Cell Culture Medium Components Research Articles

Using surface‐enhanced Raman scattering (SERS) and fluorescence spectroscopy for screening yeast extracts, a complex component of cell culture media

Yeastolate or yeast extract, which are hydrolysates produced by autolysis of yeast, are often employed as a raw material in the media used for industrial mammalian cell culture. The source and quality of yeastolate can significantly affect cell growth and production; however, analysis of these complex biologically derived materials is not straightforward. The best current method, liquid chromatography–mass spectrometry (LC‐MS), is time‐consuming and requires extensive expertise. This study describes the use of surface‐enhanced Raman scattering (SERS) and fluorescence excitation–emission matrix (EEM) spectroscopy coupled with robust principal component analysis (ROBPCA) for the rapid and facile characterization and discrimination of yeast extracts in aqueous solution. SERS using silver colloids generates time‐dependent signals, where adenine is the strongest contributor, and the spectra are stable and reproducible (< ~3%) at 180 min after mixing. Combining this spectral behavior with chemometric methods enables SERS to be used in discriminating between different yeastolate sources, for assessing lot‐to‐lot variability, and, potentially, to monitor storage‐induced compositional changes. Fluorescence EEM combined with multiway ROBPCA also provides a rapid and inexpensive method for the discrimination of yeastolate, yielding results in terms of sample discrimination very similar to that obtained with SERS. However, the EEM data does not provide the same level of chemical information that is provided by the SERS. Thus, the combination of these two methodologies has the potential to be extremely useful in biopharmaceutical manufacturing, as well as for the rapid characterization and screening of biogenic hydrolysates from animal or plant sources. Copyright © 2011 John Wiley & Sons, Ltd.

Fluorescence Excitation–Emission Matrix (EEM) Spectroscopy for Rapid Identification and Quality Evaluation of Cell Culture Media Components

The application of fluorescence excitation-emission matrix (EEM) spectroscopy to the quantitative analysis of complex, aqueous solutions of cell culture media components was investigated. These components, yeastolate, phytone, recombinant human insulin, eRDF basal medium, and four different chemically defined (CD) media, are used for the formulation of basal and feed media employed in the production of recombinant proteins using a Chinese Hamster Ovary (CHO) cell based process. The comprehensive analysis (either identification or quality assessment) of these materials using chromatographic methods is time consuming and expensive and is not suitable for high-throughput quality control. The use of EEM in conjunction with multiway chemometric methods provided a rapid, nondestructive analytical method suitable for the screening of large numbers of samples. Here we used multiway robust principal component analysis (MROBPCA) in conjunction with n-way partial least squares discriminant analysis (NPLS-DA) to develop a robust routine for both the identification and quality evaluation of these important cell culture materials. These methods are applicable to a wide range of complex mixtures because they do not rely on any predetermined compositional or property information, thus making them potentially very useful for sample handling, tracking, and quality assessment in biopharmaceutical industries.

Anti-proliferative and pro-apoptotic activities of hydroxytyrosol on different tumour cells: the role of extracellular production of hydrogen peroxide

Several recently published data suggest that the anti-proliferative and pro-apoptotic properties of hydroxytyrosol [3,4-dihydroxyphenyl ethanol (3,4-DHPEA)] on HL60 cells may be mediated by the accumulation of hydrogen peroxide (H₂O₂) in the culture medium. The aim of this study was to clarify the role played by H₂O₂ in the chemopreventive activities of 3,4-DHPEA on breast (MDA and MCF-7), prostate (LNCap and PC3) and colon (SW480 and HCT116) cancer cell lines and to investigate the effects of cell culture medium components and the possible mechanisms at the basis of the H₂O₂-producing properties of 3,4-DHPEA. The proliferation was measured by the MTT assay and the apoptosis by both fluorescence microscopy and flow cytometry. The concentration of H₂O₂ in the culture medium was measured by the ferrous ion oxidation-xylenol orange method. It was found that the H₂O₂-inducing ability of 3,4-DHPEA is completely prevented by pyruvate and that the exposure of cells to conditions not supporting the H₂O₂ accumulation (addition of either catalase or pyruvate to the culture medium) inhibited the anti-proliferative effect of 3,4-DHPEA. Accordingly, the sensitivity of the different cell lines to the anti-proliferative effect of 3,4-DHPEA was inversely correlated with their ability to remove H₂O₂ from the culture medium. With regard to the mechanism by which 3,4-DHPEA causes the H₂O₂ accumulation, it was found that superoxide dismutase increased the H₂O₂ production while tyrosinase, slightly acidic pH (6,8) and absence of oxygen (O₂) completely prevented this activity. In addition, different transition metal-chelating compounds did not modify the H₂O₂-producing activity of 3,4-DHPEA. The pro-oxidant activity of 3,4-DHPEA deeply influences its 'in vitro' chemopreventive activities. The main initiation step in the H₂O₂-producing activity is the auto-oxidation of 3,4-DHPEA by O₂ with the formation of the semiquinone, superoxide ions (O₂(-)) and 2H(+).

