Large-scale Perfusion Research Articles

Large-scale transient peri-ictal perfusion magnetic resonance imaging abnormalities detected by quantitative image analysis.

Epileptic seizures require a rapid and safe diagnosis to minimize the time from onset to adequate treatment. Some epileptic seizures can be diagnosed clinically with the respective expertise. For more subtle seizures, imaging is mandatory to rule out treatable structural lesions and potentially life-threatening conditions. MRI perfusion abnormalities associated with epileptic seizures have been reported in CT and MRI studies. However, the interpretation of transient peri-ictal MRI abnormalities is routinely based on qualitative visual analysis and therefore reader dependent. In this retrospective study, we investigated the diagnostic yield of visual analysis of perfusion MRI during ictal and postictal states based on comparative expert ratings in 51 patients. We further propose an automated semi-quantitative method for perfusion analysis to determine perfusion abnormalities observed during ictal and postictal MRI using dynamic susceptibility contrast MRI, which we validated on a subcohort of 27 patients. The semi-quantitative method provides a parcellation of 3D T1-weighted images into 32 standardized cortical regions of interests and subcortical grey matter structures based on a recently proposed method, direct cortical thickness estimation using deep learning-based anatomy segmentation and cortex parcellation for brain anatomy segmentation. Standard perfusion maps from a Food and Drug Administration-approved image analysis tool (Olea Sphere 3.0) were co-registered and investigated for region-wise differences between ictal and postictal states. These results were compared against the visual analysis of two readers experienced in functional image analysis in epilepsy. In the ictal group, cortical hyperperfusion was present in 17/18 patients (94% sensitivity), whereas in the postictal cohort, cortical hypoperfusion was present only in 9/33 (27%) patients while 24/33 (73%) showed normal perfusion. The (semi-)quantitative dynamic susceptibility contrast MRI perfusion analysis indicated increased thalamic perfusion in the ictal cohort and hypoperfusion in the postictal cohort. Visual ratings between expert readers performed well on the patient level, but visual rating agreement was low for analysis of subregions of the brain. The asymmetry of the automated image analysis correlated significantly with the visual consensus ratings of both readers. We conclude that expert analysis of dynamic susceptibility contrast MRI effectively discriminates ictal versus postictal perfusion patterns. Automated perfusion evaluation revealed favourable interpretability and correlated well with the classification of the visual ratings. It may therefore be employed for high-throughput, large-scale perfusion analysis in extended cohorts, especially for research questions with limited expert rater capacity.

A novel scale-down mimic of perfusion cell culture using sedimentation in an automated microbioreactor (SAM).

Continuous upstream processing in mammalian cell culture for recombinant protein production holds promise to increase product yield and quality. To facilitate the design and optimization of large-scale perfusion cultures, suitable scale-down mimics are needed which allow high-throughput experiments to be performed with minimal raw material requirements. Automated microbioreactors are available that mimic batch and fed-batch processes effectively but these have not yet been adapted for perfusion cell culture. This article describes how an automated microbioreactor system (ambr15) can be used to scale-down perfusion cell cultures using cell sedimentation as the method for cell retention. The approach accurately predicts the viable cell concentration, in the range of about 1 × 107 cells/mL for a human cell line, and cell viability of larger scale cultures using a hollow fiber based cell retention system. While it was found to underpredict cell line productivity, the method accurately predicts product quality attributes, including glycosylation profiles, from cultures performed in bioreactors with working volumes between 1 L and 1,000 L. The spent media exchange method using the ambr15 was found to predict the influence of different media formulations on large-scale perfusion cultures in contrast to batch and chemostat experiments performed in the microbioreactor system. The described experimental setup in the microbioreactor allowed an 80-fold reduction in cell culture media requirements, half the daily operator time, which can translate into a cost reduction of approximately 2.5-fold compared to a similar experimental setup at bench scale.

Rotating cylindrical filters used in perfusion cultures: CFD simulations and experiments.

