populations derived
from HT 29 cells) on the microcarriers that is tentatively related to the discrepancy observed in the spreading efficiency
of these clonal cells on serum-coated culture flasks. An index of spreading efficiency (IS index) has been defined to quantify
the efficiency of spreading of each cell line on microcarriers. These data gave the opportunity to develop serum-free, scale-up
methods to culture cells like HT 29 which release potentially useful products.
This work was supported by CNRS (U.A. 202 and U.A. 1186), Fédération Nationale des Centres de Lutte Contre le Cancer (FNCLCC),
INSERM (CRE, no 847006), CNAMTS-INSERM (8386), MRT (GBM 85M0564) and l'Association pour la Recherche sur le Cancer (ARC 86-234). 相似文献
12.
Experimental conditions have been defined that allow bovine corneal endothelial (BCE) cells to grow in the complete absence of serum. Low density BCE cell cultures maintained on extracellular matrix (ECM)-coated dishes and plated in the total absence of serum proliferate actively when exposed to a synthetic medium supplemented with high density lipoprotein (HDL 500 μg protein/ml), transferrin (10 μg/ml), insulin (5 μg/ml), and fibroblast (FGP) or epidermal growth factor (EGF) added at concentrations of 100 or 50 ng/ml, respectively. Omission of any of these components results in a lower growth rate and/or final cell density of the cultures. BCE cell cultures plated on plastic dishes and exposed to the same synthetic medium grow very poorly. The longevity of BCE cultures maintained on plastic versus ECM and exposed to serum-free versus serum-containing medium has been studied. The use of ECM-coated dishes extended the life span of BCE cultures maintained in serum-supplemented medium to over 120 generations, as compared to less than 20 generations for cultures maintained on plastic. Likewise, BCE cells maintained on ECM and exposed to a synthetic medium supplemented with optimal concentrations of HDL, transferrin, insulin, and FGF underwent 85 generations, whereas control cultures maintained on plastic could not be passaged. The enhancing effect of ECM on BCE cell growth and culture longevity clearly illustrates the importance of the cell substrate in the control of proliferation of these cells. 相似文献
13.
Medium conditioned by embryonic chick heart cells is known to support extensive neurite outgrowth from autonomic and sensory neurons. In the present report we describe the use of microcarrier cell culture with serum-free media to scale up the production of the nerve growth-stimulating factors. A growth medium composed of DME /F10 supplemented with insulin, transferrin, human serum albumin and fibronectin in combination with a low molecular weight (MW) fraction of fetal calf serum (FCS) or a mixture of FGF, dexamethasone, calmodulin and thrombin supported the heart cell proliferation at a rate similar to that of medium with 10% FCS. Furthermore, the level of successively accumulated nerve growth activity measured in a bioassay with sympathetic ganglia proved to be nearly equivalent to what was obtained when cells were grown in medium containing serum. The results confirm the potential of microcarrier cell culture in serum-free media for the production and subsequent recovery of a specific cell product. 相似文献
14.
Evaluation of the serum-free medium MDSS2 for the production of poliovirus on vero cells in bioreactors 总被引:2,自引:0,他引:2
The serum-free medium MDSS2 (Merten et al., 1994), was used for cultivating Vero cells as well as for producing poliovirus
(Sabin type 1) in static and in perfused micro-carrier cultures. At slightly different growth rates of 0.0120/h and 0.0106/h,
respectively, static cultures in serum-containing (SCM) and serum-free (SFM) medium produced titers of 106.75 and 106.67 TCID50 per 50 μl; signifying a specific productivity of 0.89 and 1.07 TCID50/c.
Serum-free bioreactor cultures of Vero cells on DEAE-dextran microcarriers at 6.25 g/l produced cell densities of about 1.5×106c/ml. After infection with virus (multiplicity of infection (MOI) 0.1–0.3) titers of about 6.3×108 TCID50/ml were obtained, signifying an average specific productivity of 7.1 TCID50/c.h. Although these values were 4 and
2 fold, respectively, higher than in classical resum-based production processes (Montagnon et al. Dev. biol. Stand. 1981,
47, 55), a reference culture, for which cell growth was done in SCM and only virus production was done in SFM, produced 2×109 TCID/ml with an average specific virus production rate of 18.9 TCID50/c.h. The differences between the fully serum-free and
our reference process were mainly due to physiological differences of cells grown in SCM and SFM and also due to strongly
modified consumption kinetics after virus infection leading to limitations of one or several essential medium compounds, like
glucose and amino acids. Avoiding these limitations by increasing the residual concentration of glucose, glutamine, histidine,
and SH-amino acids, led to specific virus production rates (of about 17.9 TCID59/c.h.) comparable to those found in the reference
virus production process. The optimisation of the production of the poliovirus (Sabin 1) will be described with respect to
the modification of the medium composition.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
15.
Chen Zhaolie Xiao Chengzu Liu Hong Wu Benchuan Jia Xihua 《Biotechnology Techniques》1996,10(6):449-452
Summary A novel serum-free medium for the cultivation of Vero cells on microcarriers was developed,which composed of the 1:1 mixture of Dubecco's Modified Eagle Medium: Nutrient Mixture F12, bovine serum albumin(BSA) or human serum albumin(HSA), epidermal growth factor(EGF), gelatin and Dbiotin. Both BSA and EGF were effective on cell growth, adhesion and spreading. Further addition of gelatin and biotin led to the enhanced cell adhesion and spreading without growth promoting activity. The serum-free medium was suitable for the cultivation of vero cells on several different microcarriers with cell density reached over 3×l06cells/ml. 相似文献
16.
