Treatment of mycoplasma contamination in a large panel of cell cultures

Summary Mycoplasmal contamination remains a significant impediment to the culture of eukaryotic cells. For certain cultures, attempts to eliminate the infection are feasible alternatives to the normally recommended disposal of the contaminated culture. Here, three antibiotic regimens for mycoplasmal decontamination were compared in a large panel of naturally infected cultures: a 1-wk treatment with the fluoroquinolone mycoplasma removal agent (MRA), a 2-wk treatment with the fluoroquinolone ciprofloxacin, and three rounds of a sequential 1-wk treatment with BM-Cyclin containing tiamulin and minocyclin. These antibiotic treatments had a high efficiency of permanent cure: MRA 69%, ciprofloxacin 75%, BM-Cyclin 87%. Resistance to mycoplasma eradication was observed in some cell cultures: BM-Cyclin 0%, MRA 20%, ciprofloxacin 20%. Nearly all resistant contaminants that could be identified belonged to the speciesMycoplasma arginini andM. orale. Detrimental effects of the antibiotics were seen in the form of culture death caused by cytotoxicity (in 5 to 13% of the cultures). Alterations of the cellular phenotypic features or selective clonal outgrowth might represent further untoward side effects of exposure to these antibiotics. Overall, antibiotic decontamination of mycoplasmas is an efficient, inexpensive, reliable, and simple method: 150/200 (75%) chronically and heavily contaminated cultures were cured and 50/200 (25%) cultures could not be cleansed and were either lost or remained infected. It is concluded that eukaryotic cell cultures containing mycoplasmas are amenable to antibiotic treatment and that a cure rate of three-quarters is a reasonable expectation.     

Rapid detection of mycoplasma contamination in cell cultures using sybr green-based real-time polymerase chain reaction

Summary We have developed a simple method for rapid detection of mycoplasma contamination in cell cultures using SYBR Green-based real-time polymerase chain reaction (PCR). To detect eight common contaminant mollicutes, including Mycoplasma (M. arginini, M. fermentans, M. orale, M. hyorhinis, M. hominis, M. salivarium, M. pirum) and Acholeplasma laidlawii, four primers were prepared based on the 23S rRNA regions. Using these primers and a minimum of 100 fg of mycoplasma genomic DNA, the 23S rRNA regions of these eight mycoplasma species were consistently amplified by real-time PCR. In contrast, no specific specific amplification product was observed using DNA templates prepared from various mammalian cell lines. Frozen and cultured samples of several cell lines were tested for mycoplasma contamination to evaluated the utility of this method. Of 25 samples that tested positive for mycoplasma by Hoechst staining, which requires two passages of cell cultures started from frozen samples, mycoplasma was detected by real-time PCR in 24 samples of cell extracts prepared directly from frozen samples. When cultured samples were used for this assay, the accuracy of the diagnoses was further improved. Thus, this technique, which is simple, rapid, and sensitive enough for practical application, in suitable for handling many samples and for routine screening for mycoplasma contamination of cell cultures.     

A new fluorescent dye for cell tracing and mitochondrial imaging in vitro and in vivo

Mitochondria contribute to redox and calcium balance, and apoptosis thus regulating cellular fate. In the present study, mitochondrial staining applying a novel dye, V07‐07059, was performed in human embryonic kidney cells, a human vascular endothelial cell line and primary human mononuclear cells. The new fluorescent mega Stokes dye (peak excitation: 488 nm, peak emission: 554 nm) showed superior fluorescent properties and stability. V07‐07059 stains mitochondria dependent on their membrane potential and is safe to use in vitro and in vivo. Unlike other dyes applied in this context (e.g. Tetramethylrhodamine methyl ester), V07‐07059 only marginally inhibits mitochondrial respiration and function. V07‐07059 enables real time imaging of mitochondrial trafficking and remodeling. Prolonged staining with V07‐07059 demonstrated the dyes suitability as a novel probe to track cells. In comparison to the widely used standard for cell proliferation and tracking studies 5(6)‐diacetate N‐succinimidyl ester, V07‐07059 proved superior regarding toxicity and photostability.

