A simple method for hyaluronic acid quantification in culture broth

The carbazole assay has been used for determination of the percentage of hyaluronic acid in biological fluids. However, it is difficult to measure the concentration of hyaluronic acid in culture broth because glucose and polysaccharides remaining after cultures can react with sulfuric acid and carbazole. The glucose and polysaccharide remnants must be completely removed in order to get the correct value for hyaluronic acid. The turbidity assay, another method for estimating the concentration of hyaluronic acid, is based on the formation of insoluble complexes between hyaluronic acid and cetyltrimethylammonium bromide. This method is very easy and fast compared with the carbazole assay. Because concentrations of hyaluronic acid measured by the turbidity assay were ranged around 100% of those measured by the carbazole assay, the content of hyaluronic acid in culture broth can be determined by the turbidity assay. The turbidity method also has the advantage of being safer than the carbazole assay.     

Rheological characterization of culture broth containing the exopolysaccharide PS-EDIV from Sphingomonas pituitosa

Sphingomonas pituitosa excretes the capsular exopolysaccharide PS-EDIV into the culture broth augmenting considerably its fluid viscosity. Since this change particularly affects key processes like mixing and transport during the microbial production, this work was aimed at the rheological characterization of the polymer-containing culture broth of S. pituitosa. The study included investigations on basic properties of the culture broth, but also on the dependence of the biomass–polymer-solution properties on different physicochemical post-cultivation treatment steps like variations of temperature, pH-value or concentration of salts. The essential result is the characterization of the viscoelastic behavior of the culture broth, which was more gel-like than sol-like and exhibited slight elastic properties. This rheological behavior showed that the PS-EDIV culture broth formed non-Newtonian fluids, indicating that it is a pseudoplastic biopolymer, with yield stress appearance and exhibits thixotropic properties. Rheograms were fitted to the Herschel–Bulkley model. The amplitude sweep revealed a deformation of 21% as the limiting value of the linear viscoelastic interval. Furthermore, the PS-EDIV culture broth showed a high viscosity which was strongly influenced by salt type and concentration but weakly influenced by temperature and pH-value within the investigated experimental boundaries.     

Suspension culture of hematopoietic stem cells in stirred bioreactors

Hematopoietic stem cells have applications in bone marrow transplantations for the treatment of hematopoietic disorders. When murine hematopoietic stem cells were cultured in 50 ml stirred bioreactors for 14 d, stem-cell-antigen-1 positive cells (hematopoietic primitive progenitor cells) and long-term culture-initiating cells (hematopoietic stem cells) grew by 5-fold and 4-fold, respectively. These results show the possibility of growing hematopoietic stem cells using a stirred bioreactor.     

New culture media to suppress exopolysaccharide production by Rhizobium japonicum

Summary Secretion of exopolysaccharide by Rhizobium japonicum strain USDA 110 ws measured during growth on media comprising 12.5 mM MES [2-(N-morpholino)ethanesulfonic acid] buffer, pH 5.9; 5 mM NH₄Cl; trace elements; and 25 or 100 mM of one of 23 different carbon sources. Significant amounts of exopolysaccharide were produced on all aldopentoses, polyols, organic acids, and sugar acids tested, but little or none was secreted during growth on aldohexoses or amino acids. Media suitable for routine slime-free culture of several. R. japonicum strains were made by using 100 mM d-galactose or 25 mM l-aspartate as the carbon source.     

A method using 3-O-methyl-D-glucose and phloretin for the determination of intracellular water space of cells in monolayer culture.

