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1.
Frederick J. Darfler 《In vitro cellular & developmental biology. Plant》1990,26(8):769-778
Summary A protein-free medium, termed ABC, has been developed which essentially eliminates the need for serum proteins. ABC supports
the long-term growth of murine hybridomas as well as other transformed cells of the immune system. The requirement of hybridoma
growth for transferrin has been met by substituting the soluble organo-iron compound, sodium nitroprusside. Substantial improvement
in the growth of hybridomas was afforded by the inclusion of 18 trace elements complexed to disodium ethylene diaminetetraacetate
(EDTA). The medium was further improved by the inclusion of components not found in Ham's F12 medium or by raising the concentrations
of existing low molecular weight components. Murine hybridomas can be cultured routinely in this protein-free medium in an
anchorage-independent manner with doubling times generally under 24 h. Visualized on electrophoretic gels, levels of monoclonal
antibody taken from those cultures often exceeded 80% of the total protein. The medium was also able to support the growth
of HuT 78 and H9 cells as well as certain other transformed cells of the immune system. In addition, normal human peripheral
blood lymphocytes, activated with phytohemagglutinin and cultured with 50 U/ml recombinant interleukin 2, could be grown for
2 wk with a 50-fold expansion over input cell number. 相似文献
2.
Koichi Rikimaru Hitomi Toda Noriko Tachikawa Nobuyuki Kamata Shoji Enomoto 《In vitro cellular & developmental biology. Plant》1990,26(9):849-856
Summary A novel protein-free synthetic medium has been developed for the culture of human squamous cell carcinoma cells. This medium,
designated PF86-1, supports the serial subcultivation of six out of nine human squamous cell carcinoma cell lines in a protein-free,
chemically defined condition without the adapting culture from serum-containing conditions. These cell lines growing in PF86-1
exhibited nearly equal potency to grow in massive culture without noticeable changes in morphology but presented a significantly
decreased level of colony forming efficiency when compared with the cells cultured in serum-containing media, suggesting the
implication of some autocrine mechanism. Interestingly, this medium supported the growth of normal human squamous cells of
oral mucosa and skin for more than 2 mo. in the primary explant culture in spite of high levels of calcium ion concentration,
where the overgrowth of fibroblasts as contaminant was not observed. These results suggest that PF86-1 supports the growth
of cells derived from epidermal tissues selectively and provides the same defined condition for growth of malignant and nonmalignant
human squamous cells. It seems, therefore, that PF86-1 allows investigations on the products of squamous cell carcinoma cells
or on the differences of growth mechanisms between normal and neoplastic human squamous cells. 相似文献
3.
Akira Niwa Katsuhiko Yamamoto Kenji Sorimachi Yosihiro Yasumura 《In vitro cellular & developmental biology. Plant》1980,16(11):987-993
Summary The rat hepatoma cell line, H4-II-E, was grown serially over a I-year period and about 30 passages in arginine-, glutamine-,
and tyrosine-deprived and ornithine-supplemented Eagle's mininum essential medium with no supplements other than biotin. The
adapted cel line, R-Y121B, proliferates in the above mentioned medium with a doubling time of about 4 days and maintains hepatic
“marker” enzymes such as tyrosine aminotransferase, phenylalanine hydroxylase, and all the enzymes of the urea cycle.
This work was supported in part by Grant-in-Aid for Cancer Research 301050 and Science Research Grant 337013 from the Ministry
of Education, Science and Culture, Japan. 相似文献
4.
A low-cost chemically defined protein free medium for a recombinant CHO cell line producing prothrombin 总被引:4,自引:0,他引:4
A chemically defined protein free medium, DF6S, was developed for the cultivation of a recombinant Chinese hamster ovary cell line (CHO2DS) producing human prothrombin in suspension batch culture. DF6S was formulated by optimizing DME/F12 with amino acids and supplementing the optimized DME/F12 with aurintricarboxylic acid, ethanolamine, ferric sulfate, Pluronic F68, putrescine and sodium pyruvate. From a seeding density of 2.3 × 105 cells ml–1, CHO2DS cells grown in suspension in DF6S medium reached a maximal cell density of 1.92 × 106 cells ml–1 with an accumulated prothrombin concentration of 16.7 mg l–1 after 6 days in culture. 相似文献
5.
JEROME F. LA PEYRE DORIS Y. SCHAFHAUSER ESAM H. RIZKALLA MOHAMED FAISAL 《The Journal of eukaryotic microbiology》1995,42(5):544-551
ABSTRACT. Analysis of the cell-free supernatants of Perkinsus marinus cultures by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining revealed the presence of as many as 17 bands ranging in molecular weight from 239 to 32 kDa. These bands were not present in un-inoculated medium. Moreover, P. marinus produces extracellular proteins that possess proteolytic activities; the cell-free supernatants of P. marinus cultures could digest a variety of proteins including gelatin, casein, fibronectin and laminin. Oyster plasma was also digested by cell-free culture supernatants. The proteolytic activity in cell-free culture supernatants was detected 24 h post-inoculation, while no proteolytic activity could be detected in cell lysates. The proteolytic activities were characterized using substrate-impregnated sodium dodecylsulfate-polyacrylamide gels and had approximate molecular weights ranging from 55 to 35 kDa. The proteolytic activity of cell-free culture supernatants was inhibited by the serine protease inhibitors phenylmethylsulphonyl fluoride, 3,4-dichloroisocoumarin and soybean trypsin inhibitor. In contrast, inhibitors (i.e. trans-epoxysuccinyll-leucylamido(4-guanidino)-butane, 1, 10-phenanthroline, captopril, ethylenediaminetetracetic acid, pepstatin A or diazoacetyl-DL-norleucine methyl ester) from the other three classes of proteases had no effect. It was concluded that the P. marinus proteases in cell-free culture supernatants are serine proteases. 相似文献
6.
