Evaluation of manometric temperature measurement, a process analytical technology tool for freeze-drying: Part I, product temperature measurement

This study examines the factors that may cause systematic errors in the manometric temperature measurement (MTM) procedure used to evaluate product temperature during primary drying. MTM was conducted during primary drying using different vial loads, and the MTM product temperatures were compared with temperatures directly measured by thermocouples. To clarify the impact of freeze-drying load on MTM product temperatures, simulation of the MTM vapor pressure rise was performed, and the results were compared with the experimental results. The effect of product temperature heterogeneity in MTM product temperature determination was investigated by comparing the MTM product temperatures with directly measured thermocouple product temperatures in systems differing in temperature heterogeneity. Both the simulated and experimental results showed that at least 50 vials (5 mL) were needed to give sufficiently rapid pressure rise during the MTM data collection period (25 seconds) in the freeze dryer, to allow accurate determination of the product temperature. The product temperature is location dependent, with higher temperature for vials on the edge of the array and lower temperature for the vials in the center of the array. The product temperature heterogeneity is also dependent upon the freeze-drying conditions. In product temperature heterogeneous systems, MTM measures a temperature close to the coldest product temperature, even, if only a small fraction of the samples have the coldest product temperature. The MTM method is valid even at very low product temperature (−45°C). Published: February 10, 2006     

Evaluation of manometric temperature measurement (MTM), a process analytical technology tool in freeze drying, part III: Heat and mass transfer measurement

This article evaluates the procedures for determining the vial heat transfer coefficient and the extent of primary drying through manometric temperature measurement (MTM). The vial heat transfer coefficients (K_v) were calculated from the MTM-determined temperature and resistance and compared with K_v values determined by a gravimetric method. The differences between the MTM vial heat transfer coefficients and the gravimetric values are large at low shelf temperature but smaller when higher shelf temperatures were used. The differences also became smaller at higher chamber pressure and smaller when higher resistance materials were being freeze-dried. In all cases, using thermal shields greatly improved the accuracy of the MTM K_v measurement. With use of thermal shields, the thickness of the frozen layer calculated from MTM is in good agreement with values obtained gravimetrically. The heat transfer coefficient “error” is largely a direct result of the error in the dry layer resistance (ie, MTM-determined resistance is too low). This problem can be minimized if thermal shields are used for freeze-drying. With suitable use of thermal shields, accurate K_v values are obtained by MTM; thus allowing accurate calculations of heat and mass flow rates. The extent of primary drying can be monitored by real-time calculation of the amount of remaining ice using MTM data, thus providing a process analytical tool that greatly improves the freeze-drying process design and control.     

Hibernation of mammalian cells at a living body temperature

The present study revealed that polyphenol induces the hibernation of mammalian cells at a living body temperature. It was found that polyphenol is a cytostatic-sleeping agent for mammalian cells, where almost all cells resume proliferation after the hibernation period and cell death seldom occurs. By changing the concentration of polyphenol, various mammalian cells can be stored under different conditions, such as temporary sleep, sound sleep, and hibernation conditions.     

Rapid 3D fluorescence imaging of individual optically trapped living immune cells

We demonstrate an approach to rapidly characterize living suspension cells in 4 dimensions while they are immobilized and manipulated within optical traps. A single, high numerical aperture objective lens is used to separate the imaging plane from the trapping plane. This facilitates full control over the position and orientation of multiple trapped cells using a spatial light modulator, including directed motion and object rotation, while also allowing rapid 4D imaging. This system is particularly useful in the handling and investigation of the behavior of non‐adherent immune cells. We demonstrate these capabilities by imaging and manipulating living, fluorescently stained Jurkat T cells. (© 2015 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Optimization of spray-drying conditions for the large-scale preparation of Bacillus thuringiensis var. israelensis after downstream processing