We have investigated conditions that inhibit the tendency of CHO K1 cells to form cellular bridges between microcarriers and
dense clumps of cellular overgrowth in microcarrier cultures. Microcarrier aggregation by cellular bridge formation was found
to occur only during periods of rapid cell growth. The level of microcarrier aggregation decreased with increasing agitation
intensity. Dense masses of cellular overgrowth formed inside bridges connecting the microcarriers and in clumps that protruded
off the microcarrier surface. To replace cells that were continuously sheared from the microcarriers, cell growth occurred
preferentially in areas of overgrowth after confluent microcarriers were maintained in a serum-free medium. This ultimately
led to poor surface coverage as bare spots developed on the microcarrier away from the areas of dense cellular overgrowth.
The development of bare spots was inhibited when confluent microcarriers were maintained in medium supplemented with 1% serum.
The development of cellular overgrowth was inhibited by dimethyl sulfoxide. Thus, maintaining confluent microcarriers in medium
supplemented with 1% dimethyl sulfoxide and 1% calf serum resulted in microcarriers that appeared similar to monolayer cultures.
There was also a decrease in bridging in cultures supplemented with either 1% calf serum or 1% dimethyl sulfoxide/1% calf
serum compared to serum-free cultures. 相似文献
17.
Mesothelial cells are the putative progenitors of mesotheliomas and cell lines have been used as tools to study the responses of these cells to various stimuli, including growth factors. The present study was undertaken to develop a rat mesothelial cell line capable of sustained growth under serum-free conditions with the object of avoiding the possible confounding effects of undefined serum components. Responses of mesothelial cells to epidermal growth factor were shown to differ under serum-free versus low-serum culture conditions. In contrast, a cell line, SFM1, adapted to growth in serum-free medium was characterized and found to exhibit responses to growth factors similar to the responses reported for human cell lines. This new line should prove to be a useful model for the study of these cells in vitro . 相似文献
18.
《Journal of biotechnology》1987,5(2):131-138
Mouse adrenal cortex Y-1 cell line was cultured on microcarriers in cell reactors containing 250 ml or 1500 ml culture medium; the same cell density as in the smaller vessel (0.9 × 106 cells ml−1) was obtained in the larger reactor by controlling pH and supplying the medium with O2. Consumption of glucose, oxygen, production of lactate and variation of 19 amino acids were determined with and without oxygen supply. Synthetic ACTH 1–24 induced steroidogenesis in a serum poor medium supplemented with cholesterol and albumin. Steroid production was similar to that of cell growth in usual culture dishes (2 μg 106 cells−1 24 h−1). 相似文献
19.
Jaluria P Konstantopoulos K Betenbaugh M Shiloach J 《Biotechnology and bioengineering》2008,99(6):1443-1452
Animal-derived serum is an essential media supplement for mammalian cells in cell culture. For a number of reasons including cost, regulatory concerns, lot inconsistency, potential contamination with adventitious agents, and down-stream processing it is desirable to eliminate the use of serum. Existing protocols designed to adapt cells to serum-free media (SFM) are time-consuming and provide little insight into how the cells adapt. To better understand the physiological responses associated with serum withdrawal and to expedite the adaptation process, a Human Embryonic Kidney-293 (HEK-293) cell line was propagated in 10% fetal bovine serum (FBS) and was progressively adapted to SFM and analyzed at specific serum levels by oligonucleotide microarrays. Of the differentially expressed genes two, early growth response 1 (egr1) and growth arrest specific 6 (gas6), were selected for further analysis based on their level of differential expression, overall expression patterns, and proposed functionalities. HEK-293 cells, propagated in 10% FBS were transfected with egr1 or gas6 and then adapted to SFM. Results indicated that higher expression of either gene moderately enhanced the ability of both cell lines to adapt to SFM. Egr1 appeared to have a greater impact on adaptability than gas6. Results also indicated that specific protein production was unaltered when the expression of egr1 was increased. Flow cytometric analysis revealed increased expression of egr1 was associated with an increase in the percentage of cells in the G2/M phases. These results indicate that enhanced expression of egr1 or gas6 facilitate adaptation to SFM by improving growth and viability. 相似文献
20.
Cell-mediated contraction of collagen lattices in serum-free medium: Effect of serum and nonserum factors 总被引:5,自引:0,他引:5
Steven N. Anderson Zadok Ruben Gerge C. Fuller 《In vitro cellular & developmental biology. Plant》1990,26(1):61-66
Summary This study was conducted to identify a defined, serum-free culture medium that supports cell dependent contraction of a collagen
lattice. Collagen lattices were found to contract in cultures containing human foreskin fibroblasts (HFF) or rabbit aortic
smooth muscle (RASM) cells incubated in serum-free medium. HFF and RASM cells required different supplements to contract the
collagen gels. HFF cultured in Dulbecco’s modified Eagle’s (DME) medium supplemented with bovine serum albumin (BSA) and either
endothelial cell growth supplement (EnGS), insulin (In), or platelet derived growth factor (PDGF) supported collagen lattice
contraction. Replacement of BSA with casein without the addition of other supplements improved contraction. In contrast, RASM
cells supplemented with BSA, EnGS, In, and PDGF were able to contract collagen gels only minimally. Similar to HFF, RASM cells
cultured in DME medium supplemented with casein, but without the addition of other supplements, contracted collagen lattices.
HFF-mediated collagen contraction was inhibited by prostaglandins E1 or E2, fibronectin, or ascorbic acid. The reported serum-free model provides a useful in vitro method to investigate the role of
serum and nonserum factors regulating cell mediated-contraction of insoluble collagen fibrils.
Presented in part as abstract 1963 in the 1985 Federation of American Societies for Experimental Biology, April 21–26, Anaheim,
California, and published in Fed. Proc. (44):747; 1985. 相似文献