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). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1093–1102, 2014     

Use of a spin-filter can reduce disruption of hybridoma cells in a bioreactor

Summary The possibility of damage to suspended cells arising from the introduction of a spin-filter rotating at high speed is examined experimentally and theoretically. Far from increasing damage, it is shown that a spin-filter, even when rotating at high speed, can markedly improve the survival of cells in a stirred unbuffled bioreactor. Analysis of the shear stress generated at the filter surface shows values that are orders of magnitude below those shown elsewhere to be damaging to cells. The improvement in survival seen in addition of a spin-filter to an impeller-agitated bioreactor are explained in terms of the events associated with the suppression of vortices, and bubble entrainment and disengagement.     

Silk screen based dual spin-filter module for perfusion culture of adherent and non-adherent mammalian cells

Spin-filters have been primarily used for producing therapeutic proteins from mammalian cells. However, disposability and/or high filter clogging of the existing spin-filter systems affect the process economy and productivity. Hence, to address these drawbacks a reusable dual spin-filter module for perfusion culture of adherent and non-adherent mammalian cells was designed. Two non-woven Bombyx mori silk layers were used as filter screen; the outer layer was conducive to cell attachment whilst the inner was non-conducive. Adherent cells can be cultured either in suspended mode using its inner single module or as monolayer of cells using its dual concentric module. We achieved 30 % higher urokinase productivity as compared to the stainless-steel spin-filter during perfusion experiments of adherent human kidney cells in suspended mode. This was due to the hydrophobic and negatively-charged silk screen that allows clog-free perfusion culture for prolonged periods.     

Particle image velocimetry (PIV) study of rotating cylindrical filters for animal cell perfusion processes

In the present work, the main fluid flow features inside a rotating cylindrical filtration (RCF) system used as external cell retention device for animal cell perfusion processes were investigated using particle image velocimetry (PIV). The motivation behind this work was to provide experimental fluid dynamic data for such turbulent flow using a high‐permeability filter, given the lack of information about this system in the literature. The results shown herein gave evidence that, at the boundary between the filter mesh and the fluid, a slip velocity condition in the tangential direction does exist, which had not been reported in the literature so far. In the RCF system tested, this accounted for a fluid velocity 10% lower than that of the filter tip, which could be important for the cake formation kinetics during filtration. Evidence confirming the existence of Taylor vortices under conditions of turbulent flow and high permeability, typical of animal cell perfusion RCF systems, was obtained. Second‐order turbulence statistics were successfully calculated. The radial behavior of the second‐order turbulent moments revealed that turbulence in this system is highly anisotropic, which is relevant for performing numerical simulations of this system. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Flow modeling in a novel non-perfusion conical bioreactor

We have developed a bioreactor vessel design which has the advantages of simplicity and ease of assembly and disassembly, and with the appropriately determined flow rate, even allows for a scaffold to be suspended freely regardless of its weight. This article reports our experimental and numerical investigations to evaluate the performance of a newly developed non-perfusion conical bioreactor by visualizing the flow through scaffolds with 45 degrees and 90 degrees fiber lay down patterns. The experiments were conducted at the Reynolds numbers (Re) 121, 170, and 218 based on the local velocity and width of scaffolds. The flow fields were captured using short-time exposures of 60 microm particles suspended in the bioreactor and illuminated using a thin laser sheet. The effects of scaffold fiber lay down pattern and Reynolds number were obtained and correspondingly compared to results obtained from a computational fluid dynamics (CFD) software package. The objectives of this article are twofold: to investigate the hypothesis that there may be an insufficient exchange of medium within the interior of the scaffold when using our non-perfusion bioreactor, and second, to compare the flows within and around scaffolds of 45 degrees and 90 degrees fiber lay down patterns. Scaffold porosity was also found to influence flow patterns. It was therefore shown that fluidic transport could be achieved within scaffolds with our bioreactor design, being a non-perfusion vessel. Fluid velocities were generally same of the same or one order lower in magnitude as compared to the inlet flow velocity. Additionally, the 90 degrees fiber lay down pattern scaffold was found to allow for slightly higher fluid velocities within, as compared to the 45 degrees fiber lay down pattern scaffold. This was due to the architecture and pore arrangement of the 90 degrees fiber lay down pattern scaffold, which allows for fluid to flow directly through (channel-like flow).     