Reduction of water activity in the formulations of mosquito biocontrol agent, Bacillus thuringiensis var. israelensis is very important for long term and successful storage. A protocol for spray drying of B. thuringiensis var. israelensis was developed through optimizing parameters such as inlet temperature and atomization type. A indigenous isolate of B. thuringiensis var. israelensis (VCRC B-17) was dried by freeze and spray drying methods and the moisture content and mosquito larvicidal activity of materials produced by the two methods were compared. The larvicidal activity was checked against early fourth instars Aedes aegypti larvae. Results showed that the freeze-dried powders retained the larvicidal activity fairly well. The spray-dried powder moderately lost its larvicidal activity at different inlet temperatures. Between the two types of atomization, centrifugal atomization retained more activity than the nozzle type atomization. Optimum inlet temperature for both centrifugal and nozzle atomization was 160 degrees C. Keeping the outlet temperature constant at 70 degrees C the moisture contents for the spray-dried powders through centrifugal atomization and freeze-dried powders were 10.23% and 11.80%, respectively. The LC(50) values for the spray-dried and freeze-dried powders were 17.42 and 16.18 ng/mL, respectively. Spore count of materials before drying was 3 x 10(10) cfu/mL and after spray drying through nozzle and centrifugal atomization at inlet and outlet temperature of 160 degrees C/70 degrees C were 2.6 x 10(9) and 5.0 x 10(9) cfu/mL, respectively.     

The future is cold: cryo-preparation methods for transmission electron microscopy of cells

Our knowledge of the organization of the cell is linked, to a great extent, to light and electron microscopy. Choosing either photons or electrons for imaging has many consequences on the image obtained, as well as on the experiment required in order to generate the image. One apparent effect on the experimental side is in the sample preparation, which can be quite elaborate for electron microscopy. In recent years, rapid freezing, cryo-preparation and cryo-electron microscopy have been more widely used because they introduce fewer artefacts during preparation when compared with chemical fixation and room temperature processing. In addition, cryo-electron microscopy allows the visualization of the hydrated specimens. In the present review, we give an introduction to the rapid freezing of biological samples and describe the preparation steps. We focus on bulk samples that are too big to be directly viewed under the electron microscope. Furthermore, we discuss the advantages and limitations of freeze substitution and cryo-electron microscopy of vitreous sections and compare their application to the study of bacteria and mammalian cells and to tomography.     

Deep supercooling enabled by surface impregnation with lipophilic substances explains the survival of overwintering buds at extreme freezing

The frost survival mechanism of vegetative buds of angiosperms was suggested to be extracellular freezing causing dehydration, elevated osmotic potential to prevent freezing. However, extreme dehydration would be needed to avoid freezing at the temperatures down to ?45°C encountered by many trees. Buds of Alnus alnobetula, in common with other frost hardy angiosperms, excrete a lipophilic substance, whose functional role remains unclear. Freezing of buds was studied by infrared thermography, psychrometry, and cryomicroscopy. Buds of A. alnobetula did not survive by extracellular ice tolerance but by deep supercooling, down to ?45°C. An internal ice barrier prevented ice penetration from the frozen stem into the bud. Cryomicroscopy revealed a new freezing mechanism. Until now, supercooled buds lost water towards ice masses that form in the subtending stem and/or bud scales. In A. alnobetula, ice forms harmlessly inside the bud between the supercooled leaves. This would immediately trigger intracellular freezing and kill the supercooled bud in other species. In A. alnobetula, lipophilic substances (triterpenoids and flavonoid aglycones) impregnate the surface of bud leaves. These prevent extrinsic ice nucleation so allowing supercooling. This suggests a means to protect forestry and agricultural crops from extrinsic ice nucleation allowing transient supercooling during night frosts.     

Improving AAV vector yield in insect cells by modulating the temperature after infection

Vectors based on adeno-associated viruses (AAV) are sought for therapeutic gene delivery because of their ability to transduce a variety of tissues with no significant immunological response. Production using the baculovirus expression vector (BEV)/insect cell system has the potential to meet the needs for pre-clinical and clinical trials. In this co-infection system, three baculoviruses are used to produce the AAV vector. A strategy aimed at increasing encapsidation/maturation of the viral vector involved varying the temperature over the course of the process. Cultures were subjected to temperature changes at various times pre- and post-infection (up to 24 h post-infection). It was found that raising the culture temperature to 30 degrees C at the time of infection nearly tripled the infectious titer. In fact, increasing the temperature to 30 degrees C at any time in the process investigated resulted in an increase in titer. Also, raising the culture to 33 degrees C or lowering the temperature to 24 degrees or 21 degrees C resulted in lower titers. The rise in infectious titer was also confirmed by an increase in DNase resistant particles (DRPs). Varying the temperature, however, did not affect the total amount of capsids significantly. Therefore increasing the culture temperature resulted in better encapsidation as determined by the ratio of capsids to DRPs to infectious particles. It is believed that an increase in early proteins and possibly a quicker cascade of baculovirus infection events resulted in this increased packaging efficiency.     