Metabolic profiling using HPLC allows classification of drugs according to their mechanisms of action in HL-1 cardiomyocytes

Along with hepatotoxicity, cardiotoxic side effects remain one of the major reasons for drug withdrawals and boxed warnings. Prediction methods for cardiotoxicity are insufficient. High content screening comprising of not only electrophysiological characterization but also cellular molecular alterations are expected to improve the cardiotoxicity prediction potential. Metabolomic approaches recently have become an important focus of research in pharmacological testing and prediction. In this study, the culture medium supernatants from HL-1 cardiomyocytes after exposure to drugs from different classes (analgesics, antimetabolites, anthracyclines, antihistamines, channel blockers) were analyzed to determine specific metabolic footprints in response to the tested drugs. Since most drugs influence energy metabolism in cardiac cells, the metabolite “sub-profile” consisting of glucose, lactate, pyruvate and amino acids was considered. These metabolites were quantified using HPLC in samples after exposure of cells to test compounds of the respective drug groups. The studied drug concentrations were selected from concentration response curves for each drug. The metabolite profiles were randomly split into training/validation and test set; and then analysed using multivariate statistics (principal component analysis and discriminant analysis). Discriminant analysis resulted in clustering of drugs according to their modes of action. After cross validation and cross model validation, the underlying training data were able to predict 50%–80% of conditions to the correct classification group. We show that HPLC based characterisation of known cell culture medium components is sufficient to predict a drug's potential classification according to its mode of action.

Rapid characterization and quality control of complex cell culture media solutions using raman spectroscopy and chemometrics

The use of Raman spectroscopy coupled with chemometrics for the rapid identification, characterization, and quality assessment of complex cell culture media components used for industrial mammalian cell culture was investigated. Raman spectroscopy offers significant advantages for the analysis of complex, aqueous-based materials used in biotechnology because there is no need for sample preparation and water is a weak Raman scatterer. We demonstrate the efficacy of the method for the routine analysis of dilute aqueous solution of five different chemically defined (CD) commercial media components used in a Chinese Hamster Ovary (CHO) cell manufacturing process for recombinant proteins.The chemometric processing of the Raman spectral data is the key factor in developing robust methods. Here, we discuss the optimum methods for eliminating baseline drift, background fluctuations, and other instrumentation artifacts to generate reproducible spectral data. Principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA) were then employed in the development of a robust routine for both identification and quality evaluation of the five different media components. These methods have the potential to be extremely useful in an industrial context for "in-house" sample handling, tracking, and quality control.

The growth and long term survival of Acholeplasma laidlawii in media products used in biopharmaceutical manufacturing

Acholeplasma laidlawii is a potential contaminant of bovine serum and has also been found as a contaminant in serum free cell culture media products. Anecdotal evidence of A. laidlawii contamination of tryptone soya broth circulated for a number of years before it was acknowledged that the organism could contaminate microbiological broth powders. The occasional occurrence of A. laidlawii in broth powders and possibly in powdered components of cell culture media as part of the normal bioburden poses a serious threat to routine pharmaceutical and biopharmaceutical operations where filtration is the sterilisation method of choice. Absence of visual evidence of contamination cannot be relied upon as there is variation with both organism strain and media product in the ability to produce turbidity. Strains of A. laidlawii which have been isolated from broth powders are not significantly different in temperature or media preferences from other strains. A. laidlawii is capable of growing to high titre at refrigeration and ambient temperatures in unsupplemented bacteriological sterility media or serum free cell culture media and can survive for prolonged periods in these products.