     

Detection of microbial contaminants in mammalian cell cultures using a new fluorescence-based staining method

Aims: Microbial contamination of cell culture production processes is a current concern for biopharmaceutical industries. Traditional testing methods require several days to detect contamination and may advantageously be replaced by a rapid detection method. We developed a new method combining membrane filtration to microcolonies fluorescence staining method (MFSM) and compared it to epifluorescence microscopy. Methods and Results: Both methods were used to detect bacteria in CHO cells cultures. The epifluorescence microscopy showed to be limited by filterability, media interference and nonrobustness issues, whereas MFSM enabled consistent detection of Bacillus cereus, Staphylococcus epidermidis and Propionibacterium acnes after, respectively, 8, 9 and 48 h of incubation. Thanks to the nondestructive feature of the MFSM, stained membranes could be reincubated on culture media to yield visible colonies used for identification. Conclusions: The new method described in this study showed its ability to detect microbial contaminants in cell culture samples with time‐to‐results from 2–5 times shorter than the traditional testing method. Significance and Impact of the Study: The MFSM can be used as monitoring tool for cell cultures to significantly shorten detection times of microbial contamination, while preserving the ability to identify the contaminants and their viability.     

Impacts on product quality attributes of monoclonal antibodies produced in CHO cell bioreactor cultures during intentional mycoplasma contamination events

A mycoplasma contamination event in a biomanufacturing facility can result in costly cleanups and potential drug shortages. Mycoplasma may survive in mammalian cell cultures with only subtle changes to the culture and penetrate the standard 0.2-µm filters used in the clarification of harvested cell culture fluid. Previously, we reported a study regarding the ability of Mycoplasma arginini to persist in a single-use, perfusion rocking bioreactor system containing a Chinese hamster ovary (CHO) DG44 cell line expressing a model monoclonal immunoglobulin G 1 (IgG1) antibody. Our previous work showed that M. arginini affects CHO cell growth profile, viability, nutrient consumption, oxygen use, and waste production at varying timepoints after M. arginini introduction to the culture. Careful evaluation of certain identified process parameters over time may be used to indicate mycoplasma contamination in CHO cell cultures in a bioreactor before detection from a traditional method. In this report, we studied the changes in the IgG1 product quality produced by CHO cells considered to be induced by the M. arginini contamination events. We observed changes in critical quality attributes correlated with the duration of contamination, including increased acidic charge variants and high mannose species, which were further modeled using principal component analysis to explore the relationships among M. arginini contamination, CHO cell growth and metabolites, and IgG1 product quality attributes. Finally, partial least square models using NIR spectral data were used to establish predictions of high levels (≥10⁴ colony-forming unit [CFU/ml]) of M. arginini contamination, but prediction of levels below 10⁴ CFU/ml were not reliable. Contamination of CHO cells with M. arginini resulted in significant reduction of antibody product quality, highlighting the importance of rapid microbiological testing and mycoplasma testing during particularly long upstream bioprocesses to ensure product safety and quality.     

A fluorescent screening assay for collagenase using collagen labeled with 2-methoxy-2,4-diphenyl-3(2H)-furanone

This report describes the use of the compound 2-methoxy-2,4-diphenyl-3(2H)-furanone to label collagen as a substrate for the detection of mammalian collagenase in a fluorescent assay which is suitable for screening large numbers of samples. The compound 2-methoxy-2,4-diphenyl-3(2H)-furanone presents distinct advantages over other fluorophores, since both the unbound reagent and its hydrolysis products are nonfluorescent. The labeling procedure uses commercially available collagen, is fast and simple, and gives a 90% yield of labeled substrate. The fluorescent collagen substrate is stable and retains fluorescence over a wide range of pH. The assay detects, reproducibly, metal-dependent collagenase activity in microliter volumes of conditioned media from cultured neoplastic cells or in chromatographic fractions from such media.     

Structural alterations in cell organelles induced by photodynamic treatment with chlorin p6-histamine conjugate in human oral carcinoma cells probed by 3D fluorescence microscopy

We have explored the intracellular cell organelle's structural alterations after photodynamic treatment with chlorin p₆-histamine conjugate (Cp₆-his) in human oral cancer cells. Herein, the cells were treated with Cp₆-his (10 μm) and counterstained with organelle-specific fluorescence probes to find the site of intracellular localization using confocal microscopy. For photodynamic therapy (PDT), the cells were exposed to ~30 kJ/m² red light (660 ± 20 nm) to induce ~90% cytotoxicity. We used the three-dimensional (3D) image reconstruction approach to analyze the photodynamic damage to cell organelles. The result showed that Cp₆-his localized mainly in the endoplasmic reticulum (ER) and lysosomes but not in mitochondria and Golgi apparatus (GA). The 3D model revealed that in necrotic cells, PDT led to extensive fragmentation of ER and fragmentation and swelling of GA as well. Results suggest that the indirect damage to GA occurred due to loss of connection between ER and GA. Moreover, in damaged cells with no sign of necrosis, the perinuclear ER appeared condensed and surrounded by several small clumps at the peripheral region of the cell, and the GA was observed to form a single condensed structure. Since these structural changes were associated with apoptotic cell death, it is suggested that the necrotic and apoptotic death induced by PDT with Cp₆-his is determined by the severity of damage to ER and indirect damage to GA. The results suggest that the indirect damage to cell organelle apart from the sites of photosensitizer localization and the severity of damage at the organelle level contribute significantly to the mode of cell death in PDT.     

