Cell Communication and Adhesion, 14: 9–20, 2007Copyright C© Informa HealthcareISSN: 1541-9061 print / 1543-5180 onlineDOI: 10.1080/15419060701224849

Cell Adhesion Dynamics and Actin CytoskeletonReorganization in HepG2 Cell Aggregates

GARETH OWAIN EDWARDS, DESPINA BAZOU, LARISA A. KUZNETSOVA, and W. TERENCE COAKLEYCardiff School of Biosciences, Main Building, Cardiff University, Cardiff, Wales, UK

The temporal dependence of cytoskeletal remodelling on cell–cell contact in HepG2 cells hasbeen established here. Cell–cell contact occurred in an ultrasound standing wave trap designedto form and levitate a 2-D cell aggregate, allowing intercellular adhesive interactions to proceed,free from the influences of solid substrata. Membrane spreading at the point of contact andchange in cell circularity reached 50% of their final values within 2.2 min of contact. JunctionalF-actin increased at the interface but lagged behind membrane spreading, reaching 50% of itsfinal value in 4.4 min. Aggregates had good mechanical stability after 15 min in the trap. Theimplication of this temporal dependence on the sequential progress of adhesion processes isdiscussed. These results provide insight into how biomimetic cell aggregates with some livercell functions might be assembled in a systematic, controlled manner in a 3-D ultrasound trap.

Keywords HepG2 cells, ultrasound standing wave trap, cell–cell adhesion, actin cytoskeleton

INTRODUCTION

The temporal progression of cell-contact initiatedcell morphology and F-actin cytoskeletal changesare of importance in processes such as tissue de-velopment (Carthew, 2005). Cell–cell adhesion isrequired for the formation of polarized aggregates(Carthew, 2005). Cadherin-mediated cell–cell ad-hesion initiates remodeling of the actin cytoskele-ton through catenins (D’Souza-Schorey, 2005), lead-ing to the development of a thick ring of F-actinat the cell periphery and termination of bundles ofactin fibers at regions of cell–cell contact (Bamji,2005; Zhang et al., 2005). Cell-substratum and cell-extracellular-matrix interactions through integrinscan also direct cytoskeletal remodeling (Galler et al.,

Received 26 July 2006; accepted 12 December 2006.Address correspondence to Dr. Gareth O. Edwards, Cardiff School of Biosciences, Main Building, Cardiff University, Park Place, Cardiff

CF10 3TL, Wales, UK. E-mail: edwardsgo@cardiff.ac.uk

2006; Giancotti and Ruoslahti, 1999; Sawada et al.,2003). Significant attention has been paid to thetemporal development of membrane spreading andF-actin cytoskeleton organization during integrinmediated cell-ECM adhesion of cells on artificialsubstrata (Cavalcanti-Adam et al., 2006; Chen et al.,2006; Kaido et al., 2004). The contribution of cell–cell interactions, independently of cell-ECM interac-tions, to adhesion processes has been more difficultto quantify due to the problem of identifying pairsof cells in suspension in a microscopic field that areabout to interact and then maintaining those cells infocus during the development of membrane spread-ing. An ultrasound standing wave trap (USWT) thatforms and levitates 2-D cell aggregates in suspensionas a monolayer in a microscopic field has recently

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10 G. O. EDWARDS ET AL.

Figure 1. Schematic showing the formation of an aggregate of cells in the ultrasound trap (reproduced from Coakley et al., 2004, withpermission). i: cells in suspension in trap with no ultrasound; ii: upon initiation of ultrasound exposure, cells congregate at the pressure node;and iii: the formation within a short time of a 2-D monolayer aggregate of cells.