The fate of Pluronic F-68 in chondrocytes and CHO cells

The surfactant Pluronic F-68 (PF-68) is widely used in large-scale mammalian cell culture to protect cells from shear stress that arises from agitation and gas sparging. Several studies suggested that PF-68 is incorporated into the cell plasma membrane and could enter the cells, but without providing any direct evidence. The current study has examined this question for two cell types, one of pharmaceutical interest (CHO cells) and the other of biomedical interest (chondrocytes or cartilage cells). A fluorescent derivative of PF-68 was synthesized to detect and localize internalized Pluronic with culture time. PF-68 uptake by the cells was quantified and characterized. We clearly demonstrate that PF-68 enters the cells, and possibly accumulates in the endocytic pathway. CHO cells showed an average uptake of 11.7 +/- 6.7 (SEM) microg PF-68/10(6) cells while the uptake of chondrocytes was 56.0 +/- 10.9 (SEM) microg PF-68/10(6) cells, independently of the initial PF-68 concentration (between 0.01 and 0.2%, w/v) and of cell concentration (from 1 x 10(6) to 4 x 10(6) cells/mL). These uptake values were identical for both static and agitated culture conditions. Finally, we found that CHO cells are able to eliminate intracellular fluorescent PF-68 but chondrocytes are not. These results show that the uptake of PF-68 by the cells can severely affect PF-68 concentration in the culture medium and thus shear protection effect.     

A noninvasive technique for the measurement of the energetic state of free‐suspension mammalian cells

A perfusion small‐scale bioreactor allowing on‐line monitoring of the cell energetic state was developed for free‐suspension mammalian cells. The bioreactor was designed to perform in vivo nuclear magnetic resonance (NMR) spectroscopy, which is a noninvasive and nondestructive method that permits the monitoring of intracellular nutrient concentrations, metabolic precursors and intermediates, as well as metabolites and energy shuttles, such as ATP, ADP, and NADPH. The bioreactor was made of a 10‐mm NMR tube following a fluidized bed design. Perfusion flow rate allowing for adequate oxygen supply was found to be above 0.79 mL min^?1 for high‐density cell suspensions (10⁸ cells). Chinese hamster ovary (CHO) cells were studied here as model system. Hydrodynamic studies using coloration/decoloration and residence time distribution measurements were realized to perfect bioreactor design as well as to determine operating conditions bestowing adequate homogeneous mixing and cell retention in the NMR reading zone. In vivo ³¹P NMR was performed and demonstrated the small‐scale bioreactor platform ability to monitor the cell physiological behavior for 30‐min experiments. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

A simple boronic acid-based fluorescent probe for selective detection of hydrogen peroxide in solutions and living cells

An approach of high sensitivity and selectivity for hydrogen peroxide (H₂O₂) detection is highly demanded due to its important roles in regulating diverse biological process. In this work, we introduced an easily synthesized fluorescent “turn off” probe, BNBD. It is designed based on the core structure of 4-chloro-7-nitrobenzofurazan as a fluorophore and incorporated with a specific H₂O₂-reactive group, aryl boronate, for sensitive and selective detection of H₂O₂. We demonstrated its selectivity by incubating the probe with other types of ROS, and measured the limit of detection of BNBD as 1.8 nM. BNBD is also conducive to H₂O₂ detection at physiological conditions. We thus applied it to detect both exogenous and endogenous changes of H₂O₂ in living cells by confocal microscopy, supporting its future applications to selectively monitor H₂O₂ levels and identify H₂O₂-related physiological or pathological responses from live cells or tissues in the near future.     

