A pumpless perfusion cell culture cap with two parallel channel layers keeping the flow rate constant

This article presents a novel pumpless perfusion cell culture cap, the gravity‐driven flow rate of which is kept constant by the height difference of two parallel channel layers. Previous pumpless perfusion cell culture systems create a gravity‐driven flow by means of the hydraulic head difference (Δh) between the source reservoir and the drain reservoir. As more media passes from the source reservoir to the drain reservoir, the source media level decreases and the drain media level increases. Thus, previous works based on a gravity‐driven flow were unable to supply a constant flow rate for the perfusion cell culture. However, the proposed perfusion cell culture cap can supply a constant flow rate, because the media level remains unchanged as the media moves laterally through each channel having same media level. In experiments, using the different fluidic resistances, the perfusion cap generated constant flow rates of 871 ± 27 μL h^?1 and 446 ± 11 μL h^?1. The 871 and 446 μL h^?1 flow rates replace the whole 20 mL medium in the petridish with a fresh medium for days 1 and 2, respectively. In the perfusion cell (A549 cell line) culture with the 871 μL h^?1 flow rate, the proposed cap can maintain a lactate concentration of about 2200 nmol mL^?1 and an ammonia concentration of about 3200 nmol mL^?1. Moreover, although the static cell culture maintains cell viability for 5 days, the perfusion cell culture with the 871 μL h^?1 flow rate can maintain cell viability for 9 days. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Repurposing fed-batch media and feeds for highly productive CHO perfusion processes

Perfusion is a cell culture mode that is gaining popularity for the manufacture of monoclonal antibodies and their derivatives. The cell culture media supporting perfusion culture need to support higher cell densities than those used in fed-batch culture. Therefore, when switching from a fed-batch to a perfusion mode, a new medium need to be developed which supports high cell densities, high productivity, and favorable product quality. We have developed a method for deriving perfusion culture media based on existing fed-batch media and feeds. We show that we can obtain culture media that successfully support perfusion cultures in a single-use rocking bioreactor system at cell-specific perfusion rates below 25 pL⁻¹ cell⁻¹ day⁻¹. High productivities and favorable product quality are also achievable.     

Rapid development of clone-specific,high-performing perfusion media from established feed supplements

Perfusion cultivation of recombinant CHO cells is of substantial interest to the biopharmaceutical industry. This is due to increased space–time-yields (STYs) and a short residence time of the recombinant protein in the bioreactor. Economic processes rely on cultivation media supporting rapid growth in the exponential phase and high protein production in the stationary phase at minimal media consumption rates. To develop clone-specific, high-performing perfusion media we present a straightforward and rapid two-step approach combining commercially available basal media and feed supplements using design-of-experiment. First, the best performing feed supplements are selected in batch cultures. Then, the mixing ratio of selected feed supplements is optimized in small-scale semicontinuous perfusion cultures. The final media formulation is supported by statistical response surface modeling of a set of cultivation experiments with blended media formulations. Two best performing novel media blends were finally applied to perfusion bioreactor verification runs to reach 200 × 10⁶ c/ml within 2 weeks at minimum cell-specific perfusion rates as low as 10–30 pL/c/d. Obtained STYs of 0.4–1.2 g/L/d represent a 10-fold increase compared to batch cultures. This general workflow is universally applicable to any perfusion platform combining a specific cell line, basal medium, and established feed solutions.     

An Isolated Working Heart System for Large Animal Models

Since its introduction in the late 19^th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart model, have been invaluable tools for studying cardiovascular function and disease^1-15. Although the Langendorff heart preparation can be used for any mammalian heart, most studies involving this apparatus use small animal models (e.g., mouse, rat, and rabbit) due to the increased complexity of systems for larger mammals^1,3,11. One major difficulty is ensuring a constant coronary perfusion pressure over a range of different heart sizes – a key component of any experiment utilizing this device^1,11. By replacing the classic hydrostatic afterload column with a centrifugal pump, the Langendorff working heart apparatus described below allows for easy adjustment and tight regulation of perfusion pressures, meaning the same set-up can be used for various species or heart sizes. Furthermore, this configuration can also seamlessly switch between constant pressure or constant flow during reperfusion, depending on the user’s preferences. The open nature of this setup, despite making temperature regulation more difficult than other designs, allows for easy collection of effluent and ventricular pressure-volume data.     

