Low-Frequency Ultrasound in Vitro- Changes of Cell Morphology

8/16/2019 Low-Frequency Ultrasound in Vitro- Changes of Cell Morphology

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ISSN 1855-9913

Journal of the Laser and Health Academy Vol. 2013, No.1; www.laserandhealth.com

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Low-Frequency Ultrasound in vitro : Changes of CellMorphology

Jure Jelenc1, Joze Jelenc1, Damijan Miklavcic2, Alenka Macek Lebar2 1 Iskra Medical d.o.o., Stegne 23, Ljubljana, Slovenia

2 Faculty of Electrical Engineering, Trzaska 25, Ljubljana, Slovenia

ABSTRACT

For decades, ultrasound technology has been widelyused for diagnostic imaging in various clinical fields as well as in therapeutic applications. In recent years therehas been considerable research devoted tosonoporation, a phenomenon where ultrasoundincreases cell membrane permeability. To study the

biological effects of ultrasound in an in vitro setting, wehave built a custom low-frequency ultrasoundexperimental system based on an ultrasound transducersubmerged in a waterbath. In this study we followultrasound-induced changes of cell morphology. B16-F1 cells in suspension were exposed for 300 seconds tothe continuous-wave low-frequency ultrasound (29.6kHz; 21.1 W/cm2 ). Phase contrast and fluorescencemicroscopy showed various effects of ultrasound withina single cell sample. In the cell population, cells with no visible morphological changes were present, cells thatexhibited smaller or larger blebs on the cell membrane

as well as cell debris.

Key words: sonoporation, low-frequency ultrasound,morphological changes.

Article: J. LA&HA, Vol. 2013, No.1; pp. 58-60.Received: May 7, 2013; Accepted: July 18, 2013.

© Laser and Health Academy. All rights reserved. Printed in Europe. w ww.laserandhealth.com

I. INTRODUCTION

For decades, ultrasound technology has been widely used for diagnostic imaging in various clinicalfields as well as in therapeutic applications. It wasshown that ultrasound has beneficial effects on venous ulcers, alters cell proliferation and migration,stimulates angiogenesis and arteriogenesis, altersbone fracture healing and stimulates the productionof growth factors and cytokines [1]. In all casesmentioned above, the biological effects of low-intensity ultrasound should be in the optimal rangefor each application and in within the safety limits,

which means that conditions in which damage to thecells occurs are avoided. In recent years, however

researchers in a number of studies have focused on aphenomenon where ultrasound increases cellmembrane permeability. As a result, molecules thatare otherwise unable to pass the cell membrane canbe transported across it. In this way small and largemolecules can be delivered into cells [2, 3]. Thephenomenon was named sonoporation. If the cellremains capable of repairing the damage to the

membrane and re-establishing its normal state, thephenomenon is called reversible sonoporation. Ifthe cell dies as a consequence to ultrasoundexposure, the sonoporation is irreversible. For

example, intense interest has been given toultrasound mediated DNA delivery, because itseems that sonication may be simpler to carry out incomparison with other DNA delivery methods. Butaccording to published reports it is stil not clearhow sonoporation conditions and ultrasoundparameters affect sonoporation efficiency. In somereports a satisfactory amount of successfully

sonoporated cells was demonstrated [4, 5, 6], whilea recent article has drawn attention to the lack ofefficient uptake of molecules while maintaining highcell viability after ultrasound exposure in vitro [7].

To study the biological effects of ultrasound in anin vitro setting, we have built a custom low-frequencyultrasound experimental system based on anultrasound transducer submerged in a waterbath [8]. Inthis study we follow ultrasound-induced changes ofcell morphology.

II.

MATERIALS AND METHODS

The waterbath with a length of 68 cm, width of 38cm and height of 34 cm was filled with distilled waterup to a height of 24 cm. The walls of the bath aremade from Plexiglas® and lined with the SA-J35ultrasound absorber (Hangzhou Applied AcousticsInstitute, China). In this way ultrasound reflections aresuccessfully reduced and enable experiments undercontinuous-wave ultrasound exposure [8].

Ultrasound was generated using a prototype center

bolt (Langevin type) piezoelectric ultrasoundtransducer with an operating frequency of 29.6 kHz


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(Iskra Medical, Slovenia). The transducer wassubmerged in the waterbath at a depth of 12 cm.

