A device for aeration and mixing of cell and organelle suspensions during nuclear magnetic resonance studies

A device is described which maintains homogeneous aerobic or anaerobic cell and organelle suspensions within an NMR sample tube. Line broadening due to magnetic field inhomogeneity is reduced by elimination of gas bubbles from the area of the probe receiver coils. The linewidth of the extracellular orthophosphate resonance of a yeast suspension in 31P NMR was 0.21 ppm compared with 0.4-0.7 ppm in conventionally aerated suspensions. Recirculation of the sample results in complete mixing within 90 s of addition of aliquots of acid or alkali. The maximum rate of oxygen transfer from the gaseous to the liquid phase was approximately 600 microM min-1 when aerated with 95% oxygen/5% carbon dioxide. A 60% wet weight suspension of yeast cells was recirculated for 20 h without settling of cells occurring. A method for estimating oxygen transfer rate is described.     

Microconstriction Arrays for High-Throughput Quantitative Measurements of Cell Mechanical Properties

We describe a method for quantifying the mechanical properties of cells in suspension with a microfluidic device consisting of a parallel array of micron-sized constrictions. Using a high-speed charge-coupled device camera, we measure the flow speed, cell deformation, and entry time into the constrictions of several hundred cells per minute during their passage through the device. From the flow speed and the occupation state of the microconstriction array with cells, the driving pressure across each constriction is continuously computed. Cell entry times into microconstrictions decrease with increased driving pressure and decreased cell size according to a power law. From this power-law relationship, the cell elasticity and fluidity can be estimated. When cells are treated with drugs that depolymerize or stabilize the cytoskeleton or the nucleus, elasticity and fluidity data from all treatments collapse onto a master curve. Power-law rheology and collapse onto a master curve are predicted by the theory of soft glassy materials and have been previously shown to describe the mechanical behavior of cells adhering to a substrate. Our finding that this theory also applies to cells in suspension provides the foundation for a quantitative high-throughput measurement of cell mechanical properties with microfluidic devices.     

Automated Needle‐free Injection Method for Delivery of Bacterial Suspensions into Citrus Leaf Tissues

A prototype needle‐free device was evaluated for delivery of Xanthomonas citri subsp. citri bacteria into the leaves of cultivars susceptible and resistant to citrus canker. The device delivered a precisely controlled volume of bacterial suspension through infiltration of stomata by injection with pressurized gas. The device produced a uniform inoculation of bacteria into the leaves as measured by the volume of infiltration and diameter of the infiltrated area. No damage to the leaves was observed after inoculation with the automated device, even though a higher number of canker lesions developed compared to a hand‐held needleless syringe injection method. The level of practice needed for operation of the automated device was minimal compared to considerable skill required to perform the hand‐held injection. Results from inoculations with the automated device are in accord with the results with the hand‐held syringe method that demonstrated kumquats are highly resistant to citrus canker while rough lemon and ‘Hamlin’ sweet orange are susceptible.     

Influence of serum level,cell line,flow type and viscosity on flow-induced lysis of suspended mammalian cells

An investigation was conducted of the parametric dependence of cell lysis observed when mammalian cells growing in suspension are subjected intermittently to intense hydrodynamic forces. Two flow devices were tested: one consisting of a sudden contraction into a short length of capillary tubing, in which turbulent flow is obtained, and another consisting of a smoothly converging and diverging tube, in which laminar flow is obtained. Changes in the cell line and the serum level in which the cells were grown and subjected to flow trauma both affected the specific lysis rate (fraction of cells lysed per pass through the flow device) in the turbulent flow device. The threshold value of the average wall shear stress level was approximately the same in the turbulent and laminar flow devices (1500–1800 dyn/cm²). Increasing the viscosity of the medium with 70,000 MW dextran had no effect on the specific lysis rate in either flow device.     

