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1.
A serum free medium was developed, that could be used for the large scale propagation of various cell lines in bioreactors. The medium is based on a 1:1 mixture of Iscove's Modified Dulbecco's Medium and Ham's Medium F12, supplemented with transferrin, insulin and a BSA/oleic acid complex. Several myelomas, hybridomas derived from different myelomas and spleen cells, and other lymphoid and non-lymphoid cell lines were cultivated at growth rates comparable to those observed using serum-supplemented media. There was furthermore no reduction in the formation of products such as monoclonal antibodies or recombinant human interleukin-2.Abbreviations Ag8
Mouse myeloma cell line P3-X63-Ag8.653
- BME
Basal Medium Eagle
- BSA
Bovine Serum Albumin
- DMEM
Dulbecco's Modified Eagle's Medium
- EDTA
Ethylenediaminete-traacetic Acid
- e-PC
Phosphatidyl choline from egg yolk
- FCS
Fetal Calf Serum
- FGF
Fibroblast Growth Factor
- GHL
Glycyl-histidyl-lysine
- HDL
High Density Lipoprotein
- HPL
Human Plasma Lipid
- IF
1:1 mixture of IMDM and Ham's F12
- IMDM
Iscove's Modified Dulbecco's medium
- LDL
Low Density Lipoprotein
- NS1
Mouse myeloma cell line NSI-1-Ag4-1
- PBS
Phosphate Buffered Saline
- s-PC
Phosphatidylcholine from soy beans
- s-PE
Phosphatidylethanolamine from soy beans
- s-lecithin
lecithin from soy beans 相似文献
2.
Roger H. Kennett 《In vitro cellular & developmental biology. Plant》1981,17(12):1036-1050
Summary Since the first report of hybridomas producing monoclonal antibodies by Kohler and Milstein in 1975, this technique has spread
to nearly all areas of biological, biochemical, and biomedical research. Watching the use of these methods spread from immunologists
to cell biologists, developmental biologists, biochemists and to other biological disciplines and observing the nearly logarithmic
increase in publications using these reagents has been in itself fascinating and informative. An overview of the development
of this technology and its applications is presented including the use of monoclonal antibodies to study cell surface molecules,
differentiation antigens, receptors, and histocompatibility antigens. The use of these antibodies to analyze microorganisms
and parasitic antigens as well as their use in the genetic analysis of human cell surface antigens and the detection of polymorphic
variation in enzymes and other proteins is discussed. Examples of the application of monoclonal reagents to the study of tumor
cell biology including the labeling of metastatic tumor cells and the detection of cell surface molecules implicated in the
regulation of growth control and cell division are provided.
Presented in the symposium on The Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association. Washington,
D.C., June 7–11. 1981.
This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech
Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England
Nuclear Corporation, and Ortho Pharmaceutical Corporation. 相似文献
3.
Sébastien Quesney Jacqueline Marvel Annie Marc Catherine Gerdil Bernard Meignier 《Cytotechnology》2001,35(2):115-125
The density of viable cells in a culture results from a balance between cell proliferation and cell death. The aim of this
study was to characterize and compare these two phenomena in Vero cell cultures in one serum containing medium (ScA) and one
serum free medium (SfB) in bioreactors. Cell growth was evaluated by cell counting(after crystal violet staining) and cell
cycle analysis. Necrosis and apoptosis were characterized and quantified by measuring the release of LDH, trypan blue exclusion,annex
in V-FITC/PI staining and TUNEL assay. ScA supported a higher maximal viable-cell density(2.3 × 106 vs. 1.8 × 106 cells ml-1). However, cell cycle analysis showed that cell division was more active in SfB than in ScA. LDH release in the supernatant
increased much earlier in SfB than in ScA (one vs. five days), but trypan blue counts showed no apparent difference in the
viability of the cultures. Apoptosis, evidenced by annexin V-FITC/PI staining, could be detected in the population of suspension
cells detached from microcarriers, but not among adherent cells; positivity of the TUNEL assay occurred later than that of
the annexin V-FITC/PI staining. Our data indicate that the lower cell yield in SfB,compared with that in ScA, results from
a higher cell death rate. Apparently, cells die from apoptosis followed by secondary necrosis.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
4.