been described (Bazou et al., 2004, 2005a, 2006;Coakley et al., 2004; Morgan et al., 2004; Spenglerand Coakley, 2003). The trap typically consists ofa 0.5 mm layer of cell suspension that is driven atits half wavelength resonant frequency (1.5 MHz).Acoustic standing wave radiation forces drive thecells to form a single 2-D aggregate in the center ofthe resonating volume (Figure 1). The aggregate canbe continuously monitored microscopically throughthe quartz disc that forms the reflector of the stand-ing wave device. Cell–cell adhesion and membranespreading at the cell–cell contact interface in neuralcell monolayers (Bazou et al., 2004, 2005; Coakleyet al., 2004), the distribution of adhesion-relatedmolecules NCAM and N-cadherin and F-actin inneural cells (Bazou et al., 2005a), and the devel-opment of the F-actin cytoskeleton and functioninggap junctions in chondrocytes (Bazou et al., 2006)in the USWT have been shown to change with timefrom aggregate formation. The cells remained viablethroughout experiments. The times examined in theabove studies looking at NCAM, N-cadherin, andF-actin were 1, 8, and 30 min (Bazou et al., 2005a)and 1 and 60 min for chondrocyte gap junction de-velopment (Bazou et al., 2006). It was shown thatmembrane spreading and molecular consequencesfollowing contact were beginning at 1 min, devel-oping at 8 min and completed in 30–60 min (Bazouet al., 2005a, 2006). In the above cases, membranespreading was defined as the increase in length of thecell–cell contact zone at the cell perimeter with time(Bazou et al., 2005a; Coakley et al., 2004; Coakleyand Bazou, 2005).

The work described here pays particular attentionto establishing the initial rates of change over a 10-min period following adhesion. Rates of membranespreading and cytoskeletal reorganization are estab-lished over those times. Adhesion of HepG2 cells isof interest in its own right and has additional signif-icance because of the use of alginate-encapsulatedHepG2 aggregates as a model for studying liver-specific biochemistry (Damelin et al., 2004). It isshown here that stable aggregates of HepG2 cellscan be rapidly generated, without loss of viability, inthe USWT. Cell morphology changes from an ini-tial rounded form to a more cuboidal morphology,indicating that interactions between hepatocytes di-rectly influence cell morphology. A significant redis-tribution of F-actin to the cell–cell interface occurswithin 10 min of initiation of cell contact. This ap-proach forms a basis for the development of a USWThepatocyte culture system to rapidly form “micro-spheroids” as a tool for rapid biochemical analysisand functional characterisation.

MATERIALS AND METHODS

Cell Culture

HepG2 cells (European Collection of CellCultures, Health Protection Agency, Centre forEmergency Preparedness and Response, Salisbury,Wiltshire, UK), were maintained in Minimum Es-sential Medium (Eagle) (Sigma Aldrich, Poole, UK)supplemented with 10% v/v foetal bovine serum

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CELL ADHESION IN HepG2 AGGREGATES 11

Figure 2. A: Schematic of the ultrasound trap (reproduced from Coakley et al., 2004, with permission). B: The ultrasound trap (U) onmicroscope stage and syringe used for loading and emptying the trap (S).

(Sigma Aldrich), 2 mM L-Glutamine (Invitrogen,Paisley, UK), 1% v/v non-essential amino acids(Invitrogen), 100 U/ml penicillin (Invitrogen), and100 µg/ml streptomycin (Invitrogen). Cultures wereroutinely passaged with 0.05% w/v trypsin-EDTA(Invitrogen) when 70% confluent (typically after 5to 9 days). Single cell suspensions were obtainedby washing monolayers in phosphate buffered saline(PBS, Sigma Aldrich) followed by incubation in Ac-cutase (Sigma Aldrich) for 5 min. Cells were resus-pended in serum-free medium, filtered through a 40-µm Cell Strainer (BD Biosciences, Oxford, UK) andviability assessed by Trypan Blue (Sigma Aldrich)dye exclusion.