Location of scaffolds in bioreactors modulates the hydrodynamic environment experienced by engineered tissues

Physical forces experienced by engineered-tissues during in vitro cultivation influence tissue growth and function. The hydrodynamic environment within bioreactors plays a decisive role in providing the necessary physical stimuli and nutrient transport to support tissue development. Our overall goal is to investigate interrelationships between the local hydrodynamic environment in the bioreactor and the structural and functional tissue properties in order to optimize the production of clinically relevant engineered-tissues. To this end, we used computational fluid dynamics (CFD) modeling to characterize the complex hydrodynamic environment in a wavy-walled bioreactor used for cultivation of tissue-engineered cartilage constructs and examined the changes in the flow field due to the presence of constructs. The flow-induced shear stress range experienced by engineered constructs cultivated in the wavy-walled bioreactor (0-0.67 dyn/cm(2)) was found to be significantly lower than that in the spinner flask (0-1.2 dyn/cm(2)), and to be modulated by the radial or axial position of the constructs. These CFD results are validated by experimental particle-image velocimetry (PIV) measurements previously reported by our group. Results from the present study indicate that the location of constructs in the bioreactor not only affected the magnitude and distribution of the shear stresses on the constructs, but also other hydrodynamic parameters, such as the directional distribution of the fluid velocity and the degree of fluid recirculation, all of which may differentially influence the development of tissue-engineered constructs.     

Computational fluid dynamic analysis of bioprinted self-supporting perfused tissue models

Natural tissues are incorporated with vasculature, which is further integrated with a cardiovascular system responsible for driving perfusion of nutrient-rich oxygenated blood through the vasculature to support cell metabolism within most cell-dense tissues. Since scaffold-free biofabricated tissues being developed into clinical implants, research models, and pharmaceutical testing platforms should similarly exhibit perfused tissue-like structures, we generated a generalizable biofabrication method resulting in self-supporting perfused (SSuPer) tissue constructs incorporated with perfusible microchannels and integrated with the modular FABRICA perfusion bioreactor. As proof of concept, we perfused an MLO-A5 osteoblast-based SSuPer tissue in the FABRICA. Although our resulting SSuPer tissue replicated vascularization and perfusion observed in situ, supported its own weight, and stained positively for mineral using Von Kossa staining, our in vitro results indicated that computational fluid dynamics (CFD) should be used to drive future construct design and flow application before further tissue biofabrication and perfusion. We built a CFD model of the SSuPer tissue integrated in the FABRICA and analyzed flow characteristics (net force, pressure distribution, shear stress, and oxygen distribution) through five SSuPer tissue microchannel patterns in two flow directions and at increasing flow rates. Important flow parameters include flow direction, fully developed flow, and tissue microchannel diameters matched and aligned with bioreactor flow channels. We observed that the SSuPer tissue platform is capable of providing direct perfusion to tissue constructs and proper culture conditions (oxygenation, with controllable shear and flow rates), indicating that our approach can be used to biofabricate tissue representing primary tissues and that we can model the system in silico.     

Validation of CFD predictions of flow in a 3D alveolated bend with experimental data

Verifying numerical predictions with experimental data is an important aspect of any modeling studies. In the case of the lung, the absence of direct in vivo flow measurements makes such verification almost impossible. We performed computational fluid dynamics (CFD) simulations in a 3D scaled-up model of an alveolated bend with rigid walls that incorporated essential geometrical characteristics of human alveolar structures and compared numerical predictions with experimental flow measurements made in the same model by particle image velocimetry (PIV). Flow in both models was representative of acinar flow during normal breathing (0.82ml/s). The experimental model was built in silicone and silicone oil was used as the carrier fluid. Flow measurements were obtained by an ensemble averaging procedure. CFD simulation was performed with STAR-CCM+ (CD-Adapco) using a polyhedral unstructured mesh. Velocity profiles in the central duct were parabolic and no bulk convection existed between the central duct and the alveoli. Velocities inside the alveoli were approximately 2 orders of magnitude smaller than the mean velocity in the central duct. CFD data agreed well with those obtained by PIV. In the central duct, data agreed within 1%. The maximum simulated velocity along the centerline of the model was 0.5% larger than measured experimentally. In the alveolar cavities, data agreed within 15% on average. This suggests that CFD techniques can satisfactorily predict acinar-type flow. Such a validation ensure a great degree of confidence in the accuracy of predictions made in more complex models of the alveolar region of the lung using similar CFD techniques.     