High cell density suspension culture of mammalian anchorage independent cells: Oxygen transfer by gas sparging and defoaming with a hydrophobic net

Gas sparging directly into the culture-broth is not done in cell culture, except when the gas flow rate is very small, because much foaming occurs.During screening of defoaming methods, foam was observed to be broken up effectively when it made contact with a net fabricated from hydrophobic materials. Providing a highly efficient oxygen supply to suspension culture was tried using the new defoaming method. In a 5 1 reactor equipped with the foam-eliminating net fabricated with polysiloxane, oxygen was transferred at 21 mmole/l·h equivalent to an about forty-fold higher rate than in conventional surface aeration. This was equivalent to a consumption rate of 1×10⁸ cells/ml, even at a low oxygen gas flow rate of 0.1 cm/s corresponding to a fourth of the gas flow rate when foam leaked through the net.Perfusion culture of rat ascites hepatoma cell JTC-1 was successfully carried out in the 51 scale culture system with the net and a hydrophobic membrane for cell filtration. The viable cell concentration reached 2.7×10⁷ cells/ml after twenty-seven days, in spite of the nutrient-deficient condition of the lower medium exchange rate, that is, a working volume a day, and viability was maintained at more than 90%. In a 1.21 scale culture of mouse-mouse hybridoma cell STK-1, viable cell concentration reached 4×10⁷ cells/ml. These results showed that oxygen transfer by gas sparging with defoaming was useful for high density suspension culture. A foam-breaking mechanism was proposed.Abbreviations Eagle's MEM Eagle's minimal essential medium - Dulbecco's modified Eagle MEM Dulbecco's modified Eagle minimal essential medium     

Evaluation of silicone tubing toxicity using tobacco BY2 culture

Summary Silicone tubing is frequently used for gas exchange in cell culture systems, due to its biocompatibility and high permeability to CO₂ and O₂. In cell culture chambers, medium pH and oxygen levels are often maintained by gas exchange through a coil of silicone tubing. Culture medium is recirculated between the gas exchanger and the culture chamber which contains a suspension of cells. We report that the type of agent used for silicone vulcanization (peroxide or platinum) can markedly affect its biocompatibility, and that tobacco cell culture represents a particularly sensitive indicator of tubing cytotoxicity. Under the conditions studied (cell suspension maintained with forward-reverse flow and stirring), peroxide-cured silicone tubing was toxic to the tobacco BY2 cell culture, in contrast to the platinum-cured silicone tubing that was completely biocompatible. Upon further investigation by mass spectrometry, it was determined that a component with a molecular mass of 288 Da, possibly a tetrachlorinated biphenyl, was present in culture medium in contact with peroxide-cured tubing but not in medium in contact with platinum-cured tubing. Additional curing of peroxide-cured tubing resulted in cell morphology and viability comparable to controls. These data suggest that improperly cured silicone tubing can release catalytic byproducts which can be toxic to plant cells, and that the BY2 tobacco cells represent a suitable model system for studies of materials biocompatibility.     

Constructive improvement of the ultrasonic separation device ADI 1015

The use of the ultrasonic separation deviceis a very important step in the direction forimproving animal cell bioreactor cultures. However,the normal construction of the ultrasonic separationdevice ADI 1015 has an inherent disadvantage inpumping the cell suspension continuously through thedevice by using a peristaltic pump. The cells aretaken out of the reactor and are transported to theside inlet located below the separation chamber of thedevice. This cycling leads to cell death and aconsiderable reduction of the viable cell density. Themodification of the configuration of the device (nocirculation of the cell suspension through theretention device; during approximately 9 minutescell-free supernatant is extracted; every 9 minute forabout one minute, the volume which is equivalent tothe interior volume of the chamber and the tubingconnecting the device to the reactor, is flushed backin order to return the retained cells back to thereactor) allows cell densities from 10⁶ to2.7 × 10⁶ c/ml with a viability of at least90% (tested for the shear sensitive insect cell lineHigh Five), whereas the maximal cell densitiesobtained were 0.76 × 10⁶ c/ml for the periodof continuous culture and 10⁵ c/ml at the end ofthe use of the device in the classical mode.     