A method is presented for determining the extent of methylation of tRNAs synthesized in mammalian and bacterial cell systems and is based upon determining the distribution of radioactivity associated with the guanine constituents of total cellular tRNA preparations previously labeled with [2-¹⁴C]guanosine and with [methyl]-³H or -¹⁴C]methionine. Whereas labeling with guanosine provides a means of assessing the extent of methylation of the [2-¹⁴C]guanine residues incorporated into tRNA, methionine labeling provides a measure of the percentage of [methyl-³H or -¹⁴C]methylated constituents that are methylated guanines. Analyses such as the above reveal that the tRNA of KB cells acquires approximately three times as many methyl groups as that of E. coli B tRNA. Coupled with the knowledge that both mammalian and bacterial tRNA preparations contain an average of 24 guanine residues per molecule, the above analyses further reveal that 7.2 and 2.4 methyl groups are incorporated into each tRNA molecule synthesized in exponentially growing KB- and E. coli B-cells, respectively. Additional information regarding the extent of formation of individual methylated constituents per tRNA molecule synthesized is presented.     

New method for determination of fecal sterols in urine using non-chlorinated solvents

A new method has been developed to determine a number of sterols in urine using non-chlorinated solvents, namely methyl tert.-butyl ether and methanol (the MTB method). The method involves liquid–liquid extraction, saponification, reextraction, silylation and final identification and quantification by GC–FID. The sterols determined were coprostanol, epicoprostanol, cholesterol and dihydrocholesterol. 5α-Cholestane was used as internal standard. The limit of detection for the sterols in this experiment was 2 μg l⁻¹ urine. Recovery of coprostanol over the range 5–100 μg l⁻¹ urine by this method was between 80 and 92%.     

New method for selection of hydrogen peroxide adapted bifidobacteria cells using continuous culture and immobilized cell technology

Background  

Oxidative stress can severely compromise viability of bifidobacteria. Exposure of Bifidobacterium cells to oxygen causes accumulation of reactive oxygen species, mainly hydrogen peroxide, leading to cell death. In this study, we tested the suitability of continuous culture under increasing selective pressure combined with immobilized cell technology for the selection of hydrogen peroxide adapted Bifidobacterium cells. Cells of B. longum NCC2705 were immobilized in gellan-xanthan gum gel beads and used to continuously ferment MRS medium containing increasing concentration of H₂O₂ from 0 to 130 ppm.     

A dense cell retention culture system using stirred ceramic membrane reactor

A novel reactor design incorporating porous ceramic tubes into a stirred jar fermentor was developed. The stirred ceramic membrane reactor has two ceramic tubular membrane units inside the vessel and maintains high filtration flux by alternating use for filtering and recovering from clogging. Each filter unit was linked for both extraction of culture broth and gas sparging. High permeability was maintained for long periods by applying the periodical control between filtering and air sparging during the stirred retention culture of Saccharomyces cerevisiae. The ceramic filter aeration system increased the k(L)a to about five times that of ordinary gas sparing. Using the automatic feeding and filtering system, cell mass concentration reached 207 g/L in a short time, while it was 64 g/L in a fed-batch culture. More than 99% of the growing cells were retained in the fermentor by the filtering culture. Both yield and productivity of cells were also increased by controlling the feeding of fresh medium and filtering the supernatant of the dense cells culture. (c) 1994 John Wiley & Sons, Inc.     

Novel method for detection of butanolides in Streptomyces coelicolor culture broth, using a His-tagged receptor (ScbR) and mass spectrometry

Gamma-butyrolactone derivative molecules in Streptomyces play a crucial role in cell density control, secondary metabolism, and cell differentiation. As their synthesis level in the cell is very low compared to those of similar N-acyl homoserine lactone molecules from gram-negative bacteria, it is very hard to analyze them even with several hundredfold concentration of the culture broth. We have developed a very quick and easy detection method using an affinity capture technique with His-tagged receptor proteins and electrospray tandem mass spectrometry. Using Streptomyces coelicolor as a model system, SCB1 was detected from only 100 ml of the culture broth after solvent extraction. This method can be further applied to detection and quantitative analysis of butanolides and inhibitor screening of the receptor molecules.     