The effect of ultrasound was evaluated on mousemelanoma B16-F1 cells. B16-F1 cells were cultivatedin a DMEM (Sigma-Aldrich Chemie GmbH,

Germany) cell growth medium as previously describedby Ušaj [9]. After cell detachment, a cell suspension with concentration of 106 cells/ml was introduced intoa 0.2 ml PCR tube (Invitrogen, USA). The cellsuspension was vigorously mixed before ultrasoundexposure in order to introduce gas bubbles (acting ascavitation nuclei) into the cells suspension.

The cell dish was positioned at the axial center ofthe transducer, 2.5 cm from the ultrasound transducerface. Cells were exposed to 300 seconds ofcontinuous-wave 21.1 W/cm2 ultrasound intensity [8].

A sham exposure with no applied ultrasound wasconducted with the same procedures.

Just before exposure to the ultrasound, 5 μl ofPropidium Iodide (PI) (Molecular Probes, USA) weremixed into the cell suspension. In normal conditions, acell membrane is impermeable to PI. Damage causedto the cell membrane by ultrasound enables PI toenter the cytoplasm, where it binds to the nucleus.Characteristic PI fluorescence can be used to identifycells with increased cell membrane permeability.

Within 45 second from the ultrasound exposure,cells were transferred onto a Petri dish, which wasplaced under an inverted fluorescence microscope(Zeiss AxioVert 200, Zeiss, Germany). Pictures wereacquired by a cooled CCD camera (VisiCam 1280, Visitron, Germany) using Metamorph 5.0 (MolecularDevices Corporation, PA, ZDA) software.Morphological changes of the exposed cells and PIintake-induced fluorescence were analyzed in theacquired pictures.

III. RESULTS

Morphological changes induced by ultrasound weremonitored using phase contrast microscopy (Figure1A) and Propidium Iodide (PI) intake observed usingfluorescence microscopy (Figure 1B).

Phase contrast microscopy (Figure 1A) shows various effects of ultrasound within a single sample. Inthe visual field many cells with no visiblemorphological changes are found (Figure 1, 1a). Manycells exhibit smaller or even larger blebs on the cellmembrane (Figure 1, 1c). In the population we also

see cell debris (Figure 1, 3a and 3b).

The comparison of phase contrast andfluorescence images gives us a rough classification ofthese heterogeneous effects on cells. Morphologicallyintact cells are classified into cells on which ultrasoundhad no effect (Figure 1, 1a) and reversibly sonoporatedcells which are morphologically intact cells with

observed PI intake (Figure 1, 1b). Ultrasound-induceddamage on some of the cells was so intense that webelieve they are unlikely to survive the ultrasoundexposure. These cells are grouped into necrotic (Figure1, 2) and cell debris-exhibiting (Figure 1, 3a) and without exhibiting PI fluorescence (Figure 1, 3b).

The majority of sham-exposed cells were howevermorphologically intact, with only a small fraction, lessthan 5% of cells, exhibiting PI intake-relatedfluorescence (data not shown). In the sham exposurecell population we did not notice blebs on cell

membranes.

Fig. 1: Image of B16-F1 cells after ultrasound exposure: A)phase contrast microscopy and B) fluorescent microscopy. Typical representatives of ultrasound effect are marked: 1a.intact cell, 1b. reversibly sonoporated cell, 1c. cell with blebs

on the cell membrane, 2. necrotic cell, 3a. cell debrisexibiting Propidium Iodide (PI) fluorescence, 3b. cell debris without exibiting PI fluorescence.

B


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IV. DISCUSSION

In the present study we exposed B16-F1 cells incell suspension to the continuous-wave low-frequency(29.6 kHz) ultrasound and followed its biologicaleffects. The exposure lasted 300 seconds; the

ultrasound intensity was 21.1 W/cm2. In the samesample we observed heterogeneous effects on cells,ranging from no effects to total cell destruction which was noticed as formation of small cell debris.

