- Open Access
The effect of ultrasound-related stimuli on cell viability in microfluidic channels
- Dyan N Ankrett†1Email author,
- Dario Carugo†1, 2,
- Junjun Lei1,
- Peter Glynne-Jones1,
- Paul A Townsend3,
- Xunli Zhang2 and
- Martyn Hill1
© Ankrett et al.; licensee BioMed Central Ltd. 2013
Received: 17 June 2013
Accepted: 17 June 2013
Published: 28 June 2013
In ultrasonic micro-devices, contrast agent micro-bubbles are known to initiate cavitation and streaming local to cells, potentially compromising cell viability. Here we investigate the effects of US alone by omitting contrast agent and monitoring cell viability under moderate-to-extreme ultrasound-related stimuli.
Suspended H9c2 cardiac myoblasts were exposed to ultrasonic fields within a glass micro-capillary and their viability monitored under different US-related stimuli. An optimal injection flow rate of 2.6 mL/h was identified in which, high viability was maintained (~95%) and no mechanical stress towards cells was evident. This flow rate also allowed sufficient exposure of cells to US in order to induce bioeffects (~5 sec), whilst providing economical sample collection and processing times. Although the transducer temperature increased from ambient 23°C to 54°C at the maximum experimental voltage (29 V pp ), computational fluid dynamic simulations and controls (absence of US) revealed that the cell medium temperature did not exceed 34°C in the pressure nodal plane. Cells exposed to US amplitudes ranging from 0–29 V pp , at a fixed frequency sweep period (tsw = 0.05 sec), revealed that viability was minimally affected up to ~15 V pp . There was a ~17% reduction in viability at 21 V pp , corresponding to the onset of Rayleigh-like streaming and a ~60% reduction at 29 V pp , corresponding to increased streaming velocity or the potential onset of cavitation. At a fixed amplitude (29 V pp ) but with varying frequency sweep period (tsw = 0.02-0.50 sec), cell viability remained relatively constant at tsw ≥ 0.08 sec, whilst viability reduced at tsw < 0.08 sec and minimum viability recorded at tsw = 0.05 sec.
The absence of CA has enabled us to investigate the effect of US alone on cell viability. Moderate-to-extreme US-related stimuli of cells have allowed us to discriminate between stimuli that maintain high viability and stimuli that significantly reduce cell viability. Results from this study may be of potential interest to researchers in the field of US-induced intracellular drug delivery and ultrasonic manipulation of biological cells.
In ultrasonic cell stimulation micro-devices, the inclusion of ultrasound (US) contrast agent (CA) to enhance US bioeffects or increase cell membrane permeability is common . However, CAs can initiate cavitation and streaming  local to cells, potentially compromising cell viability [3, 4]. Thus, higher cell viability is likely to be maintained in the absence of CA [5–7]. In our previous study we reported on ultrasonically induced membrane poration of a cardiac myoblast cell line (H9c2) in the absence of CA by generating an ultrasonic field within a biocompatible glass micro-capillary . Notably, high cell viability was maintained in the absence of CA . Following a similar approach, Longsine-Parker et al. recently demonstrated effective cell membrane poration in a microfluidic device by combining the action of electric fields and US waves in a CA-free environment .
Here we investigate US-“alone”-related physical stimuli of H9c2 cells. We expose suspended cells to gentle, moderate and extreme US amplitudes. Extreme amplitudes also initiate an increase in transducer temperature; therefore we also investigated the effect of US-related temperature increase on cell viability. Cell viability was also measured following infusion into the micro-device at varying flow regimes in order to optimise the flow rate. Of particular interest to us is the effect of frequency sweeping on cells as a means of controllably stressing cells and potentially increasing membrane permeability.