3-D computational modeling of media flow through scaffolds in a perfusion bioreactor

Media perfusion bioreactor systems have been developed to improve mass transport throughout three-dimensional (3-D) tissue-engineered constructs cultured in vitro. In addition to enhancing the exchange of nutrients and wastes, these systems simultaneously deliver flow-mediated shear stresses to cells seeded within the constructs. Local shear stresses are a function of media flow rate and dynamic viscosity, bioreactor configuration, and porous scaffold microarchitecture. We have used the Lattice-Boltzmann method to simulate the flow conditions within perfused cell-seeded cylindrical scaffolds. Microcomputed tomography imaging was used to define the scaffold microarchitecture for the simulations, which produce a 3-D fluid velocity field throughout the scaffold porosity. Shear stresses were estimated at various media flow rates by multiplying the symmetric part of the gradient of the velocity field by the dynamic viscosity of the cell culture media. The shear stress algorithm was validated by modeling flow between infinite parallel plates and comparing the calculated shear stress distribution to the analytical solution. Relating the simulation results to perfusion experiments, an average surface shear stress of 5x10(-5)Pa was found to correspond to increased cell proliferation, while higher shear stresses were associated with upregulation of bone marker genes. This modeling approach can be used to compare results obtained for different perfusion bioreactor systems or different scaffold microarchitectures and may allow specific shear stresses to be determined that optimize the amount, type, or distribution of in vitro tissue growth.     

Development of serum-free bioreactor production of recombinant human thyroid stimulating hormone receptor

For the detection of autoantibodies to thyroid stimulating hormone receptors (TSH-R) in Graves' disease based on a novel coated tube assay system, human TSH-R is needed in large amounts. Whereas expression of TSH-R in bacteria, yeast, or insect cells results in nonfunctional, denaturated receptor, mammalian cells such as COS, CHO, and HeLa are able to express functional TSH-R, but only in very low amounts. Furthermore, for all of these cultivations expensive standard media containing 10% fetal calf serum are needed to obtain functional receptor. Here we report on the development of a serum-free production-scale process based on a stable transformed and highly productive human leukemia cell line K562 (1). Starting with K562-TSH-R cells growing in medium containing 10% fetal calf serum the cell line was adapted to serum-free medium. The adaptation medium was optimized in regards to amino acid and protein concentrations, since the use of unadjusted medium caused cell death after 2 days. The adapted cells were stable and could be cultivated without antibiotics for more than 50 cell doublings without losing their productivity. The obtained receptor showed improved TSH binding. The process development was based on cultivations in a 2-L bench-scale bioreactor. Cultivations in batch mode and chemostat mode and perfusion cultivation with the usage of an internal microfiltration device and a spin-filter device were compared. After process optimization a continuous process using spin-filter was set up and run in a 20 L-pilot-scale bioreactor. The presented results were the prerequisite for the production of the novel assay for the diagnosis of autoantibodies to TSH-R in Graves' disease.     

可控多孔结构支架制备及灌注式体外培养

利用CAD和快速成形技术设计制造具有可控多孔结构的支架。构建灌注式生物反应器系统,实现氧气和营养物质的大量输送,同时产生一定流体剪应力,调节细胞功能的发挥。根据支架负型结构制造出相应的树脂原型,用磷酸钙骨水泥进行填充烧结,得到与设计相符的多孔支架。接种兔成骨细胞,分别采用静态和灌注式三维动态培养方法,观察不同培养条件下细胞在支架表面以及所构造微管道内的生长情况。试验结果表明,灌注式体外培养方法更有利于细胞在支架微管道内的存活和功能的发挥,此灌注式系统能够改善支架微管道内细胞生存的微环境,增强黏附在支架微管道内细胞的活性,促进细胞进一步的增殖和矿化基质的产生。     