By staining the cell plasma membrane using wheatgerm agglutinin - WGA and the intracellularmembrane using the fluorescence marker FM1-43,Schlicher et al. [10] were able to define the intracellularorigin of observed membrane blebs that were formedat sites of plasma membrane disruption after theultrasound exposure. They used low-frequency

standing wave ultrasound filed. In our experimentalsystem acoustic lining inhibits standing waveformation and allows progressive ultrasound waves. This system design difference is important since anumber of investigators have shown that cavitation, asthe main mechanism of sonoporation, can be moreeasily induced by a standing wave than by aprogressive wave [11]. During our experiments, weobserved all typical morphological changes in cellsdescribed by Schlicher et al. [10], but our experimentalreproducibility was, however, rather poor, presumablydue to the lack of control over cavitation bubbles

inside the sample.

According to current knowledge, acousticcavitation is believed to be responsible for the plasmamembrane opening [12]. In the case of inertialcavitation, tiny gas bubbles oscillate, expand andcollapse in liquid under the influence of ultrasound.Cells in the bubbles’ close vicinity experience strongmechanical stresses until the rupture of themembranes occur. Due to our current sonoporationprotocol we were unable to efficiently control thenumber of cavitation bubbles introduced by mixing

the cell suspension. To increase the reproducibility ofthe results, some other way of introducing cavitationbubbles needs to be used. Therefore we did notquantify the number of cells in certain morphologicalcharacteristic groups since their number variedsignificantly within and between samples.

V. CONCLUSIONS

A constructed experimental system that allows in- vitro experiments with continuous-wave low-frequency ultrasound exposure was successfully tested

on B16-F1 cells. Cells in suspension were exposed tothe ultrasound intensity of 21.1 W/cm2 for 300

seconds. Typical morphological changes, alreadyreported in scientific literature, were observed.

Acknowledgment

This research was carried out in collaboration with

the EU regional Competency Center for BiomedicalEngineering (www.bmecenter.com), coordinated bythe Laser and Health Academy (www.laserandhealthacademy.com), and partially supportedby the European Regional Development Fund and theSlovenian government.

REFERENCES

1. Kimmel E. Cavitation bioeffects. Critical Reviews in BiomedicalEngineering 34: 105-161, 2006.

2. Liang HD, Tang J, Halliwell M. Sonoporation, drug delivery, andgene therapy. Proc Inst Mech Eng H 224: 343 – 361, 2010.

3. Karshafian R, Bevan PD, Williams R, Samac S, Burns PN.Sonoporation by ultrasound-activated microbubble contrastagents: effect of acoustic exposure parameters on cell membranepermeability and cell viability. Ultrasound Med Biol 35: 847 – 860,2009.

4. Karshafian R, Samac S, Bevan PD, Burns PN. Microbubblemediated sonoporation of cells in suspension: clonogenic viabilityand influence of molecular size on uptake. Ultrasonics 50: 691 – 697, 2010.

5. Wei W, Zheng-zhong B, Yong-jie W, Qing-wu Z, Ya-lin M.Bioeffects of Low-Frequency Ultrasonic Gene Delivery andSafety on Cell Membrane Permeability Control. J UltrasoundMed 23: 1569 – 1582, 2004.

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IEEE Trans Biomed Eng 58: 927 – 934, 2011.7. Liu Y, Yan J, Prausnitz MR. Can Ultrasound Enable EfficientIntracellular Uptake of Molecules? A Retrospective LiteratureReview and Analysis. Ultrasound Med Biol 38: 876 – 888, 2012.

8. Jelenc J, Jelenc J, Miklavčič D, Maček Lebar A. Low-FrequencySonoporation in vitro: Experimental System Evaluation. Journalof Mechanical Engineering 58: 319 – 326, 2012.

9. Ušaj M, Trontelj K, Miklavčič D, Kandušer M. Cell-cellelectrofusion: optimization of electric field amplitude andhypotonic treatment for mouse melanoma (B16-F1) and ChineseHamster ovary (CHO) cells. J Membr Biol 236: 107 – 116, 2010.

10. Schlicher RK, Hutcheson JD, Radhakrishna H, Apkarian RP,Prausnitz MR. Changes in Cell Morphology Due to PlasmaMembrane Wounding by Acoustic Cavitation. Ultrasound MedBiol 36: 677 – 692, 2010.

11. Kinoshita M, Hynynen K. Key factors that affect sonoporation

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12. Liang HD, Tang J, Halliwell M. Sonoporation, drug delivery, andgene therapy. Proc Inst Mech Eng H 224: 343 – 361, 2010.

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Low-Frequency Ultrasound in Vitro- Changes of Cell Morphology