A cytochemical study of the calcium-activated adenosinetriphosphatase in hamster adrenal medulla: its occurrence in the golgi region of chromaffin cells

Summary Several different fixation procedures and incubation media were used in order to demonstrate the ultrastructural localisation of Ca²⁺-activated adenosinetriphosphatase (ATPase) in the hamster adrenal medulla. Fixation by perfusion with 2.5% glutaraldehyde gave the best preservation of fine structure without markedly inhibiting the enzymic activity. The localisation of Ca²⁺-activated ATPase was different from that of Mg²⁺-activated ATPase: the Mg²⁺-dependent enzyme was confined to plasma membranes. Ca²⁺-dependent ATPase also occurred on the plasma membranes of neurons and of some chromaffin cells, but the most prominent site of this enzyme was in the Golgi apparatus of chromaffin cells. Most of the reaction product was localised between Golgi lamellae, but some was found in Golgi vesicles and in prosecretory granules. The nucleus, mature chromaffin granules, roughsurfaced endoplasmic reticulum and mitochondria were usually free of reaction product. Rarely, some precipitate was found in the matrix of mitochondria and in lysosomes.Wellcome Research Fellow.J. H. Burn Research Scholar.This work was supported by a grant from the Medical Research Council.     

Measurement of diffusion coefficient and electrophoretic mobility with a quasielastic light-scattering–band-electrophoresis apparatus

We have constructed an apparatus for the simultaneous measurement of electrophoretic mobility, μ, and diffusion coefficient, D, of macromolecules and cells. It combines band electrophoresis in a vertical, sucrose-gradient stabilized column, with quasielastic laser light-scattering determination of the diffusion coefficient of the species within the band. The entire electrophoresis cell is scanned through the laser beam of the quasielastic laser light-scattering apparatus by a vertical translation stage. Total intensity light-scattering measurement at each point in the cell gives the macromolecular concentration at that point. Solvent viscosity and electrical potential are measured at each point in the cell. Application of this apparatus to resealed red blood cell ghosts and to bovine hemoglobin indicates that measurements of field, viscosity, and migration distance are reliable, and that electroosmosis is insignificant. Application to T4D bacteriophage gives μ_20,w = (?1.05 ± 0.05) × 10^?4 cm²/V sec and D_20,w = (3.35 ± 0.10) × 10^?8 cm²/sec for fiberless particles, and μ_20,w = ?(0.59 ± 0.03) × 10^?4 cm²/V sec and D_20,w = (2.86 ± 0.09) × 10^?8 cm²/sec for whole phage with 6 fibers. Approximate analysis of these results with the Henry electrophoresis theory for spheres in dicates that each fiber contributes about 193 positive charges to the phage particle, compared with 327 from amino-acid analysis. The advantages and disadvantages of this apparatus, relative to conventional electrophoresis and to electrophoretic light scattering, are discussed.     

Differential MR Delayed Enhancement Patterns of Chronic Myocardial Infarction between Extracellular and Intravascular Contrast Media

Objectives

Because the distribution volume and mechanism of extracellular and intravascular MR contrast media differ considerably, the enhancement pattern of chronic myocardial infarction with extracellular or intravascular media might also be different. This study aims to investigate the differences in MR enhancement patterns of chronic myocardial infarction between extracellular and intravascular contrast media.

Materials and Methods

Twenty pigs with myocardial infarction underwent cine MRI, first pass perfusion MRI and delayed enhancement MRI with extracellular or intravascular media at four weeks after coronary occlusion. Myocardial blood flow (MBF) was determined with microsphere measurement. The infarction histopathological changes were evaluated by hematoxylin and eosin staining and Masson''s trichrome method.