Three tank type bioreactors of very simple design were compared to a commercially available laboratory-scale bioreactor, designed
especially for mammalian cell culture, for their ability to support hybridoma growth and antibody production under batch culture
conditions. The comparison reveals quite similar numbers for maximum viable cell densities and IgG production, despite large
differences in vessel and agitator geometry and aeration mode. Furthermore, some data indicate that the hydrodynamic stress
level in the growth vessels may influence the specific production rate of the cells and thus the overall productivity of the
reactors. 相似文献
5.
研究以DMEM/F12(1:1 V/V)培养基为基础,添加不同添加剂优化一种适宜CHO DG44细胞生长的廉价培养基。以细胞密度和细胞活率为主要指标,对DMEM/F12(1:1 V/V)培养基进行了优化。通过正交试验和单因素试验筛选出了CHO DG44细胞生长的最佳培养基。正交试验结果表明添加8mg/L Insulin、10mg/L Transferrin、12mM Glutamine、9mg/L Ethanolamine、9mg/L Sodium selenite、0.5×Lipids、0.5×Vitamin,对细胞生长有较好促进作用,细胞密度从0.6×106 cells/mL上升到1.8×106 cells/mL。在此基础上添加2.5g/L Malt Peptone和2.5g/L YeastExtract可使细胞密度达到2.65×106 cells/mL,基本上达到商业培养基的培养效果,而成本降低了约60%。 相似文献
6.
Serrato JA Palomares LA Meneses-Acosta A Ramírez OT 《Biotechnology and bioengineering》2004,88(2):176-188
It is known that heterogeneous conditions exist in large-scale animal cell cultures. However, little is known about how heterogeneities affect cells, productivities, and product quality. To study the effect of non-constant dissolved oxygen tension (DOT), hybridomas were subjected to sinusoidal DOT oscillations in a one-compartment scale-down simulator. Oscillations were forced by manipulating the inlet oxygen partial pressure through a feedback control algorithm in a 220-mL bioreactor maintained at a constant agitation. Such temporal DOT oscillations simulate spatial DOT gradients that can occur in large scales. Different oscillation periods, in the range of 800 to 12,800 s (axis of 7% (air saturation) and amplitude of 7%), were tested and compared to constant DOT (10%) control cultures. Oscillating DOT decreased maximum cell concentrations, cell growth rates, and viability indexes. Cultures at oscillating DOT had an increased glycolytic metabolism that was evidenced by a decrease in yield of cells on glucose and an increase in lactate yield. DOT gradients, even several orders of magnitude higher than those expected under practical large-scale conditions, did not significantly affect the maximum concentration of an IgG(1) monoclonal antibody (MAb). The glycosylation profile of the MAb produced at a constant DOT of 10% was similar to that reported in the literature. However, MAb produced under oscillating culture conditions had a higher amount of triantennary and sialylated glycans, which can interfere with effector functions of the antibody. It was shown that transient excursions of hybridomas to limiting DOT, as occurs in deficiently mixed large-scale bioreactors, is important to culture performance as the oscillation period, and thus the time cells spent at low DOT, affected cell growth, metabolism, and the glycosylation pattern of MAb. Such results underline the importance of monitoring protein characteristics for the development of large-scale processes. 相似文献
7.
Egg yolk lipoprotein promoted growth of a wide variety of mammalian cell lines, including plasma-cytomas and epithelial cell lines, in serum-free medium. The lipoprotein was active for cell growth when used with insulin, transferrin, ethanolamine and selenite. The most active lipoprotein fraction (YLP-pI7.5) was purified to give a single peak by chromatofocusing and gel filtration, and was homogeneous on a 0.35% agarose gel electrophoretogram. The lipoprotein was characterised as a very low density lipoprotein with a protein content of only 1.3%. This lipoprotein had an optimal concentration of 300 g/ml (4 g protein/ml). It was easily separable from proteinous molecules secreted into the serum-free medium by the cells, since it floated on the surface of the medium after addition of ammonium sulfate, to precipitate protein, and centrifugation. An associated structure of lipid and protein seemed to be still necessary for the lipoprotein to exhibit a growth promoting activity. 相似文献
8.