Computational fluid dynamics simulations of particle deposition in large-scale, multigenerational lung models

Computational fluid dynamics (CFD) has emerged as a useful tool for the prediction of airflow and particle transport within the human lung airway. Several published studies have demonstrated the use of Eulerian finite-volume CFD simulations coupled with Lagrangian particle tracking methods to determine local and regional particle deposition rates in small subsections of the bronchopulmonary tree. However, the simulation of particle transport and deposition in large-scale models encompassing more than a few generations is less common, due in part to the sheer size and complexity of the human lung airway. Highly resolved, fully coupled flowfield solution and particle tracking in the entire lung, for example, is currently an intractable problem and will remain so for the foreseeable future. This paper adopts a previously reported methodology for simulating large-scale regions of the lung airway (Walters, D. K., and Luke, W. H., 2010, "A Method for Three-Dimensional Navier-Stokes Simulations of Large-Scale Regions of the Human Lung Airway," ASME J. Fluids Eng., 132(5), p. 051101), which was shown to produce results similar to fully resolved geometries using approximate, reduced geometry models. The methodology is extended here to particle transport and deposition simulations. Lagrangian particle tracking simulations are performed in combination with Eulerian simulations of the airflow in an idealized representation of the human lung airway tree. Results using the reduced models are compared with those using the fully resolved models for an eight-generation region of the conducting zone. The agreement between fully resolved and reduced geometry simulations indicates that the new method can provide an accurate alternative for large-scale CFD simulations while potentially reducing the computational cost of these simulations by several orders of magnitude.     

Radial flow hepatocyte bioreactor using stacked microfabricated grooved substrates

Bioartificial liver (BAL) devices with fully functioning hepatocytes have the potential to provide temporary hepatic support for patients with liver failure. The goal of this study was to optimize the flow environment for the cultured hepatocytes in a stacked substrate, radial flow bioreactor. Photolithographic techniques were used to microfabricate concentric grooves onto the underlying glass substrates. The microgrooves served to protect the seeded hepatocytes from the high shear stresses caused by the volumetric flow rates necessary for adequate convective oxygen delivery. Finite element analysis was used to analyze the shear stresses and oxygen concentrations in the bioreactor. By employing high volumetric flow rates, sufficient oxygen supply to the hepatocytes was possible without an integrated oxygen permeable membrane. To implement this concept, 18 microgrooved glass substrates, seeded with rat hepatocytes cocultured with 3T3-J2 fibroblasts, were stacked in the bioreactor, creating a channel height of 100 microm between each substrate. In this bioreactor configuration, liver-specific functions (i.e., albumin and urea synthesis rates) of the hepatocytes remained stable over 5 days of perfusion, and were significantly increased compared to those in the radial flow bioreactor with stacked substrates without microgrooves. This study suggests that this radial flow bioreactor with stacked microgrooved substrates is scalable and may have potential as a BAL device in the treatment of liver failure.     

Acinus-on-a-chip: A microfluidic platform for pulmonary acinar flows

Convective respiratory flows in the pulmonary acinus and their influence on the fate of inhaled particles are typically studied using computational fluid dynamics (CFD) or scaled-up experimental models. However, experiments that replicate several generations of the acinar tree while featuring cyclic wall motion have not yet been realized. Moreover, current experiments generally capture only flow dynamics, without inhaled particle dynamics, due to difficulties in simultaneously matching flow and particle dynamics. In an effort to overcome these limitations, we introduce a novel microfluidic device mimicking acinar flow characteristics directly at the alveolar scale. The model features an anatomically-inspired geometry that expands and contracts periodically with five dichotomously branching airway generations lined with alveolar-like cavities. We use micro-particle image velocimetry with a glycerol solution as the carrying fluid to quantitatively characterize detailed flow patterns within the device and reveal experimentally for the first time a gradual transition of alveolar flow patterns along the acinar tree from recirculating to radial streamlines, in support of hypothesized predictions from past CFD simulations. The current measurements show that our microfluidic system captures the underlying characteristics of the acinar flow environment, including Reynolds and Womersley numbers as well as cyclic wall displacements and alveolar flow patterns at a realistic length scale. With the use of air as the carrying fluid, our miniaturized platform is anticipated to capture both particle and flow dynamics and serve in the near future as a promising in vitro tool for investigating the mechanisms of particle deposition deep in the lung.     

