Applications of low-intensity pulsed ultrasound to increase monoclonal antibody production in CHO cells using shake flasks or wavebags.

Many technologies, such as cell line screening and host cell engineering, culture media optimization and bioprocess optimization, have been proposed to increase monoclonal antibody (mAb) production in Chinese Hamster Ovary (CHO) cells. Unlike the existing biochemical approaches, we investigated stimulation using low-intensity pulsed ultrasound (LIPUS) as a purely physical approach, offering enhanced scalability, contamination control and cost-efficiency, while demonstrating significantly increased cell growth and antibody production. It was found that daily ultrasound treatments at 40 mW/cm(2) for 5 min during cell culture increased the production of human anti-IL-8 antibody by more than 30% using 10 or 30 mL shake flasks. Further increasing the ultrasound dosage (either intensities or the treatment duration) did not appreciably increase cell growth or antibody production, however feeding the culture with additional highly-concentrated nutrients, glucose and amino acids (glutamine in this case), did further increase cell growth and antibody titer to 35%. Similar ultrasound treatments (40 mW/cm(2), 5 min per day) when scaled up to larger volume wavebags, resulted in a 25% increase in antibody production. Increased antibody production can be attributed to both elevated cell count and the ultrasound stimulation. Theoretical study of underlying mechanisms was performed through the simulations of molecular dynamics using the AMBER software package, with results showing that LIPUS increases cell permeability. The significance of this study is that LIPUS, as a physical-based stimulation approach, can be externally applied to the cell culture without worrying about contamination. By combining with the existing technologies in antibody production, LIPUS can achieve additional mAb yields. Because it can be easily integrated with existing cell culture apparatuses, the technology is expected to be more acceptable by the end users.

Ultrasonic backscatter coefficient quantitative estimates from Chinese hamster ovary cell pellet biophantoms.

A cell pellet biophantom technique is introduced, and applied to the ultrasonic backscatter coefficient (BSC) estimate using Chinese hamster ovary (CHO) cells. Also introduced is a concentric sphere scattering model because of its geometrical similarities to cells with a nucleus. BSC comparisons were made between the concentric sphere model and other well-understood models for mathematical verification purposes. BSC estimates from CHO cell pellet biophantoms of known number density were performed with 40 and 80 MHz focused transducers (overall bandwidth: 26-105 MHz). These biophantoms were histologically processed and then evaluated for cell viability. Cell pellet BSC estimates were in agreement with the concentric sphere model. Fitting the model to the BSC data yielded quantitative values for the outer sphere and inner sphere. The radius of the cell model was 6.8 ± 0.7 µm; the impedance of the cytoplasm model was 1.63 ± 0.03 Mrayl and the impedance of the nuclear model was 1.55 ± 0.09 Mrayl. The concentric sphere model appears as a new tool for providing quantitative information on cell structures and will tend to have a fundamental role in the classification of biological tissues.

Fluorescein isothiocynate-dextran uptake by chinese hamster ovary cells in a 1.5 MHz ultrasonic standing wave in the presence of contrast agent.

Uptake of fluorescein isothiocynate-dextran (FITC-dextran) by Chinese hamster ovary cells was studied after exposure to ultrasonic standing wave (USW) in presence of Optison, an ultrasound contrast agent. Confluent Chinese hamster ovary cells were harvested and suspended in phosphate-buffered saline + 0.1% bovine serum albumin containing FITC-dextran (10, 40, and 500 kDa) at 10 microM final concentration. The suspension was seeded with contrast agent (75 microL/mL) and exposed to a 1.5 MHz USW system at acoustic pressures ranging from 0.98 to 4.2 MPa. Macromolecular uptake was assessed by fluorescent microscopy and quantified by flow cytometry 10 min after exposure. FITC-dextran positive cells, as assessed by flow cytometry, were 1 +/- 0.05% and 2.58 +/- 0.27% for acoustic pressures of 1.96 and 4.2 MPa, respectively (p = 0.006). Fluorescent microscopy indicated a degree of macromolecular loading at 0.98 MPa with 46% of peripherally FITC-dextran- and/or propidium iodide-stained cells coincident with the appearance of significant frequency (f0/2 and 2 f0) emission signals. At higher pressures, high macromolecular loading with 6% peripherally stained cells at 1.96 MPa was associated with lower order emission signals and white noise. The study conclusively demonstrates macromolecular loading in an USW, a significantly higher macromolecular loading at higher pressures and indicates potential of emission signals for a feedback loop to control the acoustic power outputs and fine-tune the biologic effects associated with sonoporation.