Hybridomas lend themselves particularly well to large scale cultivation techniques since they grow as single cells in suspension without requiring attachment to a substrate. Furthermore, many cell strains have been adapted to grow in serum-free (SF) media to a similar cell density and antibody production as in serum containing media. This review will concern itself mainly with the cultivation of hybridomas in SF-media in bioreactors of various types with the ultimate goal of producing large quantities of monoclonal antibodies (mAb). 相似文献
9.
10.
Summary A monolayer culture system has recently been developed for the extended growth and serial passage of normal rat mammary epithelial
(RME) cells. In this system the cells undergo greater than 20 population doublings when grown on type I collagen-coated tissue
culture dishes in Ham's F12 medium supplemented with insulin, hydrocortisone, epidermal growth factor, prolactin, progesterone,
cholera toxin, and 5% fetal bovine serum (FBS). The purpose of the present studies was to define additional growth factors
that would allow equivalent RME cell proliferation in serum-free medium. Ethanolamine (EA) was effective at reducing the FBS
requirements for RME cell proliferation and at its optimum concentration did so by greater than 20-fold. Even with optimum
levels of EA there was essentially no cell proliferation in the absence of FBS. However, addition of bovine serum albumin
(BSA) to the hormone, growth factor, and EA-supplemented medium resulted in substantial proliferation in the absence of serum,
and the further addition of transferrin (T) potentiated this effect. Thus, in this culture system, replacement of FBS with
EA, BSA, and T resulted in RME cell proliferation in primary culture which was equivalent to that obtained in the 5% FBS-containing
medium.
This work was supported by grant RR-05529 from the Division of Research Resources, National Institutes of Health, Bethesda,
MD, and by Public Health Service grant CA40064-01 from the National Cancer Institute, Bethesda, MD. 相似文献
11.
12.
Yue Wang Fabrizio Sabba Charles Bott Robert Nerenberg 《Biotechnology and bioengineering》2019,116(10):2698-2709
Elemental sulfur (S0) can serve as an electron donor for water and wastewater denitrification, but few researchers have addressed the kinetics of S 0–based reduction of nitrate (NO 3 −), nitrite (NO 2 −), and nitrous oxide (N 2O). In addition, S 0-based denitrifying biofilms are counter-diffusional. This is because the electron donor (S 0) is supplied from the biofilm attachment surface while the acceptor, for example, NO 3 −, is supplied from the bulk liquid. No existing mathematical model for S 0-based denitrification considers this behavior. In this study, batch tests were used to determine the kinetic parameters for the reduction of NO 3 −, NO 2 −, and N 2O. Additionally, a biofilm model was developed to explore the effects of counter-diffusion on overall fluxes, that is, the mass of NO 3 − or NO 2 − removed per unit biofilm support area per unit time. The maximum specific substrate utilization rates () for NO 3 −, NO 2 −, and N 2O were 3.54, 1.98, and 6.28 g N g COD −1·d −1, respectively. The maximum specific growth rates () were 0.71, 1.21, and 1.67 d −1 for NO 3 − to NO 2 −, NO 2 − to N 2O, and N 2O to N 2, respectively. Results suggest that the observed NO 2 − accumulation during S 0-based denitrification results from a low for NO 2 − relative to that for NO 3 −. The high for N 2O, relative to that for NO 3 − and NO 2 −, suggest that little N 2O accumulation occurs during denitrification. A counter-diffusional biofilm model was used to predict trends for NO 3 − fluxes, and confirmed NO 2 − accumulation in S 0-based denitrification biofilms. It also explains the observed detrimental effects of biofilm thickness on denitrification fluxes. This study allows a more accurate prediction of NO 3 −, NO 2 −, and N 2O transformations in S 0-based denitrification. 相似文献
13.