The effect of hydrodynamic shear on 3D engineered chondrocyte systems subject to direct perfusion

Bioreactors allowing direct-perfusion of culture medium through tissue-engineered constructs may overcome diffusion limitations associated with static culturing, and may provide flow-mediated mechanical stimuli. The hydrodynamic stress imposed on cells within scaffolds is directly dependent on scaffold microstructure and on bioreactor configuration. Aim of this study is to investigate optimal shear stress ranges and to quantitatively predict the levels of hydrodynamic shear imposed to cells during the experiments. Bovine articular chondrocytes were seeded on polyestherurethane foams and cultured for 2 weeks in a direct perfusion bioreactor designed to impose 4 different values of shear level at a single flow rate (0.5 ml/min). Computational fluid dynamics (CFD) simulations were carried out on reconstructions of the scaffold obtained from micro-computed tomography images. Biochemistry analyses for DNA and sGAG were performed, along with electron microscopy. The hydrodynamic shear induced on cells within constructs, as estimated by CFD simulations, ranged from 4.6 to 56 mPa. This 12-fold increase in the level of applied shear stress determined a 1.7-fold increase in the mean content in DNA and a 2.9-fold increase in the mean content in sGAG. In contrast, the mean sGAG/DNA ratio showed a tendency to decrease for increasing shear levels. Our results suggest that the optimal condition to favour sGAG synthesis in engineered constructs, at least at the beginning of culture, is direct perfusion at the lowest level of hydrodynamic shear. In conclusion, the presented results represent a first attempt to quantitatively correlate the imposed hydrodynamic shear level and the invoked biosynthetic response in 3D engineered chondrocyte systems.     

Computational fluid dynamics simulation improves the design and characterization of a plug-flow-type scale-down reactor for microbial cultivation processes

The scale-up of bioprocesses remains one of the major obstacles in the biotechnology industry. Scale-down bioreactors have been identified as valuable tools to investigate the heterogeneities observed in large-scale tanks at the laboratory scale. Additionally, computational fluid dynamics (CFD) simulations can be used to gain information about fluid flow in tanks used for production. Here, we present the rational design and comprehensive characterization of a scale-down setup, in which a flexible and modular plug-flow reactor was connected to a stirred-tank bioreactor. With the help of CFD using the realizable k-ε model, the mixing time difference between a 20 and 4000 L bioreactor was evaluated and used as scale-down criterion. CFD simulations using a shear stress transport (SST) k-ω turbulence model were used to characterize the plug-flow reactor in more detail, and the model was verified using experiments. Additionally, the model was used to simulate conditions where experiments technically could not be performed due to sensor limitations. Nevertheless, verification is difficult in this case as well. This was the first time a scale-down setup was tested on high-cell-density Escherichia coli cultivations to produce industrially relevant antigen-binding fragments (Fab). Biomass yield was reduced by 11% and specific product yield was reduced by 20% during the scale-down cultivations. Additionally, the intracellular Fab fraction was increased by using the setup. The flexibility of the introduced scale-down setup in combination with CFD simulations makes it a valuable tool for investigating scale effects at the laboratory scale. More information about the large scale is still necessary to further refine the setup and to speed up bioprocess scale-up in the future.     