Keen MJ 《Cytotechnology》1995,17(3):193-202
Y0 is a rat x rat hybridoma cell line, which does not secrete immunoglobulin, produced using a fusion partner derived from the Y3 (Y3,Ag.1.2.3) rat myoloma cell line. Y0 and Y3 have both been widely used as fusion partners in the production of rat x rat hybridomas. Y0 has also been used in recombinant gene technology. Y0 cells grown in shake flask culture, using RPMI 1640 medium with 4mM l-glutamine and 5% foetal bovine serum, reached a maximal cell density of 1.5×106 cells ml–1 with 86% viability. Y0 cells which has been adapted to grow in ABC protein-free medium reached a maximal density, in shake flask culture, of 8.75×105 cells ml–1 with 79% viability. An improved protein-free medium, designated W38 medium, was developed. In shake flask culture, W38 medium supported Y0 cell growth to a density of 2.02×106 cells ml–1 with 96% viability. Two Y3 hybridomas, YID 13.9.4 cells and SAM 618 cells were adapted to growth in W38 medium. For both hybridomas, cell growth and product yield in shake flask culture using W38 medium was superior to that obtained with serum-containing RPMI 1640 medium.Abbreviations F12
Hams F12 medium
- DMEM
Dulbeccos medium
- RPMI
RPMI 1640 medium
- FBS
foetal bovine serum 相似文献
7.
Sodium propionate, as well as sodium butyrate, enhanced the production of recombinant B-domain-deleted, factor VIII (rFVIIIdB) by Chinese hamster ovary cells growing in a spinner-flask with a protein-free medium by more than six-fold. The two acids, however, had different cytotoxicities. 相似文献
8.
Two transfected hybridoma cell lines TB/C3-bcl2 (overexpressing the Bcl-2 protein) and TB/C3-pEF (control cell line), were
compared in batch suspension cultures using a medium supplemented either with horse serum or with a protein-free, iron-rich
supplement. The membrane intact index (percentage of cells with intact membranes determined by trypan blue staining) of the
TB/C3-bcl2 cell line decreased much slower than that of the control cell line during the dying phase of the cultures. No significant
difference in antibody, lactate and ammonia production as well as glucose and glutamine consumption was noted in the exponential
phase of the experiments. Both cell lines were also compared in batch experiments using media diluted with saline to further
investigate the effect of Bcl-2 under sub-optimal conditions. The Bcl-2 overexpressing cell line again exhibited a higher
membrane intact index at increasing dilution steps.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
9.
High-density culture of recombinant Chinese hamster ovary cells producing prothrombin in protein-free medium 总被引:2,自引:0,他引:2
A recombinant CHO cell line, CHO2DS, was immobilized on porous microcarrier Cytopore 1 and cultivated in 1 l modified Super-spinner and 2 l stirred tank bioreactor with the perfusion of a low-cost chemically defined protein-free medium DF6S. CHO2DS cells could enter into the inner space and grew both in the inner space and on the surface of Cytopore 1 in DF6S and produced prothrombin at 22 mg l–1 after 10 days. From a seeding density of 5.7 × 105 cells ml–1, the highest viable cell density of CHO2DS was 1.12 × 107 cells ml–1. 相似文献
10.
CB.Hep-1 hybridoma growth and antibody production using protein-free medium in a hollow fiber bioreactor 总被引:1,自引:0,他引:1
R. Valdés N. Ibarra M. González T. Alvarez J. García R. Llambias C. A. Pérez O. Quintero R. Fischer 《Cytotechnology》2001,35(2):145-154
The protein-free medium TurboDoma HP.1 (THP.1) was used to produce the CB.Hep-1 monoclonal antibody (mAb) in a CP-1000 hollow
fiber bioreactor (HFB). This mAb is used for the immunopurification of recombinant hepatitis B surface antigen (rHBsAg), which
is included in a vaccine preparation against the Hepatitis B Virus. By using the experimental conditions tested in this work
we were able to generate more than 433 mg of IgG in 43 days. The maximum antibody concentration obtained was about 2.4 mg
ml-1and the IgG production per day was approximately 11 mg of monoclonal antibody, which constitutes a good concentration value
in comparison to the results obtained in ascitic fluid, where concentration for this hybridoma was around 3 mg ml-1. We used different analytical methods to control the quality of mAbs, obtained from the in vitro system. They included affinity constant determination, analysis of N-glycan structures, immunoaffinity chromatography and
antigen binding properties. The results obtained suggest that no significant changes occurred in the mean characteristics
of the mAb harvested from the bioreactor during the 43 days of cultivation.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献