The production of small quantities of monoclonal antibodies and recombinant proteins was carried out using a new low cost production system, the Super Spinner. Into a 1 1 standard Duran® flask a membrane stirrer equipped with a polypropylene hollow fiber membrane was installed to improve the oxygen supply by bubble-free aeration. The aeration was facilitated by using the CO2 conditioned incubator gas, which was pumped through the membrane stirrer via a small membrane pump. The maximal oxygen transfer rate (OTRmax) of the Super Spinner was detected. For this purpose one spinner flask was equipped with an oxygen electrode. The OTRmax was measured by the dynamic method. The ratio of membrane length to culture volume was adapted corresponding to the oxygen uptake rate of the cells according to the desired cell density. A balanced nutrient supply resulted in an optimal formation and yield of products. 相似文献
14.
This paper analyses the performance of MAbMaxTM/TricentricTM, a new generation hollow fibre bioreactor, for hybridoma growth and antibody productivity, the down stream processing of
monoclonal antibody harvests throughout the run and the further control of antibody quality consistency. Handling and process
parameters were optimised using a mouse hybridoma, IgG1K secretor, and then confirmed with several other hybridomas. Cells were kept at optimal viability during an unusually long
period of time and a continuously high production of antibodies was detected over several months. Foetal bovine serum concentration
was reduced to 1\% and the effects of weaning of cells from serum were monitored in terms of cell metabolism and antibody
productivity. Antibody harvests collected at regular intervals throughout the run (2 to 12 weeks) were purified using affinity
chromatography on a recombinant protein A/G matrix and then analysed in terms of antigen binding properties, isoelectric forms
and oligosaccharide structures, in order 1) to control antibody quality consistency as a function of time and serum concentration
and 2) to compare antibody characteristics as a function of culture conditions, in vitro bioreactor cultivation versus in
vivo mouse ascite cultivation.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
15.
16.
Hormonal growth control of cells in culture 总被引:15,自引:0,他引:15
Summary Serum is the last undefined component in cell culture media. Our results indicate that the primary role of serum is to provide
hormones and that serum can be replaced by a group of hormones. A rat pituitary cell line, GH3, can grow in serum-free medium if the medium is supplemented with 3,3′,5-triiodothyronine, TSH-releasing hormone, transferrin,
parathyroid hormone, insulin and three isoelectric focusing fractions of blood meal. The blood-meal components can be replaced
by fibroblast growth factor and somatomedin C. The growth rate of GH3 cells in hormone-supplemented serum-free medium is equal to that in serum-supplemented medium, and subculture in such medium
is also possible. These results indicate that the replacement of the serum component is complete in the GH3 system. The hormonal requirements of GH3 cells and those of HeLa and mouse melanoma, M2R, were compared. Two generalizations could be made: (a) All three cell lines
require insulin and transferrin. (b) There is a requirement for a hormone which localizes in the nucleus for each cell line.
These generalizations seem to hold true for most of the other cell lines for which the hormonal requirements have been partially
worked out. Since insulin is one of the universally required hormones, its effects on GH3, HeLa and M2R were compared. Insulin stimulates glycogen synthesis in all three cell lines and facilitates fatty-acid synthesis
in GH3 and M2R. However, there is a difference in the effect of insulin on growth among the three cell lines. Insulin is an absolute
requirement for GH3 cells without which the cells cannot survive, whereas this is not the case for HeLa and M2R. The most stringent requirement
for HeLa cells is for hydrocortisone, and for M2R, it is for transferrin. These results indicate that even though the necessity
for some hormones is common, the degree of requirement may vary from one cell line to another. Whether this difference reflects
the difference in the primary mode of action of the hormone on each cell type needs further investigation.
Presented in the Opening Symposium on Nutritional Factors and Differentiation at the 28th Annual Meeting of the Tissue Culture
Association, New Orleans, Louisiana, June 6–9, 1977.
This work was supported by NIH Grant GM 17019. J. Larner was supported by Josiah Macy Foundation 相似文献
17.
