Publications de Martin Jordan | EPFL Graph Search

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Here a simple in vitro assay was used to investigate the disassembly of nanoparticles of polyethylenimine (PEI) and DNA. Particles were formed with various PEIs, allowed to mature for 10 min, and then exposed to different competitors (RNA, DNA, BSA or hepa ...

There are currently two methods for maintaining cultured mammalian cells, continuous passage at 37 degrees C and freezing in small batches. We investigated a third approach, the "pausing" of cells for days or weeks at temperatures below 37 degrees C in a v ...

An agitated 12-well microtiter plate system with a working volume of 2ml was investigated for cell culture process development. Agitation assures homogeneity in wells and enhances mass transfer between the gas and the liquid phase, thus improving maximum cell density and pH stability. The pH of the NaHCO(3)-buffered system can be adjusted by altering the carbon dioxide content of the gas phase. The non-toxic, visual pH indicator phenol red was used in combination with a spectrophotometric plate reader for r

Fluorescent proteins expressed in mammalian cells can be quantified quickly and noninvasively with a standard fluorescence plate reader. We have previously exploited this quality in cell growth assessment (Hunt et al., 1999b). In this work, different CHO cell lines constitutively expressing fluorescent proteins were evaluated as model systems for process development and optimization. Our results demonstrate that the fluorescence of these cell lines quickly reveals conditions that might improve the overall p

We present a new approach for biomass assessment in cell culture using a disposable microcentrifuge tube. The specially designed tube is fitted with an upper chamber for sample loading and a lower 5 microL capillary for cell collection during centrifugatio ...

We have developed a novel method called Calfection for gene delivery to and protein expression from suspension-cultivated mammalian cells. Plasmid DNA was simply diluted into a calcium chloride solution and then added to the cell culture for transfection. We evaluated and optimized this approach using suspension-adapted HEK293 cells grown in 12-well plates that were shaken on an orbital shaker. Highest expression levels were obtained when cells were transfected at a density of 5x10(5) cells/ml in the presen

Lactate production is monitored in industrial processes as a crucial metabolite for cultured mammalian cells. Typically lactate is strongly produced during the exponential growth phase, while its net consumption is frequently observed when cells enter into ...

NMR-microprobes based on solenoids and Helmholtz coils have been microfabricated and NMR-spectra of mammalian cells have successfully been taken. The microfabrication technology developed for these probes consists of three electroplated copper levels for l ...

Using flow cytometry, single cell sorting, confocal microscopy and fluorescent plasmids, a thorough study of DNA uptake, DNA fate and DNA expression in mammalian cells transfected with the widely used calcium-phosphate precipitation method was executed. We show for the first time that up to 100,000 plasmid molecules can be delivered into individual cells, but also that DNA transfer into cells is a dynamic process that follows a defined kinetics of uptake and intracellular processing. Analyses by flow cytome

DNA-calcium phosphate co-precipitates arise spontaneously in supersaturated solutions. Highly effective precipitates for transfection purposes, however, can be generated only in a very narrow range of physico-chemical conditions that control the initiation and growth of precipitate complexes. The concentrations of calcium and phosphate are the main factors influencing characteristics of the precipitate complex, but other parameters, such as temperature, DNA concentration and reaction time are important as w

One of the major constraints to performing large-scale transfections of cultured mammalian cells for the transient expression of recombinant proteins is the production of large quantities of purified plasmid DNA. In this report partially purified plasmid DNA was prepared by a method that combines alkaline lysis of E. coli with standard precipitation techniques. The efficiency of calcium phosphate-DNA co-precipitate formation with crude DNA was similar to that observed for pure DNA, but precipitate formed wi

Cell expansion, gene transfer and protein production were all executed with a single serum-free, animal protein-free commercial medium designed for suspension-adapted Chinese hamster ovary cells (CHO DG44). This is a most important process to consider for ...