The particle and fluid dynamics in a rotating cylindrical filtration (RCF) system used for animal cell retention in perfusion processes was studied. A validated CFD model was used and the results gave numerical evidence of phenomena that had been earlier claimed, but not proven for this kind of application under turbulent and high mesh permeability conditions, such as bidirectional radial exchange flow (EF) through the filter mesh and particle (cells) lateral migration. Taylor vortices were shown to cause EF 10-100 times higher than perfusion flow, indicating that EF is the main drag source, at least in early stages of RCF operation. Particle lateral migration caused a cell concentration reduction (CCR) near the filter surface of approximately 10%, contributing significantly to cell separation in RCF systems and giving evidence that the mesh sieving effect is not the sole phenomenon underlying cell retention in RCF systems. Filter rotation rate was shown to significantly affect both EF and CCR. A higher separation efficiency (measured experimentally at 2,000-L bioreactor scale) and an enhanced CCR (predicted by the numerical simulations) were found for the same rotation rate range, indicating that there is an optimal operational space with practical consequences on RCF performance. Experimental data of a large-scale perfusion run employing the simulated RCF showed high cell viabilities for over 100 days, which is probably related to the fact that the computed shear stress level in the system was shown to be relatively low (below 20 Pa under all tested conditions).

Optimizing capacity utilization by large scale 3000 L perfusion in seed train bioreactors

Increasing capacity utilization and lowering manufacturing costs are critical for pharmaceutical companies to improve their competitiveness in a challenging environment. Development of next generation cell lines, improved media formulations, application of mature technologies and innovative operational strategies have been deployed to improve yields and capacity utilization. This article describes a large-scale perfusion strategy for the N-1 seed train bioreactor that was successfully applied to achieve higher inoculation cell densities in the production culture. The N-1 perfusion at 3,000-L scale, utilizing a inclined settler, achieved cell densities of up to 158 × 10(5) cell mL(-1) at perfusion rates of 2950 L day(-1) and a retention efficiency of >85%. This approach increased inoculation cell densities and decreased cultivation times by ~20% in a CHO-based, fed-batch antibody manufacturing process while providing comparable culture performance, productivity, and product quality. The strategy therefore yielded significant increase in capacity utilization and concomitant cost improvement in a large scale cGMP facility. Details of the strategy, the cell retention device, and the cell culture performance are described in this article.

Influence of operating parameters of a settling-based perfusion process on expansion of VERO cells attached on microcarriers

Background The growing demand for biologicals produced by animal cells motivates the development of more efficient and reliable culture production processes. In the particular case of the industrial production of viral vaccines by Vero cells adhered on microcarriers, the cell propagation phases are mainly devoted to reach high cell density in less time while maintaining a good cell physiological state. Several papers have reported the optimization of culture conditions for microcarrier cultures [1-4]. Otherwise, perfusion bioreactors, based on continuous medium renewal and cell retention, can be a good alternative to batch systems. Among the various technologies for cell retention, gravitational settler is a promising device for large-scale perfusion culture process. This simple device takes advantage of the difference between the cell settling rate and the medium harvesting flow rate. Moreover, in the particular case of cells attached on microcarriers, it is more suitable than in the case of single suspended cells. So, the aim of this work was to evaluate the performances of adherent Vero cell cultures performed inside a perfused bioreactor using a gravitational settler as cell retention device. The study focused on the influence of two operating parameters, such as microcarrier concentration (MCs) and initial cell density (C0), on the cell growth.

High-performance liquid chromatographic total particles quantification of retroviral vectors pseudotyped with vesicular stomatitis virus-G glycoprotein

A novel and rapid method for the total particles quantification of murine leukemia virus derived retroviral vectors pseudotyped with vesicular stomatitis virus-G glycoprotein was developed using high performance liquid chromatography. Virus particles were detected by absorbance at 260 nm and quantified using a calibration curve generated from highly purified and concentrated viral stock characterized by negative stain electron microscopy. The method requires Benzonase ® digestion and concentration of the supernatant prior to analysis. The virus eluted in 12.55 min at a flow rate of 1 mL/min in 20 mM Tris–Cl, pH 7.4 + 1.1 M NaCl. The limits of detection and quantification of this assay were 4.71 × 10 8 and 1.57 × 10 9 viral particles/mL, respectively. Linearity was between 3.0 × 10 9 and 1.0 × 10 11 viral particles/mL with a correlation coefficient of 0.9923 and a slope of 6 × 10 −6. The assay precision was < 5% and <10% for intra- and inter-day analysis, respectively. This assay was used for the total particles quantification of a 7-day, large-scale perfusion culture production of a retroviral vector grown in 293 cells expressing the β-galactosidase gene.

Understanding factors that limit the productivity of suspension-based perfusion cultures operated at high medium renewal rates.