Pulse chase experiments of two mouse hybridoma lines were conducted in order to elucidate the kinetics of monoclonal antibody (mAb) production and secretion during different stages of batch cultures. The results indicate that a stock of cytoplasmic IgG exists in hybridoma cells and that the concentration of this stored IgG depends on the cell line used and the stage of the culture. This stored IgG can be released by dying cells, and a certain quantity of the secreted IgG is derived from this source. However, only between 0.3 and 9.3% of the released IgG of U0208 (average: 2.08%) and between 2.08 and 25.8% of the IgG, released from I.13.17 (average: 6.95%), were of storage origin, calculated on culture viability and intracellular IgG-stock. Comparing the accumulation of radio-labelled IgG (IgG*) in the supernatant with the reduction of cytoplasmic IgG* during the chase experiments, the percentages range between 14 and 50%, somewhat higher values probably caused by changes in the culture conditions. These changes led to a release of IgG during the chase experiments, which accounts for about 20–25% of the totally secreted IgG.It could be established that during the logarithmic growth phase of batch cultures a certain percentage of synthesized IgG was not released but stored within the cells: for U0208: 0.3–4.5%, for I.13.17: 1–7.6%. During the stationary and death phase, this percentage ranged between 1.5 and 20% for U0208 and between 0.5 and 8.1% for I.13.17. Finally, the chase experiments also revealed that the time of synthesis, assembly, and secretion of mAbs does not vary much during the different phases of batch cultures, and is within the range of 1.5 and 3 hrs. 相似文献
18.
A simple medium for the study of hepatocyte growth in culture under defined conditions 总被引:3,自引:0,他引:3
Tor-Erik Sand Thoralf Christoffersen 《In vitro cellular & developmental biology. Plant》1988,24(10):981-984
Summary The combination (1∶1) of Dulbecco's modified Eagle's medium and Waymouth's medium MAB 87/3 was found to provide favorable
conditions for serum-free culture and growth of adult rat hepatocytes. In this simple medium, a majority of hepatocytes stimulated
by epidermal growth factor plus insulin entered S phase and divided, with a normal (13 h) interval between DNA synthesis and
cell division. The proliferative response did not require extra substratum or the presence of serum, even during cell isolation
and plating.
This work was supported by the Norwegian Cancer Society. 相似文献
19.
M. Amouric J. Marvaldi J. Pichon F. Bellot C. Figarella 《In vitro cellular & developmental biology. Plant》1984,20(7):543-548
Summary Lactoferrin was examined for its effect on the growth of a human colon adenocarcinoma cell line (HT 29) in culture and its
action was compared to that produced by transferrin and two different iron solutions (ferrous sulfate and ferric chloride).
When transferrin was replaced by either iron solutions the cell grew in proportion to the quantity added and the maximal effect
obtained was identical to that produced by transferrin alone. When transferrin was replaced by lactoferrin the cells were
unable to proliferate for a long time. However, in the presence of low-concentration iron solutions, lactoferrin stimulated
the cell growth, and the effect was more pronounced with the ferric chloride solution.
This work was supported by Grants Inserum 817014 and LA CNRS 202. 相似文献
20.
Adle Martial Isabelle Gaillard Jean-Marc Engasser Annie Marc 《Enzyme and microbial technology》1995,17(12):1062-1066
A homemade serum-free medium containing a low protein level under 0.1 g l−1 has been proved to support long-term cultures of VO 208 hybridoma cells successfully up to 50 days. The low protein level was achieved by supplying the lipids through liposomes containing cholesterol, oleic acid,
- dipalmitoyl phosphatidylcholine, and bovine serum albumin. The influence of the liposome content in the feeding medium was studied in a continuous culture performed with step variations of the liposomes level, from 7.5 to 30 ml l−1. The cell density decreased at the highest liposomes content while it became higher with 7.5 or 12 ml l−1 of liposomes. For each step variation appeared a transitory activation of the specific rates of nutrient consumption, metabolite production and antibody secretion, as well as a transitory decrease of the specific cell growth rate. The overall structure of the antibodies was not affected during the culture. 相似文献