DNA-calcium phosphate coprecipitates have been used for 30 years as an efficient method to introduce genetic material into cells. The method involves simple solutions that can be prepared or purchased by the experimentalist. All the numerous variations of the protocol found in the literature are based on the same principle--a spontaneous precipitation that occurs in supersaturated solutions. When DNA is present during this process, it is readily incorporated into the forming calcium phosphate precipitate. A

This is the first report of two successful 100 l scale transienttransfections in a standard stirred bioreactor. More than half a gram of a monoclonal antibody (IgG) were produced in less than 10 days using a technology called large-scale transient gene expression(LS-TGE). Suspension adapted HEK 293 EBNA SF cells were transfectedwithin a 150 l (nominal) bioreactor by a modified calcium phosphateco-precipitation method with more than 75 mg of plasmid DNA per run.A mixture of three different plasmids, one enco

Various methods exist to transfect mammalian cells in culture. It is generally accepted that individual methods have to be optimized for each of the cell lines or cell types used. Despitethe use of optimized protocols, significant day-to-day variationsin transfection efficiency regularly occur. We postulate that the;status' of cell populations prior to transfection is involved insuch variability. This study evaluates standardized transfectionsdone at different phases of the cell cycle. Cell synchronizationw

Microinjection is a gene transfer technique enabling partial control of plasmid delivery into the nucleus or cytoplasm of cultured animal cells. Here this method was used to establish various recombinant mammalian cell lines. The injection volume was estim ...

This study correlates the fluorescent signal from stable recombinant CHO cell lines expressing the green fluorescent protein (GFP) at high levels with biomass or cell number, extending the use of fluorescent proteins to applications and assays where cell growth rates are important. Using a standard fluorometer, growth of these cells can be quantified noninvasively in multiwell plates, and because signals are obtained without preparation, the same culture samples can be measured repeatedly. Even with a small

Fluorescent protein expressing cells with very high fluorescence are described as well as a method to obtain such cells. The fluorescence of such cells can be as high as about 1000 times the auto-fluorescence of the respective host cell. Due to the high fluorescence, said cells are very suitable tools in the development, surveillance and/or optimization of production processes, for selection of culture media and process parameters and for the determination of cell growth and the detection, quantification an

For large-scale applications in biotechnology, cultivation of mammalian cells in suspension is an essential prerequisite. Typically, suspension cultures are grown in glass spinner flasks filled to less than 50% of the nominal volume. We propose a superior ...

Martin Jordan

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Disassembly of polyethylenimine-DNA particles in vitro: implications for polyethylenimine-mediated DNA delivery

Low-temperature pausing of cultivated mammalian cells

Small-scale bioreactor system for process development and optimization

Fluorescent proteins in animal cells for process development: optimization of sodium butyrate treatment as an example

New disposable tubes for rapid and precise biomass assessment for suspension cultures of mammalian cells

Calfection: a novel gene transfer method for suspension cells

Lactate metabolism shift in CHO cell culture: the role of mitochondrial oxidative activity

Microfabricated solenoids and Helmholtz coils for NMR spectroscopy of mammalian cells

Transfer of high copy number plasmid into mammalian cells by calcium phosphate transfection

Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation

Transfection of partially purified plasmid DNA for high level transient protein expression in HEK293-EBNA cells

Scalable transient gene expression in Chinese hamster ovary cells in instrumented and non-instrumented cultivation systems

Transfection of adherent and suspended cells by calcium phosphate

100-liter transient transfection

S-phase synchronized CHO cells show elevated transfection efficiency and expression using CaPi

Generation of recombinant Chinese hamster ovary cell lines by microinjection

GFP-expressing mammalian cells for fast, sensitive, noninvasive cell growth assessment in a kinetic mode

Cells, genetically modified with optical marker

Orbital shaker technology for the cultivation of mammalian cells in suspension

Polyethylenimine-based quality control assay for plasmid DNA