New method using a positively charged microporous filter and ultrafiltration for concentration of viruses from tap water

The methods used to concentrate enteric viruses from water have remained largely unchanged for nearly 30 years, with the most common technique being the use of 1MDS Virozorb filters followed by organic flocculation for secondary concentration. Recently, a few studies have investigated alternatives; however, many of these methods are impractical for use in the field or share some of the limitations of this traditional method. In the present study, the NanoCeram virus sampler, an electropositive pleated microporous filter composed of microglass filaments coated with nanoalumina fibers, was evaluated. Test viruses were first concentrated by passage of 20 liters of seeded water through the filter (average filter retention efficiency was ≥ 99.8%), and then the viruses were recovered using various salt-based or proteinaceous eluting solutions. A 1.0% sodium polyphosphate solution with 0.05 M glycine was determined to be the most effective. The recovered viruses were then further concentrated using Centricon Plus-70 centrifugal ultrafilters to a final volume of 3.3 (±0.3 [standard deviation]) ml; this volume compares quite favorably to that of previously described methods, such as organic flocculation (~15 to 40 ml). The overall virus recovery efficiencies were 66% for poliovirus 1, 83% for echovirus 1, 77% for coxsackievirus B5, 14% for adenovirus 2, and 56% for MS2 coliphage. In addition, this method appears to be compatible with both cell culture and PCR assays. This new approach for the recovery of viruses from water is therefore a viable alternative to currently used methods when small volumes of final concentrate are an advantage.     

A perfusion culture system using a stirred ceramic membrane reactor for hyperproduction of IgG2a monoclonal antibody by hybridoma cells

A novel perfusion culture system for efficient production of IgG2a monoclonal antibody (mAb) by hybridoma cells was developed. A ceramic membrane module was constructed and used as a cell retention device installed in a conventional stirred-tank reactor during the perfusion culture. Furthermore, the significance of the control strategy of perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was investigated. With the highest increasing rate (deltaD, vvd per day, vvdd) of perfusion rate, the maximal viable cell density of 3.5 x 10(7) cells/mL was obtained within 6 days without any limitation and the cell viability was maintained above 95%. At lower deltaD's, the cell growth became limited. Under nutrient-limited condition, the specific cell growth rate (mu) was regulated by deltaD. During the nonlimited growth phase, the specific mAb production rate (qmAb) remained constant at 0.26 +/- 0.02 pg/cell x h in all runs. During the cell growth-limited phase, qmAb was regulated by deltaD within the range of 0.25-0.65 vvdd. Under optimal conditions, qmAb of 0.80 and 2.15 pg/cell x h was obtained during the growth-limited phase and stationary phase, respectively. The overall productivity and yield were 690 mg/L x day and 340 mg/L x medium, respectively. This study demonstrated that this novel perfusion culture system for suspension mammalian cells can support high cell density and efficient mAb production and that deltaD is an important control parameter to regulate and achieve high mAb production.     

One-step method for plasma determination of ibuprofen by chemiluminescence-coupled ultrafiltration and application in a pharmacokinetic study

A simple one‐step method is established for plasma determination of ibuprofen and its pharmacokinetic study. The method involves simple sample pre‐treatment by dilution, rapid separation by ultrafiltration (UF) and online sensitive detection by chemiluminescence (CL) based on significant intensity enhancement of ibuprofen on the weak CL of potassium permanganate and sodium sulphite in an acidic system. The calibration curve for ibuprofen is linear in the range 0.1–50.0 µg/mL in rat plasma. Average recoveries of ibuprofen at 0.80, 12.0 and 40.0 µg/mL amounted to 98.0 ± 4.2%, 101.2 ± 3.6% and 99.3 ± 5.4%, respectively. Standard deviations of intra‐ and inter‐day measurement precision and accuracy are within ±10.0%. The detection limit for ibuprofen is 10.0 µg/L in plasma samples. Pharmacokinetic study of ibuprofen by the validated method shows that the mean plasma drug concentration–time course confirms to a classical two‐compartment open model with first‐order absorption. The proposed method will be an alternative for pre‐clinical pharmacokinetic study of ibuprofen and other non‐steroidal anti‐inflammatory drugs. Copyright © 2011 John Wiley & Sons, Ltd.