BRU59-21, a second-generation 99mTc-labeled 2-nitroimidazole for imaging hypoxia in tumors.

UNLABELLED: Hypoxia in tumors is believed to be an important cause of local failure of radiotherapy in certain types of cancer. BRU59-21 (BMS194796) is a second-generation 99mTc-labeled 2-nitroimidazole that has been shown to offer improved characteristics for imaging myocardial ischemia. It has now been evaluated in models of tumor hypoxia. METHODS: Accumulation of BRU59-21 was compared with that of BMS181321 in Chinese hamster ovary cells incubated under aerobic or hypoxic conditions. The effects of competition with unlabeled nitroimidazoles and oxygen were studied. Biodistribution studies were performed in mice bearing transplanted KHT-C tumors in the leg. RESULTS: Within 5 min, BRU59-21 partitioned into aerobic cells in vitro at a level 10 times higher than external medium with no further increase over time. In hypoxic cells this initial partitioning was followed by selective accumulation to levels 5 times higher than in aerobic cells by 4 h. Low levels of oxygen (approximately 40 ppm) inhibited the maximal accumulation rate by 50%. Unlabeled misonidazole, a 2-nitroimidazole, inhibited accumulation of radioactivity, whereas tinidazole, a 5-nitroimidazole, enhanced accumulation; similar effects had been reported with BMS181321. Biodistribution studies in mice showed rapid clearance of radioactivity from the blood, resulting in enhanced tumor-to-blood ratios compared with BMS181321. Increasing the hypoxic fraction in the tumor by injection of nitro-L-arginine resulted in increased retention of tracer in the tumor without affecting other tissues. CONCLUSION: These results suggest that BRU59-21 warrants further investigation as an agent for imaging tumor hypoxia in the clinic.

Transfection of a reporter plasmid into cultured cells by sonoporation in vitro.

Cultured Chinese hamster ovary cells were exposed to 2.25-MHz ultrasound in sterile 4.5-mL polyethylene chambers and tested for cell lysis, sonoporation and DNA transfection. Ten percent of Albunex, a gas-body-based ultrasound contrast agent, was added to ensure cavitation nucleation, and the chambers were rotated at 60 rpm to promote cavitation activity during the 1-min exposures. Uptake of large fluorescent dextran molecules by some cells was observed for spatial peak pressure amplitudes as low as 0.1 MPa, which indicates transient permeabilization and resealing, i.e., sonoporation, of these cells during exposure. Significant lysis occurred for 0.2 MPa, and increased rapidly for exposures above the apparent cavitation threshold (using the H2O2 production test) of about 0.4 MPa spatial peak pressure amplitude. In the DNA transfection tests, 20 micrograms/mL luciferase reporter plasmid was added to the suspension during exposure, and cells were assayed for proliferation ability and luciferase gene expression 2 days after exposure. Cell proliferation was greatly reduced above the cavitation threshold. Luciferase production was significant for 0.20-MPa exposure, and reached 0.33 ng per 10(6) cells at 0.8-MPa exposure. The luciferase production was great for cells exposed in medium supplemented with serum than for cells exposed in serum-free medium. Cells harvested for exposure either in the log phase or in the stationary phase of culture gave similar proliferation and transfection results. The effects essentially disappeared when the Albunex was omitted from the suspension and the tube was not rotated. Thus, sonoporation by ultrasonic cavitation in the rotating tube system yields plasmid transfection with subsequent transient gene expression.

In vitro mechanisms of chemopotentiation by tone-burst ultrasound.