Finite-element modeling of the hemodynamics of stented aneurysms

BACKGROUND: Computational fluid dynamics (CFD) simulations are used to analyze the wall shear stress distribution and flow streamlines near the throat of a stented basilar side-wall aneurysm. Previous studies of stented aneurysm flows used low mesh resolution, did not include mesh convergence analyses, and depended upon conformal meshing techniques that apply only to very artificial stent geometries. METHOD OF APPROACH: We utilize general-purpose computer assisted design and unstructured mesh generation tools that apply in principle to stents and vasculature of arbitrary complexity. A mesh convergence analysis for stented steady flow is performed, varying node spacing near the stent. Physiologically realistic pulsatile simulations are then performed using the converged mesh. RESULTS: Artifact-free resolution of the wall shear stress field on stent wires requires a node spacing of approximately 1/3 wire radius. Large-scale flow features tied to the velocity field are, however, captured at coarser resolution (nodes spaced by about one wire radius or more). CONCLUSIONS: Results are consistent with previous work, but our methods yield more detailed insights into the complex flow dynamics. However, routine applications of CFD to anatomically realistic cases still depend upon further development of dedicated algorithms, most crucially to handle geometry definition and mesh generation for complicated stent deployments.     

The in vitro growth of a three-dimensional human dermal replacement using a single-pass perfusion system

A human dermal replacement has been developed by seeding human neonatal dermal fibroblasts onto a biosorbable polyglactin (polyglycolide/polylactide) mesh and culturing in a bioreactor. The mesh provides the proper environment for the cells to attach, grow in a three-dimensional array, and establish a tissue matrix over a 2- to 3-week culture period. The dermal replacement has been characterized and found to contain a variety of naturally occurring dermal matrix proteins, including fibronectin, glycosaminoglycans, and collagen types I and III. To efficiently and reproducibly produce this dermal tissue equivalent, a closed, single-pass perfusion system was developed and compared with a static process. In the single-pas perfusion system, growth medium (containing ascorbic acid) was perfused around the 4 x 6 in. pieces of mesh at specific flow rates determined by nutrient consumption and waste production rates. The flow rates used for this system indicate that a diffusion-limited regime exists with a mean residence time greater than 1 h for essential nutrients and factors. By controlling glucose concentrations in the system to a delta of 0.70 g/L from the inlet to the outlet of the bioreactor, it took 6 fewer days to grow a tissue similar to that produced by the static system.     

Economic comparison of diagnostic antibody production in perfusion stirred tank and in hollow fiber bioreactor processes

The total operating costs of small-scale monoclonal antibody production were calculated for two different upstream options and general downstream procedure based on protein A chromatography. The upstream options were a spin-filter equipped stirred-tank bioreactor (STR) and a hollow fiber bioreactor (HFB). Both the bioreactors were operated in perfusion mode. The total operating costs of the processes were 6,900 €/g for STR option and 6,400 €/g for the HFB option. In the both systems, the costs were dominated by expenses derived from the downstream section (almost 80%) that was almost identical in the both systems. In the upstream section, the investment depreciation was the largest cost item. The lower total costs of the HFB option were a result of lower investment costs and more concentrated product that led into savings also in downstream section. This study brings out the HFB as on viable alternative for stirred-tank bioreactor, especially in small-scale diagnostic monoclonal antibody production.     

Development of a small-scale bioreactor: application to in vivo NMR measurement

A perfused bioreactor allowing in vivo NMR measurement was developed and validated for Eschscholtzia californica cells. The bioreactor was made of a 10-mm NMR tube. NMR measurement of the signal-to-noise ratio was optimized using a sedimented compact bed of cells that were retained in the bioreactor by a supporting filter. Liquid medium flow through the cell bed was characterized from a mass balance on oxygen and a dispersive hydrodynamic model. Cell bed oxygen demand for 4 h perfusion required a minimal medium flow rate of 0.8 mL/min. Residence time distribution assays at 0.8-2.6 mL/min suggest that the cells are subjected to a uniform nutrient environment along the cell bed. Cell integrity was maintained for all culture conditions since the release of intracellular esterases was not significant even after 4 h of perfusion. In vivo NMR was performed for (31)P NMR and the spectrum can be recorded after only 10 min of spectral accumulation (500 scans) with peaks identified as G-6P, F-6P, cytoplasmic Pi, vacuolar Pi, ATP(gamma) and ADP(beta), ATP(alpha) and ADP(alpha), NADP and NDPG, NDPG and ATP(beta). Cell viability was shown to be maintained as (31)P chemical shifts were constant with time for all the identified nuclei, thus suggesting constant intracellular pH.