Determination of residual cell culture media components by MEKC

Folic acid, hypoxanthine, mycophenolic acid, nicotinic acid, riboflavin, and xanthine are widely used as cell culture media components in monoclonal antibody manufacturing. These components are subsequently removed during the downstream purification processes. This article describes a single MEKC method that can simultaneously determine all the listed compounds with acceptable LOD and LOQ. All the analytes were successfully separated by MEKC using running buffer containing 40 mM SDS, 20 mM sodium phosphate, and 20 mM sodium borate at pH 9.0. The MEKC method was compared to the corresponding CZE method using the same running buffer containing no SDS. The effect of SDS concentration on separation, the pH of the running buffer, and the detection wavelength were studied and optimal MEKC conditions were established. Good linearity was obtained with correlation coefficients of more than 0.99 for all analytes. Specificity, accuracy, and precision were also evaluated. The recovery was in the range of 89-112%. The precision results were in the range of 1.7-4.8%. The experimentally determined data demonstrated that the MEKC method is applicable to the determination of the six analytes in in-process samples from monoclonal antibody manufacturing processes.

Advances in Primary Recovery: Centrifugation and Membrane Technology

Significant and continual improvements in upstream processing for biologics have resulted in challenges for downstream processing, both primary recovery and purification. Given the high cell densities achievable in both microbial and mammalian cell culture processes, primary recovery can be a significant bottleneck in both clinical and commercial manufacturing. The combination of increased product titer and low viability leads to significant relative increases in the levels of process impurities such as lipids, intracellular proteins and nucleic acid versus the product. In addition, cell culture media components such as soy and yeast hydrolysates have been widely applied to achieve the cell culture densities needed for higher titers. Many of the process impurities can be negatively charged at harvest pH and can form colloids during the cell culture and harvest processes. The wide size distribution of these particles and the potential for additional particles to be generated by shear forces within a centrifuge may result in insufficient clarification to prevent fouling of subsequent filters. The other residual process impurities can lead to precipitation and increased turbidity during processing and even interference with the performance of the capturing chromatographic step. Primary recovery also poses significant challenges owing to the necessity to execute in an expedient manner to minimize both product degradation and bioburden concerns. Both microfiltration and centrifugation coupled with depth filtration have been employed successfully as primary recovery processing steps. Advances in the design and application of membrane technology for microfiltration and dead-end filtration have contributed to significant improvements in process performance and integration, in some cases allowing for a combination of multiple unit operations in a given step. Although these advances have increased productivity and reliability, the net result is that optimization of primary recovery processes has become substantially more complicated. Ironically, the application of classical chemical engineering approaches to overcome issues in primary recovery and purification (e.g., turbidity and trace impurity removal) are just recently gaining attention. Some of these techniques (e.g., membrane cascades, pretreatment, precipitation, and the use of affinity tags) are now seen almost as disruptive technologies. This paper will review the current and potential future state of research on primary recovery, including relevant papers presented at the 234th American Chemical Society (ACS) National Meeting in Boston.

Mapping and Identification of Biopharmaceutical Grade Cell Culture Media by MEKC

This paper describes a rapid micellar electrokinetic capillary chromatographic (MEKC) method for the mapping and identification of biopharmaceutical grade cell culture media. The method has been validated and we currently use it as an identity test for raw material release testing of cell culture media. The MEKC method is capable of resolving components of cell culture medium with specificity, matching the sample profile with the expected component profile, and precision. The precision of the assay was shown by comparison of peak migration times and relative peak areas of three injections of test media on each of three days. The intra-day and inter-day peak migration times and UV214 nm responses (% total peaks areas) of the assay were within acceptable ranges. The intermediate precision (% Relative Standard Deviation) of peak migration times was less than 3%. The intermediate precision (% Relative Standard Deviation) of relative percent peak areas was less than 10% for major peaks present. A linear relationship between response (% total peaks areas) and the concentration of the cell culture medium was observed, with the average correlation coefficient of >0.96 for major peaks (each accounting for greater than 2% of total peaks areas). Based on the identification by chromatographic profile, linearity, and precision, the MEKC method for the mapping of cell culture media components is suitable for the intended use.