Synthesis of 3-[4-(dimethylamino)phenyl]alkyl-2-oxindole derivatives and their effects on neuronal cell death

Novel 3-[4-(dimethylamino)phenyl]alkyl-2-oxindole analogs were synthesized by either of the following two pathways: (1) a sequence of Knoevenagel condensation of oxindole with (4-dimethylamino)cinnamaldehyde–hydrogenation, or (2) alkylation of oxindole dianion with [(4-dimethylamino)phenyl]alkyl halides. Subsequent alkylation at C-3 and/or N-1 of the oxindole skeleton by anion-based methods provided additional substituted derivatives for structure-activity relationship studies. Their effects on neuronal cell death induced by oxidative stress were evaluated by lactate dehydrogenase assay. Compounds with the alkyl chain length of 2–4 significantly suppressed the neuronal cell death. No significant change occurred in the activity by substitution with less-polar groups. The stereochemistry at C-3 of the oxindole core was also irrelevant for the neuroprotective effects of these compounds.     

Synthesis, structures and properties of new cyclometalated iridium(III) complexes of 3-methoxy-6-methyl-2-(naphthalen-2-yl)pyridine

The reaction between 3-methoxy-6-methyl-2-(naphthalen-2-yl)pyridine 1 and IrCl₃ was performed in an attempt to synthesize a cyclometalated Ir(III) Cl-bridged dimer 2. An unexpected Ir(III) complex 3 was isolated, which was a five-coordinate bis-cyclometalated Ir(III) complex. The complexes 2 and 3 were converted to the same mononuclear complex 4 upon reacting with acetylacetonate (acac), respectively. All of the new compounds have been fully characterized by elemental analysis, IR, ¹H, ¹³C{¹H} NMR and ESI-MS. Additionally, the crystal structures and properties of these Ir(III) complexes are investigated. The most striking common features of the structures of 2 and 3 is intramolecular C-H···Cl hydrogen bonds. The complex 4 shows yellow phosphorescence with structureless emission peaks at about 556 nm.     

Fast accumulation of few polyhedra mutants during passage of a Spodoptera frugiperda multicapsid nucleopolyhedrovirus (Baculoviridae) in Sf9 cell cultures

Baculoviruses are a group of viruses that infect invertebrates and that have been used worldwide as a biopesticide against several insect pests of the Order Lepidoptera. In Brazil, the baculovirus Spodoptera frugiperda multicapsid nucleopolyhedrovirus (SfMNPV; Baculoviridae) has been used experimentally to control S. frugiperda (Lepidoptera: Noctuidae), an important insect pest of corn (maize) fields and other crops. Baculoviruses can be produced either in insect larvae or in cell culture bioreactors. A major limitation to the in vitro production of baculoviruses is the rapid generation of mutants when the virus undergoes passages in cell culture. In order to evaluate the potential of in vitro methods of producing SfMNPV on a large-scale, we have multiplied a Brazilian isolate of this virus in cell culture. Extensive formation of few polyhedra mutants was observed after only two passages in Sf9 cells.     

Variable-angle epifluorescence microscopy: a new way to look at protein dynamics in the plant cell cortex

Live-cell microscopy imaging of fluorescent-tagged fusion proteins is an essential tool for cell biologists. Total internal reflection fluorescence microscopy (TIRFM) has joined confocal microscopy as a complementary system for the imaging of cell surface protein dynamics in mammalian and yeast systems because of its high temporal and spatial resolution. Here we present an alternative to TIRFM, termed variable-angle epifluorescence microscopy (VAEM), for the visualization of protein dynamics at or near the plasma membrane of plant epidermal cells and root hairs in whole, intact seedlings that provides high-signal, low-background and near real-time imaging. VAEM uses highly oblique subcritical incident angles to decrease background fluorophore excitation. We discuss the utilities and advantages of VAEM for imaging of fluorescent fusion-tagged marker proteins in studying cortical cytoskeletal and membrane proteins. We believe that the application of VAEM will be an invaluable imaging tool for plant cell biologists.     

