共查询到20条相似文献,搜索用时 8 毫秒
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Kevin J. Gilligan Suzanne Littefild Allan P. Jarvis Jr. 《In vitro cellular & developmental biology. Plant》1988,24(1):35-41
Summary The cell growth and monoclonal antibody production characteristics of two rat x mouse heterohybridoma cell lines, designated
187.1 and M1/9.3, were investigated using a biocompatible microencapsulation technology. Both cell lines, derived from the
fusion of immunized rat spleen cells with either the NS1 or X63Ag8.653 myeloma cell lines, were found to reach a maximum intracapsular
cell density of 1.3 to 1.5×107 cells/ml during a 27-d culture period. During this period, rat monoclonal antibody accumulated in the intracapsular space
of both cultures to a final concentration of 2.0 to 2.8 mg/ml. Comparison of the concentration of rat monoclonal antibody
in the extracapsular vs. the intracapsular space of both cultures indicated that significantly less than 1% of the antibody
produced by the encapsulated hybridoma cells was capable of transiting the microcapsule membrane during the culture period.
Due to the partition of the rat monoclonal antibody within the intracapsular space, the initial purity of the antibody harvested
from 21-d microcapsule cultures of 187.1 and M1/9.3 cells was approximately 48 and 75% by weight, respectively. Analysis of
the intracapsular protein by sodium dodecyl sulfoxide gel electrophoresis at different times during the culture period demonstrated
that the principal contaminant associated with the unpurified antibody was bovine serum albumin. 相似文献
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Rapamycin was used as a medium additive to slow the progression of CRL 1606 hybridomas through the cell cycle, under the hypothesis that such a modulation might reduce cell death. Cell cycle distributions for CRL hybridomas in the G1 phase of the cell cycle ranged from 20% to 35% during batch, fed-batch, and continuous culture experiments, independent of culture time, dilution rate, growth rates, or death rates. Rapamycin, an mTOR signaling inhibitor, immunosuppressant, and G1-phase arresting agent, was identified and tested for efficacy in restraining cell cycle progression in CRL 1606 hybridoma cultures. However, in the presence of 100 nM rapamycin, the percentage of cells in the G1 phase of the cell cycle during fed-batch cultures was only increased from 28% to 31% in control cultures to 37% to 48% for those with rapamycin. Accordingly, rapamycin only slightly reduced culture growth rate. Instead, the use of rapamycin more notably kept viability higher than that of control cultures by delaying cell death for 48 h, thereby enabling viable proliferation to higher maximum viable cell densities. Furthermore, rapamycin enhanced specific monoclonal antibody production by up to 100% during high-viability growth. Thus, over the course of 6-day fed-batch cultivations, the beneficial effects of rapamycin on viable cell density and specific productivity resulted in an increase in final monoclonal antibody titer from 0.25 to 0.56 g/L (124%). As rapamycin is reported to influence a much broader range of cellular functions than cell cycle alone, these findings are more illustrative of the influence that signal transduction pathways related to mTOR can have on overall cell physiology and culture productivity. 相似文献
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We established a serum-free medium of low protein content(125g/ml) TYI 100, consisting of three hormones and five growth factors for the growth of lymphoid and hybridoma cell lines. In TYI 100 medium, mouse and human hybridomas grew equally well as in RPMI 1640 supplemented with 10% fetal bovine serum (10% FBS) without adaptation to the serum-free medium. TYI 100 medium allowed several passages of mouse hybridoma lines and the total cell number was more than in 10% FBS. TYI 100 medium also supported growth of myelomas and anchorage dependent cell lines, Bowes and CHO, well. TYI 100 medium is composed of inexpensive supplements and is therefor applicable to large scale culture.Abbreviations FBS
Fetal Bovine Serum
- IMDM
Iscove's Modification of Dulbecco's Medium
- PBS
Phosphate-Buffered Saline
- TPA
Tissue Plasminogen Activator 相似文献
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A fixed bed perfusion system for hybridoma cell immobilization is presented. The system consists of a culturing vessel (300 ml total volume) in which polyurethane (PU) sponges in the form of small cubes of about 5 mm sides are packed. Cells are immobilized by physical entrapment in the foam matrix. By entrapment of the cells in the pores of the matrix high cell concentration can be maintained in a mechanically protected environment. Medium is continuously circulated by an airlift pump mounted in the cell-free chamber (700 ml total volume).Medium flow rate, feeding rate, dissolved oxygen, pH, nutrient uptake and waste product formation can be easily monitored and controlled. Steady state conditions are established with medium dilution rates of 1.0–1.5 reactor volume per day. The steady state is characterized by a constant cell density, constant culture volume and constant glucose and lactate levels. Cell-free supernatant is collected continuously in a cold room adjacent to the 37°C culture room. Monoclonal antibodies (MAb) are produced at a concentration of 150–200 g/ml for several weeks. An important feature of the system is the capacity to maintain a population of cells after the growth phase in a non-proliferating state for extended time periods expressing high titers of MAb.Abbreviations DO