Results

Cine MRI revealed the reduced wall thickening in chronic infarction compared with normal myocardium. Moreover, significant wall thinning in chronic infarction was observed in cine MRI. Peak first-pass signal intensity didn’t significantly differ between chronic infarction and normal myocardium no matter what kinds of contrast media. At the following delayed enhancement phase, extracellular media-enhanced signal intensity was significantly higher in chronic infarction than in normal myocardium. Conversely, intravascular media-enhanced signal intensity was almost equivalent among chronic infarction and normal myocardium. At four weeks after infarction, MBF in chronic infarction approached to that in normal myocardium. Large thick-walled vessels were detected at peri-infarction zones. The cardiomyocytes were replaced by scar tissue consisting of dilated blood vessels and discrete fibers of collagen.

Conclusions

Chronic infarction was characterized by the significantly reduced wall thickening and the definite wall thinning. First-pass myocardial perfusion defect was not detected in chronic infarction with two media due to the significantly recovered MBF and well-developed collateral vessels. Infarction remodeling enlarged the extracellular compartment, which was available for extracellular media but not accessible to intravascular media. Extracellular media identified chronic infarction as the hyper-enhancement; nonetheless, intravascular media didn’t provide delayed enhancement.     

The isolation of hormone-sensitive rat hepatocytes by a modified enzymatic technique

Hepatocytes that are similar to the perfused liver in glucagon sensitivity can be obtained in a high, reproducible yield by modifications of the well-known enzymatic technique for the preparation of isolated liver cells. The major modifications are: (a) a simple, economic, and temperature-controlled apparatus for the recirculating perfusion of the isolated rat liver; (b) the use of substrate-fortified calcium-free Krebs-Henseleit bicarbonate buffer; and (c) high perfusion rates, which lead to the isolation of hepatocytes with normal ultrastructure and metabolic activities.From 4 × 10⁸ to 5 × 10⁸ cells can be routinely isolated from an 8- to 10-g liver independent of the collagenase preparations applied. The rat liver cells are viable (90–95%) by various criteria including electron microscopy and exclusion of 0.2% trypan blue. When studying various incubation techniques, it was observed that the use of gelatin in the medium is preferred as compared to albumin Fraction V or fatty acid-free albumin which tended to inhibit gluconeogenic rates from various substrates in calcium-free medium. Addition of calcium chloride to the incubation medium strikingly improved gluconeogenesis from lactate. Various procedures for calculating the number of cells corresponding to 1 g wet liver tissue are discussed in detail.     

High density culture of mouse-human hybridoma cells using a perfusion culture apparatus with multi-settling zones to separate cells from the culture medium

Mouse-human hybridoma X87X cells were cultivated using a novel perfusion culture apparatus provided with three-settling zones to separate the cells from the culture medium by gravitational settling. The maximum viable cell density in a serum-free culture medium attained 3.0×10⁷ cells/ml, when the specific perfusion rate was set to 2.3 vol day^-1, and monoclonal antibody was continuously produced. These results were almost the same as those in the perfusion culture vessel with one settling zone and revealed that the process with a plurality of settling zones is a promising one for scale-up of a gravitation type of perfusion culture vessel.     

Entrance of water into human red cells under an osmotic pressure gradient

A new technique to determine the rate of water passage through the membrane of the human erythrocyte under an osmotic gradient has been developed. It utilizes a rapid mixing apparatus of the Hartridge-Roughton type which permits measurements at short intervals after the reaction has begun. This is coupled with a light-scattering device of new design which permits the determination of very small changes in volume of the cells without disturbing them. With this technique it was possible to measure the change in volume of freshly drawn human erythrocytes after about 50, 100, 155, and 215 msec. of exposure to anisotonic media. The experimental curves were compared with theoretical curves derived from accepted equations for the process and a permeability coefficient of 0.23 ± 0.03 (cm.⁴/osm., sec.) was obtained.     

Measurement of the electrode dispersion in very low frequency DNA solutions

Based on the four electrodes technique, an apparatus is described which measures the Very Low Frequency (VLF) conductivity of ionic solutions, all electrode effects being completely eliminated. It is thus possible to measure the conductivity frequency dependence between 0.8 and 500 cps, with a relative error of 10^?4. Applying this method to DNA solutions, one always finds a conductivity dispersion in the VLF range, which disappears when the biopolymer is heat-denatured. The relaxation time is different from one solution to another, but is always greater than 10 msee. approximately, sometimes even greater than 0.1 sec., the upper limit which one can estimate with our apparatus. The different, explanations of the DNA very low frequency polarization, assuming that its relaxation is connected with the rotational diffusion of the biopolymer long axis, is discussed.     