The Phosphoinositide-Specific Phospholipase C Inhibitor U73122 (1-(6-((17β-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) Spontaneously Forms Conjugates with Common Components of Cell Culture Medium

Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in the regulation of Ca(2+) release from inositol 1,4,5-triphosphate-sensitive stores. U73122 (1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) has been extensively used as a pharmacological inhibitor of PLC to elucidate the importance of this enzyme family in signal transduction pathways. U73122 has an electrophilic maleimide group, which readily reacts with nucleophiles such as thiols and amines. In the current study the conjugation of U73122 to common components of cell culture medium, namely l-glutamine, glutathione, and bovine serum albumin (BSA), was demonstrated. The half-life of U73122 on incubation with phosphate-buffered saline (PBS), Hanks' buffered saline solution (with 2 mM glutamine), optimized basal nutrient medium (MCDB131, without BSA), complete medium, Dulbecco's modified Eagle's medium (with 2 mM l-glutamine) was approximately 150, 60, 32, 30, and 18 min, respectively. However, U73122 was not recoverable from medium supplemented with 0.5% BSA. U73122 underwent hydrolysis of the maleimide group when incubated with PBS. Glutamine conjugates of U73122 were identified in cell culture medium. Furthermore, the inhibition of epidermal growth factor-stimulated Ca(2+) release in a human epidermoid carcinoma cell line (A431) by U73122 was substantially reduced by the presence of BSA in a time-dependent manner. In complex cellular assays, the availability of U73122 to inhibit PLC may be limited by its chemical reactivity and lead to the misinterpretation of results in pharmacological assays.

Cellular Zn depletion by metal ion chelators (TPEN, DTPA and chelex resin) and its application to osteoblastic MC3T3-E1 cells

Trace mineral studies involving metal ion chelators have been conducted in investigating the response of gene and protein expressions of certain cell lines but a few had really focused on how these metal ion chelators could affect the availability of important trace minerals such as Zn, Mn, Fe and Cu. The aim of the present study was to investigate the availability of Zn for the treatment of MC3T3-E1 osteoblast-like cells and the availability of some trace minerals in the cell culture media components after using chelexing resin in the FBS and the addition of N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN, membrane-permeable chelator) and diethylenetriaminepentaacetic acid (DTPA, membrane-impermeable chelator) in the treatment medium. Components for the preparation of cell culture medium and Zn-treated medium have been tested for Zn, Mn, Fe and Cu contents by atomic absorption spectrophotometer or inductively coupled plasma spectrophotometer. Also, the expression of bone-related genes (ALP, Runx2, PTH-R, ProCOL I, OPN and OC) was measured on the cellular Zn depletion such as chelexing or TPEN treatment. Results have shown that using the chelexing resin in FBS would significantly decrease the available Zn (p<0.05) (39.4 ± 1.5 µM vs 0.61 ± 10.15 µM) and Mn (p<0.05) (0.74 ± 0.01 µM vs 0.12 ± 0.04 µM). However, levels of Fe and Cu in FBS were not changed by chelexing FBS. The use of TPEN and DTPA as Zn-chelators did not show significant difference on the final concentration of Zn in the treatment medium (0, 3, 6, 9, 12 µM) except for in the addition of higher 15 µM ZnCl2 which showed a significant increase of Zn level in DTPA-chelated treatment medium. Results have shown that both chelators gave the same pattern for the expression of the five bone-related genes between Zn- and Zn+, and TPEN-treated experiments, compared to chelex-treated experiment, showed lower bone-related gene expression, which may imply that TPEN would be a stronger chelator than chelex resin. This study showed that TPEN would be a stronger chelator compared to DTPA or chelex resin and TPEN and chelex resin exerted cellular zinc depletion to be enough for cell study for Zn depletion.

RhoA/ROCK Signaling Regulates Sox9 Expression and Actin Organization during Chondrogenesis

Endochondral ossification is initiated by the differentiation of mesenchymal precursor cells to chondrocytes (chondrogenesis). This process is characterized by a strong interdependence of cell shape, cytoskeletal organization, and the onset of chondrogenic gene expression, but the molecular mechanisms mediating these interactions are not known. Here we investigated the role of the RhoA/ROCK pathway, a well characterized regulator of cytoskeletal organization, in chondrogenesis. We show that pharmacological inhibition of ROCK signaling by Y27632 resulted in increased glycosaminoglycan synthesis and elevated expression of the chondrogenic transcription factor Sox9, whereas overexpression of RhoA in the chondrogenic cell line ATDC5 had the opposite effects. Suppression of Sox9 expression by ROCK signaling was achieved through repression of Sox9 promoter activity. These molecular changes were accompanied by reorganization of the actin cytoskeleton, where RhoA/ROCK signaling suppressed cortical actin organization, a hallmark of differentiated chondrocytes. This led us to analyze the regulation of Sox9 expression by drugs affecting cytoskeletal dynamics. Both inhibition of actin polymerization by cytochalasin D and stabilization of existing actin filaments by jasplakinolide resulted in increased Sox9 mRNA levels, whereas inhibition of microtubule polymerization by colchicine completely blocked Sox9 expression. In conclusion, our data suggest that RhoA/ROCK signaling suppresses chondrogenesis through the control of Sox9 expression and actin organization.