Dissolved Oxygen
- FBS
Fetal Bovine Serum
- FBR
Fixed Bed Reactor
- MAb
Monoclonal Antibody
- PU
polyurethane 相似文献
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It is generally accepted that the phase of cell decline observed in batch culture of mammalian cells is related to exhaustion of medium nutrients (principally glucose and glutamine) and/or to waste products accumulation. In the present paper, we have studied the influence of glutamine on the proliferation of mouse hybridoma cells. We showed that repeated addition of glutamine prolonged the life span of the culture and significantly increased the secretion of monoclonal antibody. Flow cytometry analysis suggests that this effect of glutamine is related to a delay in cell death rather than to a stimulation of proliferation.Addition of glutamine and glucose failed however to prevent the death of the culture. Determinations of amino acid consumption in glutamine-supplemented samples and experiments carried out with complementary sources of amino acids (e.g. tryptose phosphate) strongly suggest that amino acid supply is a critical factor governing cell growth and productivity. 相似文献
9.
Effect of temperature on nucleotide pools and monoclonal antibody production in a mouse hybridoma 总被引:2,自引:0,他引:2
The specific monoclonal antibody productivity (q(Mab)) of a murine hybridoma (CC9C10) increased with incubation temperature in the range 33 degrees C to 39 degrees C. q(Mab) was constant at each temperature and was independent of the phase of culture. The q(Mab) increased 97% at 39 degrees C and decreased by 21% at 33 degrees C compared with controls at 37 degrees C. Specific rates of substrate (glucose and glutamine) utilization and byproduct (lactate and ammonia) formation also increased with temperature but the yield coefficient, Y(Lac/Llc') was constant for 33 degrees C to 39 degrees C and Y(Amm/Gin) was constant for 37 degrees C to 39 degrees C. Y(Amm/Gin) at 33 degrees C was lower than the control. Changes in specific nucleotide concentrations and ratios were monitored by analysis of intracellular nucleotide pools. The NTP ratio, (ATP + GTP)/(UTP + CTP), increased and the U-ratio (UTP/UDP-GNac) decreased during the course of each culture, whereas the adenylate energy charge, (ATP + 0.5ADP)/(ATP + ADP + AMP), remained relatively constant at a value 0.8. The relative content of UDP-/N acetyl galactosamine, UDP-N acetyl glucosamine, and NAD increased with incubation temperature, whereas the relative ATP content, SA(ATP + ADP + AMP)/SU (UTP + UDP-sugars) ratio, purine/pyrimidine, ATP/GTP, and U-ratio decreased at higher incubation temperatures. It is possible that these nucleotide parameters may have a regulatory role in the changes of q(Mab) observed at the higher temperatures. (c) 1994 John Wiley & Sons, Inc. 相似文献
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At the end of a hybridoma batch culture, the cells are usually discarded after separation from the culture broth. If, however, they are aseptically recycled into the reactor, the production process can be resumed simply by the addition of fresh medium. This cycle can then be repeated several times consecutively. In a test case, with a mouse hybridoma, we found antibody yields for each cycle in the same range as for a standard batch. In a 15 1 stirred tank reactor we could, within 6 days, produce 2.8 g of monoclonal antibody (MAb). This type of reactor operation allowed a doubling in the reactor volumetric productivity (mg/l/day). 相似文献
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A new method of making microcapsules with calcium alginate gel was developed and the cultivation of the encapsulated hybridoma cells producing monoclonal antibodies against human chorionic gonadotropin was investigated. A high cell density of 2.0×108 cells/cm3 in the capsules led to a high dilution rate of 0.68 per hour and resulted in the high volumetric monoclonal antibody productivity of 652.8 mg/l/day, which was 20–30 times higher than those of traditional continuous suspension cultures. However, long-term continuous culture was not achieved with this capsule system probably because of the limitation in nutrient supply and the accumulation of waste products. Also the analysis of oxygen transfer in this system showed that oxygen supply was not enough to support such a high cell density. 相似文献
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Ronald Levy Yehudit Bergman Olivera Finn 《In vitro cellular & developmental biology. Plant》1981,17(12):1051-1057
Summary In the present paper, we will summarize studies we have performed on two distinct human lymphocyte cell surface antigens defined
by monoclonal antibodies: Leu-1 and HLA-DR.