Optimization and control of perfusion cultures using a viable cell probe and cell specific perfusion rates

Consistent perfusion culture production requires reliable cell retention and control of feed rates. An on-line cell probe based on capacitance was used to assay viable biomass concentrations. A constant cell specific perfusion rate controlled medium feed rates with a bioreactor cell concentration of ∼5 × 10⁶ cells mL^-1. Perfusion feeding was automatically adjusted based on the cell concentration signal from the on-line biomass sensor. Cell specific perfusion rates were varied over a range of 0.05 to 0.4 nL cell^-1 day^-1. Pseudo-steady-state bioreactor indices (concentrations, cellular rates and yields) were correlated to cell specific perfusion rates investigated to maximize recombinant protein production from a Chinese hamster ovary cell line. The tissue-type plasminogen activator concentration was maximized (∼40 mg L^-1) at 0.2 nL cell^-1 day^-1. The volumetric protein productivity (∼60 mg L^-1 day^-1 was maximized above 0.3 nL cell^-1 day^-1. The use of cell specific perfusion rates provided a straightforward basis for controlling, modeling and optimizing perfusion cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Magnetic Resonance Imaging Cooling-Reheating Protocol Indicates Decreased Fat Fraction via Lipid Consumption in Suspected Brown Adipose Tissue

Objectives

To evaluate whether a water-fat magnetic resonance imaging (MRI) cooling-reheating protocol could be used to detect changes in lipid content and perfusion in the main human brown adipose tissue (BAT) depot after a three-hour long mild cold exposure.

Materials and Methods

Nine volunteers were investigated with chemical-shift-encoded water-fat MRI at baseline, after a three-hour long cold exposure and after subsequent short reheating. Changes in fat fraction (FF) and R₂*, related to ambient temperature, were quantified within cervical-supraclavicular adipose tissue (considered as suspected BAT, denoted sBAT) after semi-automatic segmentation. In addition, FF and R₂* were quantified fully automatically in subcutaneous adipose tissue (not considered as suspected BAT, denoted SAT) for comparison. By assuming different time scales for the regulation of lipid turnover and perfusion in BAT, the changes were determined as resulting from either altered absolute fat content (lipid-related) or altered absolute water content (perfusion-related).

Results

sBAT-FF decreased after cold exposure (mean change in percentage points = -1.94 pp, P = 0.021) whereas no change was observed in SAT-FF (mean = 0.23 pp, P = 0.314). sBAT-R₂* tended to increase (mean = 0.65 s^-1, P = 0.051) and SAT-R₂* increased (mean = 0.40 s^-1, P = 0.038) after cold exposure. sBAT-FF remained decreased after reheating (mean = -1.92 pp, P = 0.008, compared to baseline) whereas SAT-FF decreased (mean = -0.79 pp, P = 0.008, compared to after cold exposure).

Conclusions

The sustained low sBAT-FF after reheating suggests lipid consumption, rather than altered perfusion, as the main cause to the decreased sBAT-FF. The results obtained demonstrate the use of the cooling-reheating protocol for detecting changes in the cervical-supraclavicular fat depot, being the main human brown adipose tissue depot, in terms of lipid content and perfusion.     

Neuropsychological Correlates of Brain Perfusion SPECT in Patients with Macrophagic Myofasciitis

Background

Patients with aluminum hydroxide adjuvant-induced macrophagic myofasciitis (MMF) complain of arthromyalgias, chronic fatigue and cognitive deficits. This study aimed to characterize brain perfusion in these patients.