We investigated in vitro enhancement of cytotoxicity of chemotherapeutic agents by tone-burst ultrasound. Survival of CHO cell exposed to chemotherapeutic agents in culture medium was determined with and without insonation (1.62 and 0.29 MHz, 10% duty cycle). Insonations up to 0.4 MPa peak pressure (5 kW/m2 spatial and temporal average) occurred in the middle of 1 h drug exposures. Cytotoxicity in ultrasound control groups was never observed. Ultrasound increased the clonogenic cytotoxicity of adriamycin (p = 0.00027 by paired t test) and diaziquone but not of cisplatin or mitomycin C. Potentiation of adriamycin depended on exposure time and tone-burst frequency. .OH production in water occurred at intensities as low as 0.4 kW/m2, but did not increase with added adriamycin. Ultrasound did not affect membrane fluidity, but moderately increased cellular adriamycin accumulation, possibly explaining the observed drug potentiation.

The role of cavitation in the induction of cellular DNA damage by ultrasound and lithotripter shock waves in vitro.

The induction of DNA strand breaks in Chinese hamster ovary cells was measured with the comet assay after continuous wave ultrasound or lithotripter shock wave exposure. Cell lysis and hydrogen peroxide production were measured to gauge the level of inertial cavitation activity. Significant DNA damage was found after 2.17-MHz ultrasound exposure at 37 degrees C to 0.82 MPa for 2 min or 4 min, and to 0.58 MPs for 4 min. A significant portion of the damage induced at the 0.82-MPa level was repaired by the cells when warmed. Neither exposure to 500 or 1000 shock waves at 37 degrees C in a thin-walled tube, nor exposure to 1000, 1500 or 2000 shock waves at 25 degrees C in a polyethylene pipette bulb produced a significant effect, when the flash of light from the spark-gap discharge was blocked. This finding was consistent with the generally lower lysis and hydrogen peroxide production by the shock wave exposure.

Comet assay reveals DNA strand breaks induced by ultrasonic cavitation in vitro.

The induction of DNA strand breaks in cultured Chinese hamster ovary (CHO) cells was investigated in suspensions directly exposed to 2.17-MHz ultrasound. Production of hydrogen peroxide, a DNA-damaging sonochemical, by inertial cavitation was enhanced by the use of argon-and-oxygen-bubbled media and cell survival was improved by establishing standing waves and minimizing tube rotation. Viable cells were separated from the suspension after exposure and kept on ice for evaluation with the single-cell gel electrophoresis (comet) assay. With this assay, DNA damage from as little as 2-mumol/L hydrogen peroxide treatment for 30 min could be detected, and cell survival as low as 2-5% after ultrasound exposure was adequate for assay. An ultrasound dose-response trend was noted for increasing pressure amplitude up to 0.82 MPa (free field) and increasing exposure duration up to 4 min. The cells were able to repair some of the strand breaks when warmed to 28 degrees C for 30 min. The effect was not eliminated by addition of catalase, which indicates that the DNA damage was not due to the action of residual H2O2 alone. The results confirm the hypothesis of DNA damage in cells surviving inertial cavitation.

Ultrasonic cavitation indirectly induces single strand breaks in DNA of viable cells in vitro by the action of residual hydrogen peroxide.

Direct exposure of cells to vigorous ultrasonic cavitation results predominantly in mechanical cell lysis, but latent effects due to production of toxic sonochemicals can also be present. Phosphate buffered saline (PBS) was exposed to 1.61 MHz ultrasonic cavitation at 20 degrees C in a rotating tube exposure system to build up sonochemical products. Single strand DNA breaks (SSBs) were then induced by treating Chinese hamster ovary (CHO) cells with the cavitated PBS for 30 min on ice. The SSBs resided in viable cells, as evidenced by their ability to repair the breaks when warmed. This indirect effect could be explained by the action of cavitation-generated hydrogen peroxide that had built up (e.g., to 16 microM after 30 min exposure) in the PBS. Dissolution of argon gas in the PBS before exposure enhanced the SSB effect and the H2O2 production. Addition of catalase to the cavitated PBS before cell treatment eliminated the H2O2 and the SSB gamma effect. Tests with hydrogen peroxide showed that 16 microM H2O2 treatment for 30 on ice was as effective as 1 Gy dose of 60Co gamma rays in producing single strand breaks. The SSB effect of H2O2 and gamma rays was reduced by addition of the radical scavenger cysteamine to the cells before treatment, but cysteamine did not reduce the SSB effect of direct exposure to ultrasonic cavitation. These results help to clarify the potential for genetic effects from ultrasonic cavitation. These effects help to clarify the potential for genetic effects from ultrasonic cavitation.