One of the key parameters in perfusion culture is the rate of medium replacement (D). Intensifying D results in enhanced provision of nutrients, which can lead to an increase in the viable cell density (X(v)). The daily MAb production of hybridoma cells can thus be increased proportionally without modifying the bioreactor scale, provided that both viable cell yield per perfusion rate (Y(Xv/D)) and specific MAb productivity (q(MAb)) remain constant at higher D. To identify factors prone to limit productivity in perfusion, a detailed kinetic analysis was carried out on a series of cultures operated within a D range of 0.48/4.34 vvd (volumes of medium/reactor volume/day) in two different suspension-based systems. In the Celligen/vortex-flow filter system, significant reductions in Y(Xv/D) and q(MAb) resulting from the use of gas sparging were observed at D > 1.57 vvd (X(v) > 15 x 10(6) cells/mL). Through glucose supplementation, we have shown that the decrease in Y(Xv/D) encountered in presence of sparging was not resulting from increased cellular destruction or reduced cell growth, but rather from glucose limitation. Thus, increases in hydrodynamic shear stress imparted to the culture via intensification of gas sparging resulted in a gradual increase in specific glucose consumption (q(glc)) and lactate production rates (q(lac)), while no variations were observed in glutamine-consumption rates. As a result, while glutamine was the sole limiting-nutrient under non-sparging conditions, both glutamine and glucose became limiting under sparging conditions. Although a reduction in q(MAb) was observed at high-sparging rates, inhibition of MAb synthesis did not result from direct impact of bubbles, but was rather associated with elevated lactate levels (25-30 mM), resulting from shear stress-induced increases in q(lac), q(glc), and Y(lac/glc). Deleterious effects of sparging on Y(Xv/D) and q(MAb) encountered in the Celligen/vortex-flow filter system were eliminated in the sparging-free low-shear environment of the Chemap-HRI/ultrasonic filter system, allowing for the maintenance of up to 37 x 10(6) viable cells/mL. A strategy aimed at reducing requirements for sparging in large-scale perfusion cultures by way of a reduction in the oxygen demand using cellular engineering is discussed.

Mammalian cell retention devices for stirred perfusion bioreactors.

Within the spectrum of current applications for cell culture technologies, efficient large-scale mammalian cell production processes are typically carried out in stirred fed-batch or perfusion bioreactors. The specific aspects of each individual process that can be considered when determining the method of choice are presented. A major challenge for perfusion reactor design and operation is the reliability of the cell retention device. Current retention systems include cross-flow membrane filters, spin-filters, inclined settlers, continuous centrifuges and ultrasonic separators. The relative merits and limitations of these technologies for cell retention and their suitability for large-scale perfusion are discussed.

Large-scale perfusion culture process for suspended mammalian cells that uses a centrifuge with multiple settling zones

Large-scale perfusion culture process for suspended mammalian cells that uses a centrifuge with multiple settling zones

Large-scale perfusion culture process for suspended mammalian cells that uses a centrifuge with multiple settling zones.

A high-cell-density perfusion culture process, using a novel centrifuge, was developed. The centrifuge has spiral multiple settling zones to separate cells from culture medium. Because of the multiple zones, the separation area can be efficiently increased without enlarging the diameter of the centrifuge. The centrifuge used in this study had a separation capacity of 2600 ml culture medium min -1 at 100 g of the centrifugal force. A new cell separation and withdrawal method was also developed. The cells separated in the centrifuge can be withdrawn easily from the centrifuge with no cell clogging by feeding a liquid carrier such as a perfluorocarbon into the centrifuge and pushing the cells out with the liquid carrier. By this culture process, monoclonal antibodies were produced with mouse-human hybridoma X87X at a cell density of about 8 x 10(6) cells ml -1 for 25 days. This centrifuge culture shows promise as a large-scale perfusion culture process. (Himmelfarb et al. 1969; Feder and Tolbert 1983) usually have the problem of filter clogging, and perfusion culture processes with gravitational cell settling (Tokashiki and Arai 1989) are limited in scale-up because of low separation efficiency. Recently centrifugal separation of cells from culture medium has been used for perfusion culture processes (Hamamoto et al. 1989; Tokashiki et al. 1990; Apelman 1992; Jäger 1992). This process essentially has a high separation efficiency and can avoid further operational problems, such as filter clogging, by the type of filtration. To investigate the scale-up feasibility of the centrifuge culture system, we have developed a new type of centrifuge with four layers of spiral cell-settling zones to increase the separation area, and a novel cell separation and withdrawal method using a perfluorocarbon.