Performance trade-offs in in situ chemostat NMR

Investigating cell cultures with NMR requires high cell densities to provide adequate signal-to-noise, or scans must be summed over long time periods and short-term events are lost. The mixing within a chemostat can be used to shorten the time required to acquire informative in situ NMR spectra from cell cultures. However, performance trade-offs can occur between net signal, spectral resolution, and oxygenation due to sampling volume, conductivity, gas bubble, and fluid flow effects. These trade-offs and the effect of different mixing regimes were theoretically analyzed to quantify how device design decisions impact performance. The results were found to concur with data from cell-free NMR experiments performed in 18 mS/cm conductivity medium. The results also guided the redesign of an NMR bioreactor in terms of relative radio frequency (rf) coil and sample dimensions and other factors. The design, which entails using chemostat mixing to shunt sample through a rf coil in ca. 0.4 s, provides adequate oxygenation for the 4-16% (v/v) cell suspensions examined. Gains realized include lower conductive losses, better magnetic field homogeneity, and the exclusion of gas bubbles from the sampling zone. These gains enable the acquistion of spectra from dilute (3-4% v/v) Saccharomyces cerevisiae chemostat cultures in 6.9 min with high resolution in both the orthophosphate and the beta-NTP regions. Samples with 16% (v/v) cells also yield useful spectra within 0.5-1.0 min.     

In vivo monitoring of alkaloid metabolism in hybrid plant cell cultures by 2D cryo-NMR without labelling

Non-invasive measurements of alkaloid metabolism in plant cell suspension cultures of a somatic hybrid from Rauvolfia serpentina Benth. ex Kurz and Rhazya stricta Decaisne were carried out. When cell samples were taken sequentially from a stock feeding experiment, measuring times for in vivo NMR of 40 min were sufficient for following conversions of alkaloids at the natural abundance of 13C. Degradation of ajmaline added to the cells at 1.6 mM concentration to raumacline could be monitored after 96 h on a standard 800 MHz NMR instrument (Avance 800). Feeding vinorine an intermediate of ajmaline biosynthesis at 1.8 mM showed with a 500 MHz CryoProbe that the alkaloid enters two metabolic routes. Vinorine is intracellularly transformed on route I through vellosimine and 10-deoxysarpagine into sarpagine. On route II, the alkaloid is converted by hydroxylation through vomilenine into the glucoside raucaffricine. Intracellular alkaloid concentrations of approximately 500 microM are measurable in vivo with cryogenic NMR technology.     

A convenient on-line device for reagent addition, sample mixing, and temperature control of cell suspensions in flow cytometry

We describe a simple and inexpensive device which permits the addition of up to three different solutions into a cell suspension which is on-line in a flow cytometer. The mixing chamber houses a disposable plastic cuvette stirred with a magnetic stirrer. The sample chamber is attached to a circulating water bath, hence accurate temperature control is achieved. Because the system is prepressurized and the sample line is very short, the delay time-between the point of sample modification and the point of analysis is reduced to a few seconds. Thus reagents may be added rapidly, and kinetic measurements of high temporal resolution are possible. Because the temperature of the sample chamber is regulated, binding can be observed over longer time periods than was previously possible. We demonstrate the usefulness of this device in determining the binding of fluoresceinated hexapeptide to human neutrophils under conditions where the stimulus is infused into the cell suspension while on-line in the cytometer.     

Red blood cells under flow show maximal ATP release for specific hematocrit

ATP release by red blood cells (RBCs) under shear stress (SS) plays a pivotal role in endothelial biochemical signaling cascades. The aim of this study is to investigate through numerical simulation how RBC spatiotemporal organization depends on flow and geometrical conditions to generate ATP patterns. Numerical simulations were conducted in a straight channel by considering both plasma and explicit presence of RBCs, their shape deformation and cell-cell interaction, and ATP release by RBCs. Two ATP release pathways through cell membrane are taken into account: pannexin 1 channel, sensitive to SS, and cystic fibrosis transmembrane conductance regulator, which responds to cell deformation. Several flow and hematocrit conditions are explored. The problem is solved by the lattice Boltzmann method. Application of SS to the RBC suspension triggers a nontrivial spatial RBC organization and ATP patterns. ATP localizes preferentially in the vicinity of the cell-free layer close to channel wall. Conditions for maximal ATP release per cell are identified, which depend on vessel size and hematocrit Ht. Increasing further Ht beyond optimum enhances the total ATP release but should degrade oxygen transport capacity, a compromise between an efficient ATP release and minimal blood dissipation. Moreover, ATP is boosted in capillaries, suggesting a vasomotor activity coordination throughout the resistance network.     

