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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.
Human-human hybridoma SH-76 cells were found to produce a factor that supported the growth of lymphocytic cells at low densities. The factor was purified from serum-free conditioned medium of the hybridoma cells by a successive application of ammonium sulfate precipitation, DEAE-Toyopearl, TSK G3000 SW and DEAE-5PW column chromatograph. The purified factor was a 72K single protein. The factor showed marked growth stimulating effect on lymphocytic cell lines, but had no effect on the growth of human adhesive cancer cell lines. Thus, the factor is a lymphocytic clonal growth factor (LCGF), as found previously in human plasma (Miyata, 1988). The LCGF of SH-76 cells could be produced in growth factor-free RPMI medium and purified easily from the conditioned medium. The factor is inactivated by heating at over 80°C, but is much more stable than the LCGF in human plasma. 相似文献
3.
Charles R. Ill Tammy Brehm Bjorn K. Lydersen Rachel Hernandez Karen G. Burnett 《In vitro cellular & developmental biology. Plant》1988,24(5):413-419
Summary Studies with Human x Human (HxH), Human x Mouse (HxM), and Mouse x Mouse (MxM) hybridomas have enabled us to define specific
factors that affect hybridoma growth in a species-specific manner. Three transferrins and three lipophilic iron chelates have
been tested for their ability to support hybridoma proliferation and antibody production. The results of these studies demonstrate
that HxH hybridomas do not respond to bovine transferrin a+ concentrations up to 100 μg/ml and are approximately 100-fold less responsive to mouse transferrin than to human transferrin.
HxM and MxM hybridomas respond equally to human or mouse transferrin but are 100-fold less sensitive to bovine transferrin.
An antibody to the human transferrin receptor inhibited the growth-promoting activity of human or mouse transferrin on HxH
hybridomas but was ineffective on HxM hybridomas. This semonstrated the functionality of the human transferrin receptor in
HxH hybridomas and that human, mouse, and bovine transferrin were interacting through the mouse transferrin receptor in HxM
hybridomas. HxH and HxM hybridomas respond similarly to three different iron chelates exhibiting 80 to 110% of the growth
response to human transferrin. MxM hybridomas fail to respond to the iron chelates at similar concentrations, suggesting that
the human genome present in the other hybridoma species confers a unique ability for utilizing iron when delivered in this
form. 相似文献
4.
It has been known for several decades that cultured murine cells undergo a defined series of changes, i.e., anin vitro evolution, which includes crisis, spontaneous transformation (immortalization), aneuploidy, and spontaneous neoplastic transformation. These changes have been shown to be caused by thein vitro environment rather than an inherent instability of the murine phenotype or genotype. Serum amine oxidases were recently identified as a predominant cause of crisis. These enzymes generate hydrogen peroxide from polyamine substrates that enter the extracellular milieu. This finding implicates free-radical toxicity as the underlying cause ofin vitro evolution. We propose an oxyradical hypothesis to explain each of the stages ofin vitro evolution and discuss its significance for cytotechnology and long-term cultivation of mammalian cell types.ORR, CDER, FDA Mod-1, Room 2023, 8301 Muirkirk Road, Laurel MD 20708, USA 相似文献
5.
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
6.
Hybridomas were cultured under steady-state conditions in a series of two continuous stirred-tank reactors (CSTRs), using a serum-free medium. The substrate not completely converted in the first CSTR, was transported with the cells to the second one and very low growth rates, high death rates, and lysis of viable cells were observed in this second CSTR. These conditions are hardly accessible in a single vessel, because such experiments would be extremely time-consuming and unstable due to a low viability. In contrast to what is often observed in literature, kinetic parameters could thus be derived without the neccessity for extrapolation to lower growth rates. Good agreement with literature averages for other hybridomas was found. Furthermore, showing that the reactor series is a valuable research tool for kinetic studies under extreme conditions, the possibility to observe cell death under stable and defined steady-state conditions offers interesting opportunities to investigate apoptosis and necrosis. Additionally, a model was developed that describes hybridoma growth and monoclonal antibody production in the bioreactor cascade on the basis of glutamine metabolism. Good agreement between the model and the experiments was found.Abbreviation MAb
Monoclonal antibody
Nomenclature
C
AConcentration of any (mol m-3) component A
D Dilution rate (s-1)
K
dDeath-rate constant (mol m-3)
K
lLysis-rate constant (mol m-3)
K
sMonod constant (mol m-3)
m Maintenance coefficient (mol cell-1 s-1)
q Specific consumption (mol cell-1 s-1) or production rate
t Time (s)
X Cell concentration (cell m-3)
Y Yield coefficient (cell mol-1)
Greek symbols
d
Specific death rate (s-1)
l
Specific lysis rate (s-1) of viable cells
net
Net specific growth (s-1) rate
true
True specific growth (s-1) rate 相似文献
7.
Michael Ma Shuenn-Jue Wu Maureen Howard Alexej B. Borkovec 《In vitro cellular & developmental biology. Plant》1984,20(9):739-742
Summary We report here that the use of murine thymoma cell EL-4 conditioned medium enhances hybridoma yield in a low-antigen dose
in vitro immunization protocol. This improved protocol allowed the production of a panel of monoclonal antibodies toDrosophila yolk proteins using less than 1 nanomole of antigen. We believe this refinement will be valuable for the application of hybridoma
technology to biologically active materials that are hard to isolate and purify due to their low concentration in the biological
fluids.
This research was supported by the College of Agriculture and Life Sciences, University of Maryland, USDA-University of Maryland
Cooperative
Editor's Statement This observation should simplify in vitro immunization approaches and shed new light on the factors required
for the in vitro immune response. Wallace L. McKeehan 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.
T cell hybridomas were raised against the glycopeptide S72 (Core-1) containing the tumor-associated disaccharide Gal (1–3) GalNAc (Core-1) O-linked to serine at position 72 in the mouse hemoglobin derived decapeptide Hb (67–76). All hybridomas recognized the glycopeptide S72 (Core-1). Two of the selected hybridomas responded, however, much better to the S72 (Tn) glycopeptide containing the monosaccharide GalNAc O-linked to serine. In addition, one hybridoma cross-responded to the glycopeptide T72 (Core-1) having a threonine at position 72 instead of a serine. No cross-responses were found to other glycopeptides consisting of the same hemoglobin peptide with different glycans attached or to the unglycosylated peptides. The T cell receptor V and V usage was clearly diverse. The CDR3 regions demonstrated moreover a predominance of small polar amino acid side chains, and three hybridomas contained a common sequence motif. All the sequenced CDR3 regions contained furthermore a conserved proline-glycine motif. In conclusion, immunization with the disaccharide containing glycopeptides S72 (Core-1) created a heterogeneous population of glycopeptide specific T cells with the ability of cross-responding toward related glycopeptides. 相似文献
10.
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