Methods

Brain perfusion SPECT was performed in 76 consecutive patients (aged 49±10 y) followed in the Garches-Necker-Mondor-Hendaye reference center for rare neuromuscular diseases. Images were acquired 30 min after intravenous injection of 925 MBq ^99mTc-ethylcysteinate dimer (ECD) at rest. All patients also underwent a comprehensive battery of neuropsychological tests, within 1.3±5.5 mo from SPECT. Statistical parametric maps (SPM12) were obtained for each test using linear regressions between each performance score and brain perfusion, with adjustment for age, sex, socio-cultural level and time delay between brain SPECT and neuropsychological testing.

Results

SPM analysis revealed positive correlation between neuropsychological scores (mostly exploring executive functions) and brain perfusion in the posterior associative cortex, including cuneus/precuneus/occipital lingual areas, the periventricular white matter/corpus callosum, and the cerebellum, while negative correlation was found with amygdalo-hippocampal/entorhinal complexes. A positive correlation was also observed between brain perfusion and the posterior associative cortex when the time elapsed since last vaccine injection was investigated.

Conclusions

Brain perfusion SPECT showed a pattern of cortical and subcortical changes in accordance with the MMF-associated cognitive disorder previously described. These results provide a neurobiological substrate for brain dysfunction in aluminum hydroxide adjuvant-induced MMF patients.     

Critical Parameters of the In Vitro Method of Vascular Smooth Muscle Cell Calcification

Background

Vascular calcification (VC) is primarily studied using cultures of vascular smooth muscle cells. However, the use of very different protocols and extreme conditions can provide findings unrelated to VC. In this work we aimed to determine the critical experimental parameters that affect calcification in vitro and to determine the relevance to calcification in vivo.

Experimental Procedures and Results

Rat VSMC calcification in vitro was studied using different concentrations of fetal calf serum, calcium, and phosphate, in different types of culture media, and using various volumes and rates of change. The bicarbonate content of the media critically affected pH and resulted in supersaturation, depending on the concentration of Ca²⁺ and Pi. Such supersaturation is a consequence of the high dependence of bicarbonate buffers on CO₂ vapor pressure and bicarbonate concentration at pHs above 7.40. Such buffer systems cause considerable pH variations as a result of minor experimental changes. The variations are more critical for DMEM and are negligible when the bicarbonate concentration is reduced to ¼. Particle nucleation and growth were observed by dynamic light scattering and electron microscopy. Using 2mM Pi, particles of ~200nm were observed at 24 hours in MEM and at 1 hour in DMEM. These nuclei grew over time, were deposited in the cells, and caused osteogene expression or cell death, depending on the precipitation rate. TEM observations showed that the initial precipitate was amorphous calcium phosphate (ACP), which converts into hydroxyapatite over time. In blood, the scenario is different, because supersaturation is avoided by a tightly controlled pH of 7.4, which prevents the formation of PO₄ ^3--containing ACP.

Conclusions

The precipitation of ACP in vitro is unrelated to VC in vivo. The model needs to be refined through controlled pH and the use of additional procalcifying agents other than Pi in order to reproduce calcium phosphate deposition in vivo.     

Interferometric Studies of Growth Kinetics and Surface Morphology in Macromolecular Crystal Growth: Canavalin, Thaumatin, and Turnip Yellow Mosaic Virus

In situ laser Michelson interferometry was utilized to investigate mechanisms of growth and surface morphology in protein and virus crystallization, These included plant proteins canavalin and thaumatin and turnip yellow mosaic virus. The experimental apparatus allowed us to obtain interferometric patterns and investigate growth kinetics from growing macromolecular crystals as small as 20 μm. For the crystallization of canavalin, dislocations are the sources of growth steps on the surfaces. Supersaturation and time dependencies of the normal growth rates, tangential growth step velocities, and the slopes of the dislocation hillocks were measured. The kinetic coefficient β (rate of incorporation of protein molecules into the growing crystal) was estimated for canavalin to be 9 × 10^-4 cm/sec. This is among the first estimates of such fundamental kinetic parameters for macromolecular crystallization. The change in the activities of dislocation sources under different growth conditions was also analyzed. Michelson interferometry was clearly demonstrated to be a useful tool for quantitative studies of macromolecular crystal growth.     

