High viable cell concentration fed-batch cultures of hybridoma cells through on-line nutrient feeding

SummaryA laboratory scale bioreactor was used for continuous acidification and inoculation of milk with a proteinase-negative, lactose-fermenting strain,Lactococcus lactissubsp.lactisC2S. Calcium alginate-entrapped cells were immobilized on a spiral stainless steel mesh incorporated into a column bioreactor and used to acidify and inoculate reconstituted skim milk. Characteristics of the immobilized cell bioreactor (ICB) were compared with those of a free cell bioreactor (FCB) during challenge with a virulent phage. Steady state biomass and lactate productivities were respectively 25-fold and 12-fold larger with the ICB than with the FCB. The ICB and the FCB were inoculated with the prolate phage c2 at multiplicities of infection of 0·25 and 0·02 respectively. Within 90 min of the infection, the FCB viable cell concentration dropped by five orders of magnitude and never recovered, while the plaque forming units/ml increased dramatically. In the ICB, released cells decreased immediately after infection, but subsequently increased, while the plaque forming units/ml steadily declined, indicating that phage were being washed out of the bioreactor. Productivity of FCB decreased to zero, whereas productivity of the ICB only decreased ∼ 60% and subsequently recovered to its initial steady state value.

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Microbes in deionized water: Implications for maintenance of laboratory water production system

10.7287/peerj.preprints.181v3 ◽

2016 ◽

Author(s):

Wenfa Ng ◽

Yen-Peng Ting

Keyword(s):

Ion Exchange ◽

Microbial Diversity ◽

Production System ◽

Cell Concentration ◽

Tap Water ◽

Viable Cell ◽

Just In Time ◽

Water Production ◽

Viable Cell Concentration ◽

Salt Solutions

Microbes, with their diverse metabolic capabilities and great adaptability, occupy almost every conceivable ecological niche on Earth – thus, could they survive in the oligotrophic (i.e., nutrient-poor) deionized (DI) water that we use for our experiments? Observations of white cauliflower-like lumps and black specks in salt solutions after months of storage in plastic bottles prompted the inquisition concerning the origin and nature of the “contaminants”. Hypothesizing that the “contaminants” may be microbes from DI water, a series of growth experiments was conducted to detect and profile the microbial diversity in fresh DI water - produced on a just-in-time basis by a filter-cum-ion-exchange system with tap water as feed. While microbes could also be present on the surfaces and headspace of the unsterilized polyethylene bottles, investigating whether microbes are present in freshly produced DI water provides a more stringent performance test of the production system. Inoculation of DI water on R2A agar followed by multi-day aerobic cultivation revealed the presence of a wide variety of microbes (total viable cell concentration of ~103 colony forming units (CFU) per mL) with differing pigmentations, growth rates as well as colony sizes and morphologies. Additionally, greater abundance and diversity of microbes was recovered at 30 oC relative to 25 and 37 oC; most probably due to adaptation of microbes to tropical ambient water temperatures of 25 to 30 oC. Comparative experiments with tap water as inoculum recovered a significantly smaller number and diversity of microbes; thereby, suggesting that monochloramine residual disinfectant in tap water was effective in inhibiting cell viability. In contrast, possible removal of monochloramine by adsorption onto ion-exchange resins – and thus, alleviation of a source of environmental stress - might explain the observed greater diversity and abundance of viable microbes in DI water. Collectively, this study confirmed the presence of microbes in fresh DI water – and suggested a possible source of the “contaminants” in prepared salt solutions. Propensity of microbes for forming biofilm on various surfaces suggested that intermittent flow in just-in-time DI water production provided opportunities for cell attachment and biofilm formation in the system during water stagnation, and subsequent dislodgement and resuspension of cells upon water flow. Thus, regular maintenance and cleaning of the production system should help reduce DI water’s microbial load. Additionally, simple and low-cost culture experiments on agar medium can provide a qualitative and semi-quantitative estimate of microbial diversity and viable cell concentration in DI water, respectively, and along with regular monitoring of water resistivity or conductivity, comprise a trio of tests useful for detecting possible contamination, or deterioration of DI water’s chemical and microbiological quality.

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Effect of the Purine Derivative Myoseverin and of Its Analogues on Cultured Hybridoma Cells

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc20020257 ◽

2002 ◽

Vol 67 (2) ◽

pp. 257-266 ◽

Cited By ~ 6

Author(s):

František Franěk ◽

Věra Siglerová ◽

Libor Havlíček ◽

Miroslav Strnad ◽

Tomáš Eckschlager ◽

...

Keyword(s):

Monoclonal Antibody ◽

Cell Concentration ◽

Viable Cell ◽

Hybridoma Cells ◽

Purine Derivatives ◽

Monoclonal Antibody Production ◽

Purine Derivative ◽

Methoxy Groups ◽

Benzene Rings ◽

M Phase

Two 2,6,9-trisubstituted purine derivatives, 9-isopropyl-2,6-bis[(4-methoxybenzyl)amino]-9H-purine (myoseverin, PMYO, 1) and 9-isopropyl-2,6-bis[(2-methoxybenzyl)amino]-9H-purine (OMYO, 2), and two 6,9-disubstituted derivatives, 9-isopropyl-6-[(4-methoxybenzyl)amino]-9H-purine (3) and 9-isopropyl-6-[(2-methoxybenzyl)amino]-9H-purine (4), were synthesized with the aim to examine their cell proliferation inhibiting activity, and possible additional effects in cultures of hybridoma cells producing monoclonal antibody. The substances were tested over a concentration range from 0.003 to 30 μmol l-1. The most active compound 1 caused a total loss of cell viability at 1 μmol l-1, while its isomer 2 showed the same effect at 10 μmol l-1 concentration. In the presence of compound 1, but not of compound 2, the character of the cell cycle phases profile changed dramatically, most cells being arrested in the G2/M phase. At intermediate concentrations of compound 2 a substantially higher viable cell concentration was observed, relative to control. These differences demonstrated the principal significance of the position of the methoxy groups on the benzene rings for the biological effect. The 6,9-disubstituted derivatives 3 and 4 were without significant effect in the whole range of concentrations tested. The enhancement of monoclonal antibody production, observed in certain concentration intervals of added substances, was of marginal character.

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A Kinetic Model of Macromolecular Metabolism in Batch and Fed-Batch Cultures of Hybridoma Cells Producing Monoclonal Antibody

Animal Cell Technology: Challenges for the 21st Century ◽

10.1007/0-306-46869-7_13 ◽

2005 ◽

pp. 69-73

Author(s):

J. D. Jang ◽

J.P Barford

Keyword(s):

Monoclonal Antibody ◽

Kinetic Model ◽

Hybridoma Cells ◽

Fed Batch ◽

Batch Cultures

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