Ultrasound Exposure

The USWT employed here has been describedpreviously (Bazou et al., 2004, 2005a). Its general

structure (Figure 2A) is based on a transducer witha nominal resonance frequency of 1.5 MHz (Ferrop-erm, Kvistgard, Denmark) attached to a steel plate,above which there is a sample space (to the depthof 0.5 mm, which equals λ/2, where λ represents thewavelength of sound in water) covered with a 1-mm-thick quartz glass reflector allowing optical accesswith a microscope. The transducer was driven bya sine wave generated by a Hewlett Packard 33120A function generator. A 2-ml disposable syringe andsmall length of silicon tubing were employed to gen-tly introduce cell suspensions into the USWT and tocarefully remove aggregates from the trap at speci-fied times after formation. The whole assembly wasmounted on an Olympus BX41M reflection epiflu-orescence microscope (Figure 2B). The aggregationprocess was monitored using a CCD F-view cam-era mounted via a 0.3 × TV adapter. Images werecaptured with the analySIS 3.1 software package

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12 G. O. EDWARDS ET AL.

(Soft Imaging System GmbH, Munster, Germany).Sonication and thus levitation of cells commencedimmediately following introduction of cells into theUSWT and microscope focusing, which was takenas the zero timepoint in all experiments. The work-ing acoustic pressure amplitude (110 kPa) was de-termined by trapping a single 25-µm latex particlein the USWT and measuring the threshold voltagerequired to levitate the particle against gravity; thisvalue was used to determine the working pressureon the basis of linear extrapolation (Coakley et al.,2004).

Aggregate Area and Cell Death Assay

Ultrasound exposure has previously been used asa cell disruption tool resulting in cell death by ruptur-ing cell membranes (Borthwick et al., 2006); hence,cells were incubated with Ethidium Homodimer, aDNA-binding fluorophore capable of passing acrossmembranes with compromised integrity (Decherchiet al., 1997). Cells (3 × 105 cells · ml−1) in serum-free medium containing 1 µM Ethidium Homod-imer (Invitrogen) were suspended in the USWT for30 min, and aggregate micrographs were analyzedwith Image-J 1.37b (Rasband, 1997–2006). Micro-graphs were converted from 8-bit grayscale bright-field to binary images by applying a threshold. Us-ing the Wand tool, a region of interest (ROI) wasselected around an aggregate. The area of this re-gion (Aw) was measured. The cell-free area (Acf)within the region was determined (See Morphologyand Void Analysis). The area of the aggregate (Aa)was given by Aw–Acf. Using the ‘ROI Manager,’ thisROI was applied to a fluorescence micrograph of thesame aggregate, and the area of fluorescent cells (Afl)within the aggregate measured with the Analyze Par-ticles tool. A measure of cell death was calculatedas Afl/Aa and converted to a percentage. Differencesin the area of fluorescing cells at 3 min and 30 minwere determined with a one-tailed paired t-test inGraphPad Prism (GraphPad Software Incorporated,San Diego, CA); all experiments were carried outin triplicate unless otherwise stated and standard er-ror of the mean was calculated. Membrane perme-

ability of cells incubated in 1% v/v Triton X-100 at4◦C for 30 min and then suspended with EthidiumHomodimer in the USWT for 3 min was assessedas a positive control for reduced membrane perme-ability, and compared (t-test in GraphPad Prism) tountreated cells sonicated for 3 min.

Morphology and Void Analysis

Cells (3 × 105 cells · ml−1) in serum-free mediumwere loaded into the USWT and aggregated. Usingthe Freehand tool, a ROI was selected around a celland circularity was measured (defined in Image-J as4Pi × [area/perimeter2]; a value of 1.0 representsa perfect circle) and differences were analyzed byanalysis of variance (ANOVA) in Microsoft Excel.Interfacial length, i.e., the length of interfacial mem-brane spreading at the tangential contact area be-tween two cells (Bazou et al., 2005a), was measuredand differences were analyzed by ANOVA. To assessintercellular void space (Bazou et al., 2004), a ROIwas selected around a binary image of an aggregateusing the Wand tool, area was measured, and the An-alyze Particles tool used to determine the void areawithin the ROI. A void index was established by di-viding the void area within the ROI by the total ROIarea (Bazou et al., 2004). The significance of corre-lation coefficients was assessed in GraphPad Prismusing the F-test.