Experience in scale-up of homogeneous perfusion culture for hybridomas

A perfusion system for production of monoclonal antibodies was developed using an externally-mounted, hollow-fibre cartridge. The experimental apparatus was operated for 420 h and demonstrated increased steady-state viable cell concentration with increase in perfusion rate. Antibody titres were up to three times those measured for batch cultures and specific antibody productivity was doubled.The procedure was successfully scaled to a 10 dm³ system which produced antibody under conditions of Good Manufacturing Practice (GMP). A calculation of productivity between the scaled perfusion system and 260 dm³ batch cultures resulted in comparable antibody production, whereas the perfusion allowed a halving in medium utilisation. Reactivity assays conducted on the purified antibody from both batch and perfusion cultures showed no evidence of proteolysis or altered antibody activity in the final perfusion product. This study provides additional support for the use of homogeneous perfusion cultures in production of monoclonal antibodies under GMP conditions.     

Peptone,a low-cost growth-promoting nutrient for intensive animal cell culture

The effect of addition of peptone to serum-free and serum supplemented media for the growth of hybridoma cells in various systems was studied. Supplementation of defined medium with either proteose peptone or meat peptone resulted in significant increases in cell number and specific monoclonal antibody production in batch culture system. Other peptones were either inactive or less effective. In continuous culture, using medium supplemented with new born calf serum, the addition of peptone resulted in 125% and 150% increases in cell and antibody concentrations respectively. Similar increase in cell number (128%) was also obtained in spin-filter perfusion culture when medium was supplemented with peptone. By comparison, the substitution of a defined 1xMEM amino acids mixture resulted in only a 50% increase. At higher perfusion rates the cell number maintained in steady state using peptone supplement could be increased to 1.3×10⁷ cells ml^–1 while the serum concentration was reduced from 5% to 1% at a perfusion rate of 2.5 volumes per day.     

A comparison of different culture methods for hybridoma propagation and monoclonal antibody production

A major variable to consider in the production of biologicals from mammalian cell cultures is the mode of operation, be it a batch, continuous, perfusion, fed-batch or other production method. The final choice must consider a number of fundamental and economic issues. Here we present some antibody production data from different cell lines using different modes of production and discuss the important factors for consideration in choosing a production strategy. It was found that the productivity of batch cultures was lower than that obtained in continuous and perfused cultures, but that productivity could be improved by implementing suitable feeding strategies. The antibody productivity of one cell line, MCL1, during exponential phase was not affected by media type or glucose level. The maximum productivity of two cell lines in continuous culture was found to occur at dilution rates below the maximum, from 0.019 to 0.030 hr^–1.     

The Nuclear Pore Complex Function of Sec13 Protein Is Required for Cell Survival during Retinal Development

Sec13 is a dual function protein, being a core component of both the COPII coat, which mediates protein trafficking from the endoplasmic reticulum to the Golgi apparatus, and the nuclear pore complex (NPC), which facilitates nucleo-cytoplasmic traffic. Here, we present a genetic model to differentiate the roles of these two functions of Sec13 in vivo. We report that sec13^sq198 mutant embryos develop small eyes that exhibit disrupted retinal lamination and that the mutant retina contains an excessive number of apoptotic cells. Surprisingly, we found that loss of COPII function by oligonucleotide-mediated gene knockdown of sec31a and sec31b or brefeldin A treatment did not disrupt retinal lamination, although it did result in digestive organ defects similar to those seen in sec13^sq198, suggesting that the digestive organ defects observed in sec13^sq198 are due to loss of COPII function, whereas the retinal lamination defects are due to loss of the NPC function. We showed that the retinal cells of sec13^sq198 failed to form proper nuclear pores, leading to a nuclear accumulation of total mRNA and abnormal activation of the p53-dependent apoptosis pathway, causing the retinal defect in sec13^sq198. Furthermore, we found that a mutant lacking Nup107, a key NPC-specific component, phenocopied the retinal lamination phenotype as observed in sec13^sq198. Our results demonstrate a requirement for the nuclear pore function of Sec13 in development of the retina and provide the first genetic evidence to differentiate the contributions of the NPC and the COPII functions of Sec13 during organogenesis.