Validation of cell culture media components

It is difficult to predict the influence that a particular combination of processes and culture media may have on cells and tissues manipulated in vitro. Accordingly, it is important that the procedures, media and reagents used in the manipulation of cells for clinical application are specified and have been appropriately assessed for safe and reliable use. This presents an approach to the validation of cell culture media and reagents which is a vital process in the overall quality assurance that is increasingly demanded for cells and tissues used in humans.

Effect of serum from chickens treated with clenbuterol on myosin accumulation, beta-adrenergic receptor population, and cyclic AMP synthesis in embryonic chicken skeletal muscle cell cultures.

Broiler chickens at 35 d of age were fed 1 ppm clenbuterol for 14 d. This level of dietary clenbuterol led to 5-7% increases in the weights of leg and breast muscle tissue. At the end of the 14-d period, serum was prepared from both control and clenbuterol-treated chickens, and was then employed as a component of cell culture media at a final concentration of 20% (v/v). Muscle cell cultures were prepared from both the leg and the breast muscle groups of 12-d chick embryos. Treatment groups included control chicken serum to which 10 nM, 50 nM, and 1 microM clenbuterol had been added, as well as cells grown in media containing 10% horse serum. Cultures were subjected to each treatment for 3 d, beginning on the seventh d in culture. Neither the percent fusion nor the number of nuclei in myotubes was significantly affected by any of the treatments. The quantity of myosin heavy chains (MHCs) was not increased by serum from clenbuterol-treated chickens in either breast or leg muscle cultures; however, the MHC quantity was 50-150% higher in cultures grown in control chicken serum to which 10 and 50 nM clenbuterol had also been added. The beta-adrenergic receptor (betaAR) population was 4000-7000 betaARs per cell in cultures grown in chicken serum, with leg muscle cultures having approximately 25-30% more receptors than breast muscle cultures. Receptor population was not significantly affected by the presence of clenbuterol or by the presence of serum from clenbuterol-treated chickens. In contrast, the betaAR population in leg and breast muscle cultures grown in the presence of 10% horse serum was 16,000-18,000 betaARs per cell. Basal concentration of cyclic adenosine 3':5'monophosphate (cAMP) was not significantly affected by the treatments. When cultures grown in chicken serum were stimulated for 10 min with 1 microM isoproterenol, limited increases of 12-20% in cAMP concentration above the basal levels were observed. However, when cultures grown in the presence of horse serum were stimulated with 1 microM isoproterenol, cAMP concentration was stimulated 5- to 9-fold above the basal levels. Thus, not only did cells grown in horse serum have a higher betaAR population, but also each receptor had a higher capacity for cAMP synthesis following isoproterenol stimulation. Finally, the hypothesis that clenbuterol exerts its action on muscle protein content by changes in cAMP concentration was tested. No correlation was apparent between basal cAMP concentration and MHC content.

Role of vitamins in determining apoptosis and extent of suppression by bcl-2 during hybridoma cell culture.

The identification of cell culture media components that may instigate apoptosis in cell lines used for the production of commercial antibodies and recombinant proteins, is crucial to aid the development of improved media for reduced cell death and to understand the role of nutrient components in cell survival and maintenance. Here we determine the impact of depriving all or individual B-group media vitamins either, D-CaPantothenate (DCaP), choline chloride (CC), riboflavin (Rb), i-inositol, nicotinamide (NAM), pyridoxal hydrochloride (PyrHCl), folic acid (FA), or thiamine hydrochloride (ThHCl) on hybridoma cell growth and viability using fluorescence microscopy techniques. Cultivation in media deprived of all these vitamins prevented cell proliferation from reaching maximum capacity while increasing cell death rate, predominantly via apoptosis. Deletion of either DCaP, CC, or Rb showed that these components were most likely responsible for the development of apoptosis. Exclusion of either i-inositol, NAM or PyrHCl failed to inhibit cell growth and viability, while marginal improvements in viability were noted by ThHCl deprivation and more so by FA exclusion. Over-expression of the anti-apoptotic gene bcl-2 suppressed cell death initiated by all or single vitamin (either DCaP, CC or Rb) deprivation. The involvement of bcl-2 activity, established a close association between small vitamin molecules particularly DCaP, CC or Rb and the biochemical activation of apoptosis.