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 work was supported by USPHS-NIH Grants CA-21223, AI-11313, and CA-09302.
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
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The effects of several different substances, including insulin, transferrin, ethanolamine, selenite and butyrate on the growth of murine hybridoma 2F7 cells, which secrete monoclonal antibody against small cell lung cancer, were investigated, and a serum-free medium SFMI was formulated. The effects of taurine, spermidine, progesterone and adenine on the cell growth were tested further on the basis of the medium SFMI, and a modified serum-free medium SFM II was established. On the basis of medium SFM II, the substitution tests of ferric citrate for transferrin were carried out, and it was found that transferrin could be replaced. The experiments suggested that the formulated serum-free medium was suitable for 2F7 cell growth and monoclonal antibody secretion, and thus facilitated subsequent purification of monoclonal antibody.Abbreviations BSA
bovine serum albumin
- CS
calf serum
- DMEM
Dulbecco's modified Eagle's medium
- ELISA
enzyme-linked immunosorbant assay
- McAb
monoclonal antibody
- PEG
polyethylene glycol
- SFM
serum-free medium 相似文献
14.
Effect of temperature on hybridoma cell cycle and MAb production 总被引:3,自引:0,他引:3
The kinetics of growth and antibody formation of an anti-interleukin-2 producing hybridoma line were studied in suspension culture at temperatures ranging from 34 degrees C to 39 degrees C. Flow cytometry was used to determine the effect of temperature on the cell cycle. Maximum cell density and monoclonal antibody yield were observed at 37 degrees C. The specific monoclonal antibody production rate was approximately constant throughout each batch experiment. Lower temperatures caused cells to stay longer in the G(1)-phase of the cell cycle, but temperature had only a marginal effect on the specific antibody production rate. Arresting of cells in the G(1)-phase by means of temperature was, therefore, not suited for enhanced monoclonal antibody production. Rather, antibody production for this hybridoma was directly linked to viable cell concentration. (c) 1992 John Wiley & Sons, Inc. 相似文献
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A general weaning procedure is described which allowed a range of hybridomas to be weaned readily off serum without loss of antibody production. Initial work was carried out with one cell line only (SPO1 cells) and one serum substitute containing a final protein concentration of 40 mg l-1. The SPO1 cells were first adapted to a range of readily available basal media and then weaned off serum by a range of protocols. From this work an optimal weaning protocol and basal medium for weaning were determined. These were then used to wean the SPO1 cells and two other cell lines off serum with a second, protein free, serum substitute with varying concentrations of defined proteins added. All three cell lines investigated were readily weaned off serum by this protocol at protein concentrations as low as 1 mg l-1. No loss of antibody production was observed with any of the cell lines. The weaning procedure outlined is both simple and rapid and has been successfully adopted in our laboratory by relatively inexperienced cell culture technicians. 相似文献
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Electron microscopy of mouse hybridoma cell lines shows that the major difference between non, low and high producer cell lines is the amount of endoplasmic reticulum. Vesicular-tubular or cavernous structures of endoplasmic reticulum, which can survive long after cell death, are particularly abundant in producer cell lines. Immunogold labelling with anti-mouse IgG reveals that antibodies are predominantly located in these structures. The cell membrane undergoes structural changes during the late stages of batch culture with the disappearance of microvilli and the appearance of blebs and deep indentations. Necrosis disrupts the cytoplasmic structures and the nucleus is last to degrade. 相似文献
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Béatrice Mathiot Angelo Perani Dominique Dumas Michel Maugras Jacques Didelon Jean-François Stoltz 《Cytotechnology》1993,11(1):41-48
Hybridoma cell growth and monoclonal antibody production were investigated with a laboratory-made system in which cells were grown in dialysis tubing (MW cut-off 25 kD). The dialysis system contained 10 ml of cell suspension and was immersed in 200 ml of culture medium which when replaced or was at 4-day intervals. With this system, monoclonal antibody concentrations similar to those observed in ascites (concentrations in the order of one gramme per liter) were obtained. With no medium replacement, the antibody production was 3.3 g/l and the cell productivity 3.2×10–8 g of IgM produced per cell in one minute. With medium replacement the antibody production was higher, 4.4 g/l but the cell productivity was lower, 1.49×10–8 g per cell in one minute. Cells cultivated in non-optimized conditions were better producers than cells growing in a good environment.Abbreviations FCS