Temperature effects on alpha-crystallin structure probed by 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone, an environmentally sensitive two-wavelength fluorescent dye covalently attached to the single Cys residue

The single Cys residue in the C-terminal domain of bovine eye lens alpha-crystallin was covalently labelled with 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone. This novel SH-reactive two-band ratiometric fluorescent dye is characterized by excited state intramolecular proton transfer reaction yielding two highly emissive N* and T* bands separated by more than 100 nm. Their relative intensities are known to be highly sensitive to the H-bonding ability of the environment. Properties of the environment of the dye attached to the protein were studied under native-like conditions and at a range of elevated temperatures that are known to facilitate alpha-crystallin chaperone-like activity. We observe that on heating, the environment of the dye becomes more flexible and the H-bonding of the dye with the protein vicinity decreases. The spectroscopic properties observed on heating were partially restored after cooling, but the initial values were not reached on the time scale of our experiments (up to 3 h). This suggests that the changes of the dye microenvironment are connected with the rearrangements of alpha-crystallin quaternary structure. Since there is only one Cys residue in alphaA subunit of alpha-crystallin (whereas alphaB subunit contains no Cys), we attributed the observed temperature-induced changes of the dye's microenvironment to the particular site within alpha-crystallin molecule.     

Synthesis and physical properties of phosphatidylcholine labelled with 9-(2-anthryl)nonanoic acid,a new fluorescent probe

Synthesis and physical properties of a new anthracene fatty acid, 9-(2-anthryl)nonanoic acid, and the corresponding anthracene-phosphatidylcholines which were obtained by condensing the acid with sn-1-palmitoyl-lysophosphatidylcholine (PAPC) and with egg lysophosphatidylcholine (EAPC) are described. Differential scanning calorimetry experiments show that these lipids can undergo a liquid-crystal to gel phase transition at temperatures of 15°C and 18°C for EAPC and PAPC, respectively. In monolayers, PAPC exhibits a compression curve nearly superimposable to that of dipalmitoylphosphatidylcholine (DPPC), with a molecular area of 0.48 nm² at π = 30 mN m^?1. The data indicate that in these lipids, the anthracene group is only slightly more bulky than a normal acyl chain and that it does not significantly affect the regular phospholipid molecular packing. In ethanol solutions or when incorporated into egg phosphatidylcholine liposomes in a molar ratio of 1%, these lipids display UV absorption spectra and fluorescence emission spectra similar to those of 2-methyl anthracene. For EAPC liposomes, a broad and structureless fluorescence emission spectrum centered at around 450 nm, was recorded, suggesting the occurrence of anthracene excimers. As ascertained by UV spectrophotometry, differential scanning calorimetry, fluorescence polarization and anthracene photodimerization experiments, EAPC displays good miscibility properties with lipids in the liquid state (egg phosphatidylcholine) or in the gel state (distearoylphosphatidylcholine (DSPC)). The potential of these anthracene derivatives for studying the dynamics and the topological distribution of lipids in biomembranes is discussed.     

The effect of 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline on cell growth, cell cycle, induction of DNA fragmentation, and activity of caspase 3 in murine leukemia L1210 cells and fibroblast NIH-3T3 cells

Quinazolines are multitarget agents, which have broad spectrum of biological activity, and some of them are now in cancer clinical testing. 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline is a new synthetically prepared derivative, which in our previous study showed cytotoxic effects on cancer cell lines HeLa and B16. Quinazoline, at micromolar concentrations, induced morphological changes and necrosis of B16 cells, and at nanomolar concentrations it produced changes of F-actin cytoskeleton. It did not cause changes in the cell cycle, did not induce apoptotic cell death in B16 cells, did not have a mutagenic effect, and did not even behave as a typical intercalating agent. Little significant reduction of tumor volume in intramuscular transplanted B16 cells was observed. The aim of the present study was to examine the cytotoxic effect of 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline on murine leukemia L1210 cells and fibroblast NIH-3T3 cells. Induction of cell morphology and cell cycle changes, induction of apoptosis and caspase 3 activity were studied. Quinazoline acted cytotoxically on both cell lines. The sensitivity of leukemia L1210 cells to the quinazoline was higher than that of fibroblast NIH-3T3. The IC(100) was 12 microM for L1210 cells and 24 microM for NIH-3T3 cells. No effect of quinazoline on the cell cycle profile of L1210 and NIH-3T3 was detected, however, quinazoline induced an increase of the sub-G(0) cell fraction, apoptotic DNA fragmentation, and apoptotic morphological changes at a concentration of 12 microM. This quinazoline concentration induced caspase 3 activity. Our results demonstrated that induction of apoptotic cell death via activation of caspase 3 contributed to the cytotoxic effects of 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline in murine leukemia L1210 cells.     