Contribution of serum and cellular semicarbazide-sensitive amine oxidase to amine metabolism and cardiovascular toxicity.

Semicarbazide-sensitive amine oxidase (SSAO) plays a role in the in vivo and in vitro toxicity of several environmental and endogenous amines. We investigated the role of SSAO as a component of cell culture medium (through addition of fetal calf serum (FCS)) compared to intracellular SSAO in the in vitro cytotoxicity of three amines and metabolites. Smooth muscle cells and beating cardiac myocytes were grown in 96-well plates and exposed to various concentrations and combinations of FCS in medium, amines (allylamine, AA; benzylamine, BZA; and methylamine, MA), and amine metabolites (aldehydes: acrolein, benzaldehyde, and formaldehyde; hydrogen peroxide, H2O2; ammonia, NH3). Amine and amine metabolite cytotoxicity was quantified by monitoring cell viability. SSAO activity was measured in FCS, cardiovascular cells, or rat plasma by a radioenzymatic assay using [14C]BZA. Our data show that AA and its aldehyde metabolite, acrolein, were the most toxic compounds to both cell types. However, AA toxicity was FCS-dependent in both cell types, while BZA, MA, and amine metabolite (i.e., aldehydes, H2O2, and NH3) cytotoxicity showed little FCS dependence. In these experiments, medium containing 10% FCS had a calculated amine metabolic capacity that was 30- to 50-fold that of the cultured smooth muscle cellular content in a single well of a 96-well plate. Our study demonstrates that SSAO in FCS contributes to amine metabolism and cytotoxicity to rat cardiovascular cells in vitro and how critical it is to evaluate serum for its role in mechanisms of amine toxicity in vitro and in vivo.

Targeted gene transfer in eucaryotic cells by dye-assisted laser optoporation

The blue beam of an Argon laser (488 nm) has been focused on the cell membrane in the presence of phenol-red, an usual component of cell culture media, through a 100 × objective. At the site of the beam impact, due probably to local temperature changes, the cell membrane modifies its permeability. As a consequence of the hit, circular areas, whose radius may be apparently regulated by changing the irradiation time and/or the radiation intensity (energy), appear on the wall, last for a short time and fade spontaneously within 1–2 minutes. No evident signs of cell injury or hurt have been observed afterward. Plasmid DNA, purposely added to culture fluid, easily slips in the cytoplasm; utilizing such approach, thereafter indicated as ‘optoporation’, we have successfully transfected two genes, namely β-galactosidase and chloramphenicol-acetyl-transferase in murine NIH 3T3 fibroblasts. Therefore optoporation represents an additional procedure for gene transfer with several advantages over already available methods: (1) it only takes advantage of the presence of phenol-red, a normal cell medium component, with no need of addition of extraneous substances; (2) it is a very mild treatment virtually suitable for any cell type and (3) it allows transfection of selected cells even in the presence of cells of different type (providing that they are morphologically distinguishable).

Growth of cultured rabbit renal tubular cells does not require exogenous glutamine

Following the pioneering work of Eagle et a! [1], glutamine, which is the most abundant amino acid in plasma (0.5 to 0.6 mM) [2] and an important source of energy and nitrogen for cell metabolism and growth [3], is considered to be a mandatory component of cell culture media. In renal epithelial cell cultures, it is added often at a final concentration of 2 to 5 mrvi [4—10]. However, this is not necessarily useful in primary cultures of renal proximal tubular cells from the rabbit, the most widely used species for renal cell cultures. Indeed, glutamine synthetase is present in the renal cortex of this species; in addition, the activity of phosphate-activated glutaminase, the enzyme which initiates glutamine metabolism and yields glutamate and ammonium [3], is low in the rabbit kidney [2]. Moreover, freshly isolated rabbit proximal tubules do not or hardly remove glutamine in net amounts when they are incubated in the presence of glutamine concentrations varying from 1 to 5 mtvi [11]. In an attempt to clarify this point, we cultured rabbit proximal tubular cells on a permeable collagen membrane and investigated whether the addition of glutamine to the culture medium influences cellular growth and glycolysis. The results obtained show that omission of exogenous glutamine does not alter the growth of rabbit renal proximal tubular cells in primary culture, and that the provision of glutamine by the glutamine synthetase reaction is sufficient and mandatory for their growth and DNA synthesis. Addition of glutamine, which stimulated glycolysis in freshly isolated proximal tubules, failed to do so in cultured rabbit renal cells.

