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Biomedical subjects

G Blüml

Publications and source records attributed to G Blüml.

11 recordsLinked to original sources
PubMed2003

Scalable inoculation strategies for microcarrier-based animal cell bioprocesses.

Scalability is a major demand for high-yield, stable bioprocess systems in animal cell culture-based biopharmaceutical production. Increased yields can be achieved through high-density cell culture, such as in the combination of microcarrier and fluidized bed bioreactor technology. To minimize inocula volume in industrial applications of fluidized bed fermentation systems, it is crucial to increase the bed volume in the reactor during the fermentation process. We tested scale-up strategy for the production of recombinant human arylsulfatase B (ASB) enzyme used in enzyme replacement therapy in patients afflicted with mucopolysaccharidosis type VI (MPS VI). This enzyme was derived from Chinese hamster ovary (CHO) cells cultivated as adherent cell culture on Cytoline macroporous microcarriers (Amersham Biosciences, Uppsala, Sweden) using a Cytopilot Mini fluidized bed bioreactor (FBR; Amersham Biosciences, Vogelbusch, Austria). Both 1:2 expansion (herein referred to as the addition of fresh, not-yet-colonized microcarriers) and 1:6 expansion of the carrier bed were performed successfully; the cells restarted to proliferate for colonizing these newly added carriers; and the stability of the culture was not negatively affected.

Animals↗
PubMed1994

Two-dimensional gel electrophoresis for controlling and comparing culture supernatants of mammalian cell culture productions systems.

A recombinant Chinese hamster ovary cell line, producing human erythropoietin, was cultivated in a continuous mode in a stirred tank reactor applying different dilution rates. In order to monitor the stability of this expression system, product and non-product proteins of the cell culture supernatant were analyzed by two-dimensional electrophoresis. The consistency of the isoforms of the recombinant product was determined by western blot combined with specific staining. The same cell line was propagated in a high cell density cultivation system based on macro-cell-aggregates. The patterns of secreted proteins of the cell line cultivated in the different systems were compared in order to detect modifications in protein expression of the product and of non product proteins relevant for cell culture supernatant. Hardly any alterations in two-dimensional pattern were detectable. The isoforms of erythropoietin, as well as the overall pattern of secreted proteins, detectable with the two-dimensional electrophoresis method were remarkably stable under different cultivation conditions.

Animals↗
PubMed1994

Large-scale mammalian cell culture.

Over the past year, progress has been made toward a better understanding of the physiological and biochemical responses of cell cultures to various environmental changes. Much of the theoretical and experimental work that has been reported will help improve expression in mammalian cell culture, which is one of the key steps in biopharmaceutical manufacturing.

Animals↗
PubMed1992

Flow cytometry and two-dimensional electrophoresis (2-DE) for system evaluation of long term continuous perfused animal cell cultures in macroporous beads.

Immobilization of r-CHO cells at high density using macroporous polyethylene carriers in a modular fluidized bed reactor is demonstrated. Specific growth rates of the cells are measured by incorporation of BrdU. At a cell density of about 10(8) cells/ml a stable growth rate of 0.004 h-1 was established. Total release of proteins into the culture supernatant during protein-free perfusion was analyzed by 2-DE in various phases of the long-term culture showing very similar patterns indicating a constant pattern of gene expression.

Animals↗
PubMed1992

Determination of division rates of rCHO cells in high density and immobilized fermentation systems by flow cytometry.

As most high density and immobilized fermentation systems do not allow the direct quantitative determination of cell density, two flow cytometric methods (the determination of incorporation of bromodeoxyuridine into newly synthesized DNA and the increase in mitotic cells by colchicine blockage) were evaluated as to their suitability to measure true division rates of cells in bioreactors. The BrdU method gave division rates identical to the growth rates measured by cell count, while the colchicine block method gave values that were lower and varied with the cell line. This is due to the cytotoxicity of colchicine and makes a calibration of the method for each cell line necessary. Both methods have been successfully used to measure division rates of rCHO cells immobilized in an alginate matrix as well as in macroporous carriers in a fluidised bed system and in dialysis culture.

Animals↗
PubMed1992

Two-dimensional electrophoresis as a tool for control of quality and consistency in production systems using animal cells. Two-dimensional electrophoresis in animal cell culture technology.

A nonrecombinant human melanoma cell line and recombinant chinese hamster ovary (CHO) cells were used as examples for long-term in vitro cultivation in protein-free media. The method used to monitor the consistency of protein release by these mammalian cells was two-dimensional electrophoresis with immobilized pH gradient. Secreted proteins from a melanoma cell line cultivated in a continuous fermentation system over a period of 22 months were monitored. Two-dimensional patterns of secreted proteins were compared and the stability of their composition was determined over a period of nearly 14 months, with significant pattern variation being observed after 14 months. The protein pattern from this extended in vitro culture was compared to those of the very same melanoma cell line recultivated after being frozen in liquid nitrogen for more than 2 years. Due to the high resolution of complex polypeptide mixtures and the possibility to detect even minor differences in the composition of protein patterns, we propose the two-dimensional electrophoresis as a tool for quality assessment in animal cell culture technology.