Radiolabeled guanine derivatives for the in vivo mapping of O(6)-alkylguanine-DNA alkyltransferase: 6-(4-[(18)F]Fluoro-benzyloxy)-9H-purin-2-ylamine and 6-(3-[(131)I]Iodo-benzyloxy)-9H-purin-2-ylamine

Two radiolabeled analogues of 6-benzyloxy-9H-purin-2-ylamine (O(6)-benzylguanine; BG) potentially useful in the in vivo mapping of O(6)-alkylguanine-DNA alkyltransferase (AGT) were synthesized. Fluorine-18 labeling of the known 6-(4-fluoro-benzyloxy)-9H-purin-2-ylamine (FBG; 6) was accomplished by the condensation of 4-[(18)F]fluorobenzyl alcohol with 2-aminopurin-6-yltrimethylammonium chloride (4) or 2-amino-6-chloropurine in average decay-corrected radiochemical yields of 40 and 25%, respectively. Unlabeled 6-(3-iodo-benzyloxy)-9H-purin-2-ylamine (IBG; 7) was prepared from 4 and 3-iodobenzyl alcohol. Radioiodination of 9, prepared from 7 in two steps, and subsequent deprotection gave [(131)I]7 in about 70% overall radiochemical yield. The IC(50) values for the inactivation of AGT from CHO cells transfected with pCMV-AGT were 15 nM for IBG and 50 nM for FBG. The binding of [(18)F]6 and [(131)I]7 to purified AGT was specific and saturable with both exhibiting similar IC(50) values (5-6 microM).     

Stereospecific synthesis of l-2-amino-3-(7-methoxy-4-coumaryl)propionic acid, an alternative to tryptophan in quenched fluorescent substrates for peptidases

A high-yielding synthesis of the highly fluorescent amino acid l-2-amino-3-(7-methoxy-4-coumaryl)propionic acid (l-Amp) has been developed via (2R)-bornane-10,2-sultam glycinate. l-Amp facilitates the synthesis of sensitive fluorogenic proteinase substrates with N-terminal solubilising or reactive groups.     

Stereospecific synthesis of l-2-amino-3-(7-methoxy-4-coumaryl)propionic acid,an alternative to tryptophan in quenched fluorescent substrates for peptidases

Summary A ligh-yielding synthesis of the highly fluorescent amino acidl-2-amino-3-(7-methoxy-4-coumaryl)propionic acid (l-Amp) has been developed via (2R)-bormane-10,2-sultam glycinate.l-Amp facilitates the synthesis of sensitive fluorogenic proteinase substrates with N-terminal solubilising or reactive group.     

Covalent binding of 1-nitro-9-(3-dimethyl-n-propylamino) acridine, a new antitumor drug, to DNA of Ehrlich ascites tumor cells in vivo.

After exposing a line of rat liver epithelial cells to a single dose of the carcinogen N-acetoxy-2-acetylaminofluorene (N-acetoxy-AAF), a dose-dependent decrease in [³H]uridine incorporation into total cellular RNA was found. Approx. 50% inhibition occurred with 0.5 μg/ml of the compound. The kinetics of the response, the effects of actinomycin D, and the fractionation of the newly synthesized RNA by polyacrylamide gel electrophoresis indicated preferential inhibition of the synthesis of 45S ribosomal RNA precursor and a relative sparing of the synthesis of heterogeneous nuclear RNA.     

Methyl 2-methoxy-7-(4-methylbenzoyl)-4-oxo-6-p-tolyl-4H-furo[3,2-c]pyran-3-carboxylate:A combined experimental and theoretical investigation

The title compound, methyl 2-methoxy-7-(4-methylbenzoyl)-4-oxo-6-p-tolyl-4H-furo[3,2-c]pyran-3-carboxylate (C₂₅H₂₀O₇), was prepared and characterized by IR and single-crystal X-ray diffraction (XRD). The compound crystallizes in the triclinic space group P ?1 with a?=?8.9554(9) Å, b?=?10.0018(10) Å, c?=?12.7454(13) Å, α?=?67.678(7)°, β?=?89.359(8)° and γ?=?88.961(8)°. In addition to the molecular geometry from X-ray experiment, the molecular geometry and vibrational frequencies of the title compound in the ground state have been calculated using semiempirical AM1 and PM3 methods, as well as Hartree-Fock (HF) and density functional (B3LYP) levels of theory with 6–31G(d) basis set. To determine conformational flexibility, molecular energy profile of the title compound was obtained by semi-empirical (AM1) calculations with respect to two selected degrees of torsional freedom, which were varied from ?180° to +180° in steps of 10°. Besides, frontier molecular orbitals (FMO) analysis and thermodynamic properties of the title compound were performed by the B3LYP/6–31G(d) method.