Liarozole potentiates the cancer chemopreventive activity of and the up-regulation of gap junctional communication and connexin43 expression by retinoic acid and beta-carotene in 10T1/2 cells.

Liarozole has been reported to inhibit P450 enzymes responsible for the catabolism of retinoic acid. This suggests that it may increase the effectiveness of cancer chemopreventive agents, such as retinoic acid, and pro-vitamin A carotenoids, such as beta-carotene, which may yield retinoids. To test this we have utilized the 10T1/2 cell assay system of neoplastic transformation. Simultaneous treatment with Liarozole (10(-5) M) potentiated by a factor of 1000 the ability of low concentrations of retinoic acid (10(-10) M) to inhibit carcinogen-induced neoplastic transformation, to up-regulate gap junctional communication and to increase connexin43 expression. When tested under conventional culture conditions, Liarozole itself partially suppressed neoplastic transformation and up-regulated gap junctional communication; this ability appears due to the presence of retinol in the serum component of cell culture medium. When assays for junctional communication and of connexin43 expression were performed under defined conditions, in the absence of serum, Liarozole was ineffective alone, yet still augmented the effects of retinoic acid. HPLC analysis of cell culture medium demonstrated that Liarozole (10(-5) M) completely protected retinoic acid (10(-6) M) from catabolism over a 48 h period. Potential effects of Liarozole on the activities of carotenoids were also examined. Inhibition of neoplastic transformation by the pro-vitamin A carotenoid beta-carotene, but not by the non-pro-vitamin A carotenoid canthaxanthin, was moderately potentiated by Liarozole. The augmentation of response to beta-carotene was more apparent when tested under defined conditions; here Liarozole strongly increased junctional communication and Cx43 expression. In contrast, Liarozole did not potentiate the activity of canthaxanthin under defined conditions, while increasing the activity of 4-keto retinoic acid, a likely metabolite. Liarozole also elevated connexin43 expression in early passage human fibroblasts. These data indicate that rapid catabolism of retinoic acid limits its in vitro activities, that a portion of the action of beta-carotene as a cancer preventive agent is due to its conversion to retinoic acid and that canthaxathin exerts its chemopreventive action largely independent of conversion to 4-keto retinoic acid.

Antibacterial Silver

The antibacterial activity of silver has long been known and has found a variety of applications because its toxicity to human cells is considerably lower than to bacteria. The most widely documented uses are prophylactic treatment of burns and water disinfection. However, the mechanisms by which silver kills cells are not known. Information on resistance mechanisms is apparently contradictory and even the chemistry of Ag+ in such systems is poorly understood. Silver binds to many cellular components, with membrane components probably being more important than nucleic acids. It is difficult to know whether strong binding reflects toxicity or detoxification: some sensitive bacterial strains have been reported as accumulating more silver than the corresponding resistant strain, in others the reverse apparently occurs. In several cases resistance has been shown to be plasmid mediated. The plasmids are reported as difficult to transfer, and can also be difficult to maintain, as we too have found. Attempts to find biochemical differences between resistant and sensitive strains have met with limited success: differences are subtle, such as increased cell surface hydrophobicity in a resistant Escherichia coli. Some of the problems are due to defining conditions in which resistance can be observed. Silver(I) has been shown to bind to components of cell culture media, and the presence of chloride is necessary to demonstrate resistance. The form of silver used must also be considered. This is usually water soluble AgNO3, which readily precipitates as AgCl. The clinically preferred compound is the highly insoluble silver sulfadiazine, which does not cause hypochloraemia in burns. It has been suggested that resistant bacteria are those unable to bind Ag+ more tightly than does chloride. It may be that certain forms of insoluble silver are taken up by cells, as has been found for nickel. Under our experimental conditions, silver complexed by certain ligands is more cytotoxic than AgNO3, yet with related ligands is considerably less toxic. There is evidently a subtle interplay of solubility and stability which should reward further investigation.