Summary of the experiments performed to investigate the effect of US-related stimuli on H9c2 cell viability
Flow rate through the micro-capillary
Inlet flow rate: 1.3–13.0 mL/h
US-induced thermal variations
PZT temperature measurements and CFD simulations of fluid temperature distribution
Controls (correspondent PZT temperatures, absence of US)
Driving voltage: 6–29 V pp
Sweep period variations
Sweep period: 0.02–0.50 sec
The effect of individual US-related physical parameters (fluid flow rate, US heat generation, amplitude and frequency sweep period) on H9c2 cell viability was assessed within a microfluidic device. The optimised flow rate did not inflict any detectable mechanical stress, and thus high cell viability was maintained. Moreover cells were allowed sufficient exposure to US in order to elicit bioeffects, whilst providing economical sample processing times and minimising cell trapping. High cell viability was maintained at amplitudes where streaming was not evident. However, when more extreme amplitudes were employed, streaming velocities increased and cell viability significantly decreased. Extreme amplitudes also initiated an increase in PZT temperature, however cell viability was unaffected by this increase due to heat dissipation, confirmed by controls and CFD simulations. Longer duration frequency sweeps were identified to have little or no effect on cell viability, whereas short sweeps resulted in reduced cell viability. This effect may be attributed to mechanical stress generated by rapid oscillatory movements of the cell within the fluidic domain . Notably, experiments with fluorescent tracer beads revealed that bead oscillation frequency increased with reducing the sweep interval, which may explain the reduction in cell viability at the shorter tsw. However, an in depth investigation into the effects of frequency sweeping on cell viability is currently underway in our laboratories.
Our CA-free investigation into the effects of US on cell viability has enabled us to discriminate between US-related stimuli that do not compromise cell viability and stimuli that significantly reduce cell viability within our micro-device. Our findings may be of potential interest to researchers in the field of US-induced intracellular drug delivery and ultrasonic manipulation of biological cells.
H9c2 cardiac myoblasts were grown in Dulbecco’s Modified Eagle Medium (DMEM) culture medium supplemented with 10% (v/v) foetal calf serum and 1% (v/v) penicillin-streptomycin (media and supplements purchased from Fisher Scientific, Loughborough, UK). Cells were maintained at 37°C, 5% CO2 in air with 95% humidity. Cells were routinely harvested and suspended at a density of 2×106 cells/mL in serum free DMEM within a 1 mL sterile, plastic syringe (BD Bioscience, Oxford, UK). Cells were infused into the device using a syringe pump (KD100, KD Scientific Inc., Holliston, USA) and subjected to ultrasound-related physical stimuli. Cells were captured in 1 mL sterile tubes, followed by counting and viability assessment using a Neubauer haemocytometer (depth: 0.1 mm, area: 0.04 mm2) and trypan blue exclusion dye. All viability measurements were in triplicate or greater.
To optimise the flow rate, cell viability was measured following infusion into the device at a range of flow rates (1.3-13.0 mL/h), which were prior calculated in order to: i) provide sufficient exposure of cells to US, ii) provide economical cell collection and processing times, iii) minimise flow-induced mechanical stress on cells and iv) minimise cell trapping.
To assess US-related thermal effects on cell viability, cells were infused into the device at a fixed flow rate (2.6 mL/h) and exposed to US (6–29 V pp ), whilst thermocouples were attached to the transducer, and temperatures recorded using a thermometer (HH11 Omega®, Manchester, UK). Controls were produced in the absence of US by replacing the transducer with a hot plate (Fisher Scientific, Loughborough, UK) at identical temperatures to the recorded transducer temperatures. Additionally, computational fluid dynamic (CFD) simulations were performed to predict the transfer of heat from the transducer to the cell medium within the capillary.
The effect of US amplitude on cell viability was investigated by varying the V pp , ranging from 0–29 V pp , using a fixed frequency sweep period of 0.05 sec in the frequency range 2.13-2.40 MHz. Additionally, flow visualisation experiments, using 1 μ m diameter fluorescent tracers (Polysciences, Inc., Warrington, USA), were performed to characterise the fluid dynamic environment under “gentle” (6 V pp ) to “extreme” (29 V pp ) US amplitudes. The acoustic pressure within the capillary was measured through drop-voltage analysis , using 20 μ m diameter fluorescent polystyrene beads. A fixed resonance frequency of 2.18 MHz was set in this case, due to the difficulty in obtaining acoustic pressure values during frequency sweeping.