Increase in macrophages in the testis of cathepsin a deficient mice suggests an important role for these cells in the interstitial space of this tissue

Cathepsin A (PPCA) is a lysosomal carboxypeptidase that functions as a protective protein for alpha-neuraminidase and beta-galactosidase in a multienzyme complex. In the present study, the testes of PPCA -/- mice from 2 to 10 months of age were compared with those of their wild type counterparts. While germ and Sertoli cells appeared comparable in appearance and distribution, the mean profile area of seminiferous tubules showed a significant decrease between wild type and PPCA -/- mice, suggesting changes to the seminiferous tubules and their contents. In addition, macrophages in the interstitial space (IS) of PPCA -/- mice were large, spherical, and filled with pale lysosomes, unlike those seen in wild type mice, and a quantitative analysis of their frequency per unit area of IS in PPCA -/- mice revealed a significant increase compared to that of wild type mice; this was also the case for their mean profile area. Absence of mitotic figures, cycling cells, or degenerating figures in the IS suggests that the major recruitment of macrophages appears to be from the circulation. In the IS, Leydig cells also showed an accumulation of large pale lysosomes in PPCA -/- mice, and their frequency also increased significantly as compared to wild type mice. In the electron microscope, a close association of Leydig cell microvilli with the surface of macrophages was pronounced in PPCA -/- mice. Since macrophages and Leydig cells interact by secreting various factors between each other, and considering the fact that Leydig cells show an accumulation of large pale lysosomes in PPCA -/- mice, it is suggested that macrophages accumulate as a result of abnormalities occurring in Leydig cells. Taken together, the data on increase in frequency of macrophages suggests important functions for these cells in both wild type and PPCA -/- mice.     

Media for cultivation of animal cells: an overview

The increasing interest in products from animal cells has caused an extensive research effort towards development of media for cell cultivation.The basic components in the media used for cultivation of animal cells vary depending upon the characters of the cells and the cultivation method. Basic components consist of an energy source, nitrogen source, vitamins, fats and fatty soluble components, inorganic salts, nucleic acid precursors, antibiotics, oxygen, pH buffering systems, hormones, growth factors and serum. Extensive efforts are directed towards developing serum-free or chemically defined media. Among the serum substitutes is a long list of hormones and growth factors.     

Metabolism of Klebsiella pneumoniae freeze‐dried cultures for the design of BOD bioassays

Aims: The survival rate of freeze‐dried cultures is not enough information for technological applications of micro‐organisms. There could be serious metabolic/structural damage in the survivors, leading to a delay time that can jeopardize the design of a rapid biochemical oxygen demand (BOD) metabolic‐based bioassay. Therefore, we will study the metabolic activity (as ferricyanide reduction activity) and the survival rate (as colony‐forming units, CFU) of different Klebsiella pneumoniae freeze‐dried cultures looking for stable metabolic conditions after 35 days of storage. Method and Results: Here, we tried several simple freeze‐drying processes of Kl. pneumoniae. Electrochemical measurements of ferrocyanide and survival rates obtained with the different freeze‐dried cultures were used to choose the best freeze‐drying process that leads to a rapid metabolic‐based bioassay. Conclusions: The use of milk plus monosodium glutamate was the best choice to obtain a Kl. pneumoniae freeze‐dried culture with metabolic stable conditions after storage at ?20°C without the need of vacuum storage and ready to use after 20 min of rehydration. We also demonstrate that the viability and the metabolic activity are not always directly correlated. Significance and Impact of the Study: This study shows that the use of this Kl. pneumoniae freeze‐dried culture is appropriate for the design of a rapid BOD bioassay.     

A transient decrease of electrochemical gradient stabilizes DNA structural change in single mitochondria of living cells

The effect of controlled and reversible perturbation of the electrochemical gradient on the structural changes of mitochondrial DNA has been studied in living cells by fluorescence microscopy. Electrochemical gradient perturbations were induced by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and quantified by measuring the mitochondrial membrane potential using tetramethyl rhodamine methyl ester. Under our experimental conditions, we have shown that ethidium fluorescence was mainly due to ethidium molecules intercalated in mtDNA. Ethidium fluorescence variations have been used to probe DNA structural changes. This showed that: i) electrochemical gradient perturbations induced mtDNA structural change; ii) this change was readily reversible following a total but short collapse of the electrochemical gradient; iii) in contrast, a short and weak perturbation of the electrochemical gradient stabilized the mtDNA structural change; and iv) the degree of weak depolarization varied from cell to cell, showing the necessity of studying the effect of energetic perturbations at the level of an individual cell.