Animals↗
PubMed1991

Modular integrated fluidized bed bioreactor technology.

We describe the design and demonstrate the application of a modular integrated fluidized bed bioreactor system. Basically the system is a reactor vessel equipped with an extending cylinder and a liquid distributor plate. Instead of an external recirculation loop, as used in existing fluidized bed systems, a low shear stress impeller is used as the recirculation pump. The system has several unique features, such as modular exchangeable elements, efficient oxygenation and the option of operating as a stirred tank-, a packed bed- or a fluidized bed reactor. An example of a fluidized bed run using CHO-K1 cells is shown. Under standard culture conditions a 100-fold increase in cell density (up to 1.2 x 10(8) cells/ml) was achieved.

Animals↗
PubMed1991

Limitations of flow detection by color Doppler: in vitro comparison to conventional Doppler.

There is little awareness of the limitations of flow detection with the commercially available color Doppler flow mapping system. The influence of flow velocity, ultrasound attenuation, and penetration depth on flow detection in color Doppler (Toshiba SSH 65A) were therefore studied in vitro and compared with conventional Doppler. The flow model had physiological flow volumes and laminar flow with parabolic velocity profile in a horizontal tube of Lucite with less than 3 degrees of coincidence. Conventional Doppler flow velocity measurements correlated highly with laser Doppler anemometry results (r = 0.99, SEE = 3 cm/sec). Signal strength of color Doppler and pulsed Doppler was semi-quantitatively graded using a scale from 0 to 5. Scale 1 (sparse signals) was useless for any assessment in color Doppler but just allowed velocity measurement in pulsed Doppler. Using 19-dB attenuation, flow velocities greater than 100 cm/sec had good scores with moderate gain, 60-100 cm/sec needed increasing gain, and velocities less than 40 cm/sec were not detectable with color Doppler but readily so with pulsed Doppler. With increasing attenuation (1-29 dB) and also with increasing penetration depth, flow detection was reduced significantly (P less than 0.001) more in color Doppler than in the pulsed technique (P less than 0.01). In conclusion, low flow velocities, high attenuation, and greater than 8 cm penetration depth may hamper flow detection in color Doppler and, thus, diagnostic accuracy. Conventional Doppler with its superior accuracy and sensitivity should therefore consolidate diagnostic ultrasound assessment.

Blood Flow Velocity↗
PubMed1990

The use of macroporous gelatin carriers for the cultivation of mammalian cells in fluidised bed reactors.

A fluidised bed system for the cultivation of mammalian cells on a new type of macroporous gelatin microcarrier is described. The volumetric cell densities achieved under controlled conditions for two 'standard cell lines' (VERO, CHO) were one order of magnitude higher compared to conventional techniques using spherical microcarriers. The system can be potentially used for both anchorage dependent and independent cells.

Animals↗
PubMed1990

[Accuracy of various Doppler technics in recording blood flow velocity. Studies in vitro].

Conventional and color-coded Doppler techniques were studied as to their accuracy in displaying flow and velocity using an in vitro model and a Laser-Doppler-anemometer. Furthermore, the estimation of pressure gradients as determined by Doppler ultrasound was compared to measurements by manometers under a variety of hemodynamic conditions. Pulsed and continuous wave Doppler had good reproducibility. There was an excellent correlation for measurements of flow velocity as determined by Doppler ultrasound and by Laser-Doppler anemometer (r = 0.98, SEE = 3 cm/s). The well-known underestimation of flow velocity due to an increasing angle of incidence (greater than 25 degrees) was confirmed in vitro. However this error was smaller than the actual overestimation resulting from angle correction for the apparent cosine. Doppler gradients correlated strongly with manometer gradients for orifice areas 12-80 mm2 and flow volumes 0.9-12.8 l/min (r = 0.98, SEE = 7 mm Hg) using continuous as well as pulsatile flow. Some overestimation of the Doppler gradient occurred with increasing flow rates (r = 0.66). Color-Doppler has poor spatial resolution. Display of velocities was therefore assessed using a qualitative score (0-5), the variability of which was 13 +/- 30% of the initial value. Display of faintest quality (score 1) was useless for clinical assessment in color-Doppler technique, but allowed quantitative measurement of velocity in conventional Doppler. Reduction of flow velocities limited display in color-Doppler (5-20 cm/s) but not in pulsed-Doppler technique. Thus, conventional Doppler has better sensitivity and accuracy of displaying flow when compared to color-Doppler, particularly in conditions of poor imaging. As reproducibility and accuracy of velocity determination are excellent, this technique should be used in all diagnostic procedures involving ultrasound. The Doppler gradient as derived from the modified Bernoulli equation provides accurate results in vitro which may also be concluded for use in the clinical situation.

Blood Flow Velocity↗