Hydrodynamic behaviour of animal cell microcarrier suspensions in split-cylinder airlift bioreactors

Hydrodynamic characteristics of suspensions of microcarriers used for culturing anchorage dependent animal cells are reported in split-cylinder internal-loop airlift bioreactors. Cell culture media are simulated using salt solutions that duplicate the ionic strengths of typical media. Effects of solids loading (0–30 kg·m^–3), microcarrier particle size (150–300×10^–6 m diameter) and density (1030–1050 kg·m^–3) on gas induced circulation of the slurry, mixing time, gas holdup and gas velocity requirements to attain complete suspension of solids are discussed for two reactors with aspect ratios of 7.6 and 14.5, but equal riser-to-downcomer cross-sectional area ratios of 1.0, aerated at low air flow rates (0–8×10^–6 m³·s^–1) through a sintered glass sparger with 110×10^–6 m diameter pores. The study covers the ranges of solids concentrations, types, densities, particle sizes and aeration rates that are of relevance in animal cell culture applications.Airlift bioreactors displayed suitable hydrodynamic characteristics for potentially supporting anchorage dependent cell cultures on microcarriers at carrier loadings similar to those that are currently used in stirred tank bioreactors. The minimum gas flow rates and the induced liquid circulation rates necessary to achieve and maintain suspension of the heaviest and the largest microcarriers were well within practicable limits, limits which have been shown to be withstood by animal cells in non-anchorage dependent suspension culture in airlift bioreactors. No floatation problems were encountered with the carriers, nor was sedimentation a problem so long as the identified minimum suspension criteria were met. Chisti's liquid circulation equation, originally intended for two-phase flow, applied to the three-phase gas-liquid-microcarrier systems.     

Use of Surface Enhanced Blocking (SEB) Electrodes for Microbial Cell Lysis in Flow-Through Devices

By simultaneously subjecting microbial cells to high amplitude pulsed electric fields and flash heating of the cell suspension fluid, effective release of intracellular contents was achieved. The synergistic effect of the applied electric field and elevated temperature on cell lysis in a flow-through device was demonstrated for Gram-negative and Gram-positive bacteria, and Mycobacterium species. The resulting lysate is suitable for downstream nucleic acid amplification and detection without requiring further preparation. The lysis chamber employs surface enhanced blocking electrodes which possess an etched micro-structured surface and a thin layer of dielectric metal oxide which provides a large effective area and blocks transmission of electrical current. The surface enhanced blocking electrodes enable simultaneous suppression of the rapid onset of electric field screening in the bulk of the cell suspension medium and avoidance of undesired electrochemical processes at the electrode-electrolyte interface. In addition the blocking layer ensures the robustness of the cell lysis device in applications involving prolonged flow-through processing of the microbial cells.     

Combined 13C NMR- and mass-spectrometry for non-invasive monitoring of metabolism

Summary A method for the long-term incubation of cell suspensions at measured dissolved gas concentrations during acquisition of NMR spectra is described: a mass spectrometer probe in the cell suspension provides continuous monitoring of gases. Its usefulness is shown for Trichomonas vaginalis where fermentation products from glucose in 72 M O₂ are different from those under strictly anaerobic conditions.     

Portable Device for Preparation and Delivery of Gas Mixtures

A simple, portable device for the preparation and delivery of gas mixtures has been designed and constructed. The basic feature of the device is the use of gas flow controllers to maintain stable flow rates over a wide range of downstream pressures, instead of the capillary tubes and water-filled barostats commonly used in gas-mixing devices. Elimination of the barostat avoids problems such as water leakage, the loss of gases through the barostat, and changes in gas pressure due to evaporative loss of water from the barostat. The absence of a barostat also provides a closed system, allowing the use of the device for mixing and delivering of toxic gases. The prototype of the device has been used to prepare mixtures of different gases for more than 1 year and has been found to operate consistently and reproducibly. The actual concentrations of O₂, CO₂, and N₂ in gas mixtures (determined by gas chromatography) immediately after mixing were between 2.2 and 6.6% of the desired values in four performance tests. Fluctuations in concentration of gases in mixtures after 9 days of continuous gas delivery was less than 2% in four performance tests.     