The effect of frequency sweep duration on cell viability was investigated by varying the sweep period (0.02-0.50 sec) at a fixed voltage (29 V pp ).
We are very grateful to Agilent Technologies, Santa Clara, CA, USA for funding this research (University Relations Grant 2012, Gift# 2700).
- Le Gac S, Zwaan E, Van Den Berg A, Ohl C-D: Sonoporation of suspension cells with a single cavitation bubble in a microfluidic confinement. Lab Chip. 2007, 7 (12): 1666-1672. 10.1039/b712897p.View ArticleGoogle Scholar
- Collis J, Manasseh R, Liovic P, Tho P, Ooi A, Petkovic-Duran K, Zhu Y: Cavitation microstreaming and stress fields created by microbubbles. Ultrasonics. 2010, 50 (2): 273-279. 10.1016/j.ultras.2009.10.002.View ArticleGoogle Scholar
- Carugo D, Ankrett DN, Glynne-Jones P, Capretto L, Boltryk RJ, Zhang X, Townsend PA, Hill M: Contrast agent-free sonoporation: the use of an ultrasonic standing wave microfluidic system for the delivery of pharmaceutical agents. Biomicrofluidics. 2011, 5 (4): 044108-10.1063/1.3660352.View ArticleGoogle Scholar
- Brayman AA, Azadniv M, Cox C, Miller MW: Hemolysis of albunex-supplemented, 40% hematocrit human erythrocytes in vitro by 1-MHz pulsed ultrasound: Acoustic pressure and pulse length dependence. Ultrasound Med Biol. 1996, 22 (7): 927-938. 10.1016/0301-5629(96)00108-1.View ArticleGoogle Scholar
- Hultström J, Manneberg O, Dopf K, Hertz HM, Brismar H, Wiklund M: Proliferation and viability of adherent cells manipulated by standing-wave ultrasound in a microfluidic chip. Ultrasound Med Biol. 2007, 33 (1): 145-151. 10.1016/j.ultrasmedbio.2006.07.024.View ArticleGoogle Scholar
- Wiklund M: Acoustofluidics 12: Biocompatibility and cell viability in microfluidic acoustic resonators. Lab Chip. 2012, 12: 2018-2028. 10.1039/c2lc40201g.View ArticleGoogle Scholar
- Evander M, Johansson L, Lilliehorn T, Piskur J, Lindvall M, Johansson S, Almqvist M, Laurell T, Nilsson J: Noninvasive acoustic cell trapping in a microfluidic perfusion system for online bioassays. Anal Chem. 2007, 79 (7): 2984-2991. 10.1021/ac061576v.View ArticleGoogle Scholar
- Longsine-Parker W, Wang H, Koo C, Kim J, Kim B, Jayaraman A, Han A: Microfluidic electro-sonoporation: a multi-modal cell poration methodology through simultaneous application of electric field and ultrasonic wave. Lab Chip. 2013, 13: 2144-2152. 10.1039/c3lc40877a.View ArticleGoogle Scholar
- Glynne-Jones P, Boltryk RJ, Hill M, Zhang F, Dong L, Wilkinson JS, Melvin T, Harris NR, Brown T: Flexible acoustic particle manipulation device with integrated optical waveguide for enhanced microbead assays. Anal Sci. 2009, 25 (2): 285-291. 10.2116/analsci.25.285.View ArticleGoogle Scholar
- Sorando AC, Hawkes JJ, Fielden PR, González I: Patterns of particles aggregation and streaming in resonating fluids. AIP Conference Proceedings. 2012, 1433: 757.View ArticleGoogle Scholar
- Apfel RE, Holland CK: Gauging the likelihood of cavitation from short-pulse, low-duty cycle diagnostic ultrasound. Ultrasound Med Biol. 1991, 17 (2): 179-185. 10.1016/0301-5629(91)90125-G.View ArticleGoogle Scholar
- Radel S, McLoughlin A, Gherardini L, Doblhoff-Dier O, Benes E: Viability of yeast cells in well controlled propagating and standing ultrasonic plane waves. Ultrasonics. 2000, 38 (1): 633-637.View ArticleGoogle Scholar
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