Fluorescent Localization of F-Actin

Cells (1 × 106 cells · ml−1) in serum-free mediumwere loaded into the USWT and exposed to ul-trasound. While continuing sonication, aggregateswere removed from the trap and placed on Histobondmicroscope slides (Raymond A Lamb Ltd., East-bourne, UK), where they immediately sedimented.The supernatant was removed with a pipette, andthe cells were fixed with 90% v/v ethanol within60 s of removal from the USWT. After 20 min fix-ation, the cells were washed in PBS before beingpermeabilized for 30 min in PBS containing 0.1%v/v Triton X-100 (Sigma Aldrich), and then blocked

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CELL ADHESION IN HepG2 AGGREGATES 13

for 30 min in PBS containing 1% w/v BSA. Actinwas stained with 10 U/ml of Phalloidin-Alexa488conjugate (Invitrogen) for 20 min in the dark. Slideswere washed three times in PBS and mounted inVectaShield Mounting Medium (Vector Laborato-ries Ltd., Peterborough, UK). Images were collectedas described previously.

A rectangular ROI was selected around a chosenarea at the interface of a pair of interacting cellsand used to generate a 3-D surface plot to visual-ize F-actin intensity (Barthel, 2004). A linear ROIwas selected with the midpoint (0 µm) over the cen-ter of the cell–cell boundary, halfway along the tan-gential length of the cell–cell interface, and fluores-cence intensity values were obtained using the PlotProfile tool. Five fields of view were captured perreplicate treatment. F-actin intensity was measuredat five cell–cell interfaces from each field of view.Each dataset followed a Gaussian distribution (as-sessed in GraphPad Prism). These 25 measurementsgave a mean value for each replicated experiment.The mean F-actin intensity over the distance 2 to3 µm on each side of the cell–cell interface wassubtracted (as a background intensity level ‘correc-tion’) from all intensity values over the range 0 to2 µm from the interface. The area under the result-ing curves was calculated in GraphPad Prism. Thesemeasurements were repeated on unsonicated cells.The resulting ‘corrected’ values over 0–2 µm fromthe cell rim were determined. A time zero controlF-actin distribution was constructed from this dataand its mirror image about the cell rim. To assesschanges in the thickness of peripheral F-actin, thehalf-height of plot profile peak intensity was calcu-lated and used to determine the half-width of F-actinat the cell–cell boundary.

RESULTS

Aggregate Formation and Strength

A cell aggregate was established from a suspen-sion of HepG2 cells at a concentration of 3 × 105

cells · ml−1 within 30 s of commencing sonication

(Figure 3A). The aggregate rapidly increased insize over 3 min (Figure 3B) and had stabilizd by15 min (Figure 3C). As the aggregate formed, itsarea showed a steep rise between 30 s and 3 min witha gradual increase beyond this point until 15 min(Figure 3D). Mean aggregate area at 15 min was0.558 ± 0.003 mm2. The mean area of a HepG2 cellwas 113 ± 12 µm2 (n = 5), therefore aggregatescontained approximately 5000 cells. To assess celladhesion in the aggregate, levitation was terminatedfollowing 15 min and the aggregate allowed to sedi-ment to the trap surface. The area of the sedimentedaggregate, measured after 3 min on the trap base(0.568 mm2), was not significantly different (P =0.83) from that of the suspended aggregate.

Cell Death Assay

The membrane permeability cell death assayshowed that 14.0 ± 0.24% (n = 3) and 15.0 ± 0.69%(n = 3) of the aggregate area contained fluorescingcells (i.e., those with compromised membrane in-tegrity) at 3 and 30 min, respectively. A one-tailedpaired-control t-test showed that this increase wasnot significant (P = 0.15). Following 3 min treatmentwith Triton X-100, 91.6 ± 3.0% of the aggregatecontained fluorescing cells with compromised mem-brane integrity, which was significantly greater thanthe 14.0% for non-Triton-treated cells (P = 1.43 ×10−5, n = 3).