fetal calf serum
- Ig
immunoglobulin
- MAb
monoclonal antibody
- MW
molecular weight
- MWCO
molecular weight cut off
- RM
replaced medium
- NRM
non replaced medium 相似文献
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Stacey Spencer Deidra Bethea T. Shantha Raju Jill Giles-Komar Yiqing Feng 《MABS-AUSTIN》2012,4(3):319-325
The successful development of antibody therapeutics depends on the molecules having properties that are suitable for manufacturing, as well as use by patients. Because high solubility is a desirable property for antibodies, screening for solubility has become an essential step during the early candidate selection process. In considering the screening process, we formed a hypothesis that hybridoma antibodies are filtered by nature to possess high solubility and tested this hypothesis using a large number of murine hybridoma-derived antibodies. Using the cross-interaction chromatography (CIC) method, we screened the solubility of 92 murine hybridoma-derived monoclonal antibodies and found that all of these molecules exhibited CIC profiles that are indicative of high solubility (>100mg/mL). Further investigations revealed that variable region N-linked glycosylation or isoelectric parameters are unlikely to contribute to the high solubility of these antibodies. These results support the general hypothesis that hybridoma monoclonal antibodies are highly soluble. 相似文献
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《MABS-AUSTIN》2013,5(3):319-325
The successful development of antibody therapeutics depends on the molecules having properties that are suitable for manufacturing, as well as use by patients. Because high solubility is a desirable property for antibodies, screening for solubility has become an essential step during the early candidate selection process. In considering the screening process, we formed a hypothesis that hybridoma antibodies are filtered by nature to possess high solubility and tested this hypothesis using a large number of murine hybridoma-derived antibodies. Using the cross-interaction chromatography (CIC) method, we screened the solubility of 92 murine hybridoma-derived monoclonal antibodies and found that all of these molecules exhibited CIC profiles that are indicative of high solubility (>100mg/mL). Further investigations revealed that variable region N-linked glycosylation or isoelectric parameters are unlikely to contribute to the high solubility of these antibodies. These results support the general hypothesis that hybridoma monoclonal antibodies are highly soluble. 相似文献
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Immobilization offers several intrinsic advantages over free suspension cultures for the production of monoclonal antibodies. An important advantage of immobilization is the improved specific monoclonal antibody (MAb) productivity (q(MAb)) that can be obtained. However, there are conflicting reports in the literature on the enhancement of the q(MAb) with immobilization. The discrepancies between these reports can be attributed to the different to either the cultivation methods used for immobilized cell or to difference between the cell lines used in the various studies. We show that these differences may be attributed to the different cultivation methods used for one model hybridoma cell line. S3H5/Upsilon2bA2 hybridoma cells entrapped in different sizes of calcium alginate beads were cultivated in both T- and spinner flasks in order to determine whether cultivation methods (T- and spinner flasks) and bead size influence the q(MAb) Free-suspended cell cultures inoculated with cells recovered from alginate beads were also carried out in order to determine whether changes in the q(Mab) of the entrapped cells are reversible.The cultivation methods was found to influence significantly the q(MAb) of the entrapped cells. When the entrapped cells in 1-mn diameter beads were cultivated in T-flasks, the q(MAb) was not increased by 200% as previously observed in an entrapped cell culture using 1-mm-diameter alginate beads in spinner flasks. The q(MAb) of the entrapped cell was approximately 58% higher than that of the free-suspended cells in a control experiment. Unlike the cultivation method, the bead size in the range of 1- to 3-mm diameter did not significantly influence the q(MAb), regardless of cultivations methods. The changes in q(MAb) of an entrapped cells were reversible. When the free-suspended cells recovered from the T- and spinner flasks were sub-cultured in T- and spinner flasks enhanced q(MAb) of the entrapped cells in both cases decreased to the level of the free-suspended cell in a control experiments. Taken together, these results shows that the method of cultivation of hybridoma cells immobilized in alginate beads determines the extent of enhancement of the q(MAb). (c) 1993 John Wiley & Sons, Inc. 相似文献