Use of biocarrier beads and flow cytometry for single-cell studies of fibronectin gene regulation in dibutyrl cyclic AMP reverse transformed CHO-K1 cells

The protease sensitivity of a number of cell surface or cytoskeletal components and the relationship of these to the substratum in attached cells has prevented the quantitative measurement of their expression by flow cytometry. Using traditional cell sorting techniques, cells must be treated with a protease to detach them from a substrate in order to produce a single-cell suspension. Unfortunately, proteolytic treatment alters or destroys a number of cellular proteins. Fibronectin either on the cell surface or as part of the substratum laid down by the cell is particularly sensitive to proteases, preventing its quantitative study by flow cytometry. To circumvent these problems and produce a single cell suspension necessary for flow cytometric analysis, CHO-K1, a Chinese hamster ovary cell line, were grown in suspension on specially-treated 25 μm biocarrier beads. The CHO-K1 cell line is composed of transformed epithelial-like cells that have lost the fibronectin deposit around their cell membranes. To restore the typical fibroblastic deposit of fibronectin, the cells attached to beads were induced by dibutyrl cAMP to undergo a reverse transformation reaction to restore fibroblastic morphology and the typical fibroblastic deposite of fibronectin on the cell surface and substratum. The cells attached to beads were then immunofluorescently labeled for the protease-sensitive, extracellular matrix component, fibronectin, and examined on a flow cytometer. Cell surface fibronectin heterogeneity was then examined on a cell-by-cell basis as a function of cell cycle using Hoechst 33342 dye that binds to AT base pairs of cellular DNA. Dual laser measurement and multiparameter list mode data analysis were used to study the relationship between cell surface fibronectin of biocarrier bead attached cells and cell cycle.     

A tube-cutting device for fractionating preparative gradients from the ultracentrifuge

A device is described which cuts off the bottom of a polyallomer (plastic) centrifuge tube, allowing collection of a preparative gradient from the bottom through a large-diameter opening. The gradient flows downward through a funnel-shaped portal positioned below the tube, and the cross-sectional area of flow is gradually restricted. This device resolved into separate peaks DNA sample components differing in density by only 0.007 g/ml in neutral cesium chloride.     

CFD simulation coupled with population balance equations for aerated stirred bioreactors

Mammalian cells have been widely used to produce therapeutic proteins in stirred bioreactors in suspension culture. Local hydrodynamics can have a great impact on cell proliferation and protein synthesis, but there are few reports on spatial heterogeneity of nutrients, gas bubbles, and mass transfer coefficients. We have employed computational fluid dynamics (CFD) coupled with population balance equations to study local hydrodynamics in a 20 L stirred bioreactor. The flow patterns, energy dissipation rates, gas volume fraction, gas bubble size distribution and local mass transfer coefficient have been displayed throughout the whole bioreactor. Their implications for mammalian cell culture have been discussed. This study provides an insight into rational design and optimum operation conditions in a stirred bioreactor for mammalian cell cultivation.     

Preparation of single rice chromosome for construction of a DNA library using a laser microbeam trap

We report the development of a laser micromanipulation system and its application in the isolation of individual rice chromosomes directly from a metaphase cell. Microdissection and flow sorting are two major methods for the isolation of single chromosome. These methods are dependent on the techniques of chromosome spread and chromosome suspension, respectively. In the development of this system, we avoided using chromosome spread and cell suspension was used instead. The cell wall of metaphase rice cell was cut by optical scissors. The released single chromosome was captured by an optical trap and transported to an area without cell debris. The isolated single chromosome was then collected and specific library was constructed by linker adaptor PCR. The average insert size of the library was about 300 bp. Two hundred inserts of chromosome 4 library were sequenced, and 96.5% were aligned to the corresponding sequences of rice chromosome 4. These results suggest the possible application of this method for the preparation of other subcellular structures and for the cloning of single macromolecule through a laser microbeam trap.     

NMR tools for biotechnology

Recent developments in NMR spectroscopy verify that NMR continues to be an exciting area of research. These advances can be placed into three general categories: new hardware; new techniques; and novel applications. The hardware developments include many advances in the area of flow NMR and some new probe designs. The new techniques include several ways to edit the NMR spectra of mixtures without using chromatographic separation. These new NMR tools are now allowing us to analyze complex mixtures, combinatorial-chemistry libraries, bound drugs, unstable compounds, very small samples, and heterogeneous samples.