Abstract:
:We have recently shown that the semi-continuous cultivation of a mouse hybridoma line in spinner flasks, with a basal defined medium (BDM) devoid of serum and protein, increases the secretion of the immunoreactive monoclonal antibody (MAb) by a factor of ca. 2.4, compared to culture in flasks with serum-containing medium (Schneider, 1989). To further optimise MAb production, we have now investigated the composition of BDM and the mode of cultivation. Hybridoma cells were inoculated at 0.3 x 10(6) cells/ml in 200 ml of BDM containing 4, 6 or 8 mM glutamine; after 3-4 days (when the cell density reached greater than or equal to 10(6) cells/ml) 20, 40, 60 or 100% of the culture medium were replaced daily by fresh nutritive BDM with or without 33, 66 or 100% cell recycling. Daily, over a total period of 15-16 days, viable cells were counted and the concentration of the MAb, of glucose and glutamine (the main nutrients) and of lactate, alanine and ammonia (the main metabolites) were assayed. These experiments indicate that to provide optimal cell growth and MAb production: (i) the concentration of glutamine in the nutritive medium should be maintained at 8 mM, otherwise it falls on some days to 0 with a significant amount of cell death; at 25 mM, glucose concentration is not a limiting factor, whatever the culture conditions; (ii) with a semi-continuous mode of cultivation, a perfusion rate of 40%/day appears optimal; without cell recycling, a rate of 20% does not provide enough nutrients and/or does not remove enough metabolites, whereas a rate of 60% washes over the cells; with cell recycling, rates of 60 (moderately) or 100% (considerably) increase cellular metabolism without concurrent augmentation of MAb secretion; (iii) the recycling of the cells increases the mean cell densities and the rate of production of the MAb, as well as the rate of consumption of nutrients and of production of metabolites; recycling of 33-66% is optimal, since the total recycling progressively raises the number of dead cells and debris; (iv) there are maximal values for cell densities (ca. 2.5 X 10(6) cells/ml) and MAb production (ca. 26 micrograms/10(6) cells X day) as well as for nutrient consumption and metabolite production (except with a very high perfusion rate).(ABSTRACT TRUNCATED AT 400 WORDS)
journal_name
J Immunol Methodsjournal_title
Journal of immunological methodsauthors
Schneider YJ,Lavoix Adoi
10.1016/0022-1759(90)90446-3subject
Has Abstractpub_date
1990-05-25 00:00:00pages
251-68issue
2eissn
0022-1759issn
1872-7905pii
0022-1759(90)90446-3journal_volume
129pub_type
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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abstract::A method is described for the study of lymphokine-mediated cellular interactions using triple wells, which permits co-culture of cell subpopulations without direct physical contact. The triple wells are constructed by slitting the walls to half height between three adjacent wells of a 96-well microtiter plate. The cel...
journal_title:Journal of immunological methods
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doi:10.1016/0022-1759(94)90279-8
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abstract::This paper describes a novel method for following the changes in mouse tumour-infiltrating immune cell populations by repeated sampling of tumours by fine needle aspiration (FNA), followed by flow cytometry. Using this technique we were able to collect samples from P815 mouse mastocytomas, and identify and enumerate s...
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journal_title:Journal of immunological methods
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(94)90231-3
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(89)90031-8
更新日期:1989-09-29 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(99)00087-3
更新日期:1999-08-31 00:00:00
abstract:OBJECTIVE:The central mechanism for establishing a self-tolerant and functional T cell repertoire includes the promiscuous expression of otherwise tissue-restricted proteins by medullary thymic epithelial cells (TEC). We here demonstrate a novel and highly efficient method for isolating this rare key cell type. METHOD...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2019.02.001
更新日期:2019-04-01 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(91)90051-g
更新日期:1991-10-25 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(92)90211-b
更新日期:1992-09-18 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(99)00114-3
更新日期:1999-11-19 00:00:00
abstract::The epitope specificities of monoclonal antibodies against carcinoembryonic antigen (CEA) were determined in a solid-phase, biotin-avidin enzyme immunoassay. Constant amounts of biotin-labeled antibodies were incubated with the immobilized antigen in the presence of decreasing amounts of unlabeled antibodies. The biot...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(84)90157-1
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2016.02.012
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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更新日期:2001-06-01 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2013.12.005
更新日期:2014-02-01 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(85)90484-3
更新日期:1985-01-21 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(82)90051-5
更新日期:1982-07-30 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(84)90352-1
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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journal_title:Journal of immunological methods
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pub_type: 杂志文章
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(89)90206-8
更新日期:1989-01-17 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(02)00213-2
更新日期:2002-10-15 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(82)90384-2
更新日期:1982-01-01 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
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更新日期:1988-04-06 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(84)90401-0
更新日期:1984-10-26 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/0022-1759(90)90053-x
更新日期:1990-07-03 00:00:00
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journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/s0022-1759(02)00025-x
更新日期:2002-04-01 00:00:00
abstract:INTRODUCTION:Peripheral blood mononuclear cells (PBMCs) are frequently used for genomic analyses, but several factors can affect the yield and integrity of nucleic acids, including the methods of cell collection and isolation. The goal of this work was to analyze the utility of systematic isolation of different immune ...
journal_title:Journal of immunological methods
pub_type: 杂志文章
doi:10.1016/j.jim.2016.12.005
更新日期:2017-03-01 00:00:00