Cell Morphology and Void Analysis

Cells entered the USWT with a rounded morphol-ogy characteristic of cells in suspension. Cells son-icated for 30 s (Figure 4A) showed the same roundmorphology as the unsonicated cells (not shown).By 10 min, the loss of rounded morphology was dis-tinguishable as a flattening of sides due to membranespreading and overlapping (Figure 4B). Circularitywas significantly reduced (P = 3.63 × 10−8, n = 12)from 0.939 ± 0.003 at 30 s to 0.886 ± 0.006 at 10 min(Figure 4C). This occurred most extensively withinthe first 5 min of cell–cell contact during sonication.

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Figure 3. Development of HepG2 cell aggregate in the USWT at (A) 30 s, (B) 3 min, and (C) 15 min. Mean aggregate area increases withaggregate age until 3 min (D). Bar in micrographs represents 200 µm.

The interfacial length, an index of membrane spread-ing, increased significantly (P = 1.31 × 10−6, n =8) from 2.46 ± 0.25 µm at 30 s to 5.86 ± 0.28 µmat 10 min (Figure 4D). Void index measurementsdemonstrated an initial reduction in void area withtime as cells formed a more closely packed aggre-gate (Figure 4E). This proceeded for 6 min until aplateau was reached. Void index was plotted againstinterfacial length at each time point (Figure 4F). Thecorrelation index (r2 = 0.699) was significantly dif-ferent from zero (P < 0.0001).

F-Actin Distribution

F-actin distribution was monitored as a function oftime (the reported times were the time in the trap plus

a 1 min delay for fixation). The distribution showedno difference at 2 min (Figure 5A) from cells fixedand stained immediately following preparation (notshown). 3-D surface plots of F-actin intensity fromfluorescence micrographs showed that F-actin wasdistributed towards the cell periphery after 2 min(Figure 5B) but was localized at regions of cell–cellcontact after 4 and 11 min (Figure 5C, E). This tran-sition in staining at sites of contact was sharp as timeincreased from 2 to 4 min (Figure 5B, D) and becameeven more defined at 11 min (Figure 5F). F-actin dis-tribution at the cell–cell boundary increased in peakintensity and decreased in width (Figure 6A). F-actinwidth perpendicular to the contact interface was 1.98µm at 2 min and 1.43 µm at 11 min. The % change inthe area under the curve in Figure 6A showed a rapid

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CELL ADHESION IN HepG2 AGGREGATES 15

Figure 4. Representative images of (A) the circular morphology of HepG2 cells at 30 s following introduction into the USWT and (B) thedeparture from circular morphology following 10 min sonication in the trap. Cells show overlapping (o) and membrane spreading at pointsof contact (s) leading to an increase in the interfacial length (i) and an overall reduction of voids (v) within the aggregate. Aggregate agedependence of (C) Cell circularity; (D) Membrane spreading (as assessed by length of contact interface); and (E) void index. (F) Changes incircularity and void index correlate with membrane spreading (interfacial length). Bar in micrographs represents 10 µm.

increase until 6 min, beyond which there was littlefurther change (Figure 6B). The mean peak inten-sity of F-actin fluorescence increased more slowlythan area under the curve as aggregate age increased(Figure 6B).

DISCUSSION

HepG2 cells introduced into the USWT hadclearly established 2-D aggregates within 30 s, a time

comparable to the 20 and 30 s periods previouslyreported for neural cells (Bazou et al., 2005a) andchondrocytes, respectively (Bazou et al., 2006). Thespeed of aggregate formation results in essentiallysynchronous initiation of cell–cell adhesive interac-tions across the aggregate. The cells retained via-bility, in agreement with previous results for neuralcells following 60 min ultrasound exposure in theUSWT (Bazou et al., 2005a). Indices of cell mor-phology demonstrated rapid changes following the

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16 G. O. EDWARDS ET AL.

Figure 5. Fluorescence micrographs and 3-D surface intensity plots (inset shows magnified region of micrograph used for construction ofsurface plot) of F-actin stained with Phalloidin-Alexa488 in aggregates aged 2 min (A, B), 4 min (C, D), and 11 min (E, F), demonstratingthat an increase in F-actin at regions of contact between HepG2 cells can be detected in aggregates aged 4 min. Bar in micrographs represents20 µm, and bar in magnified insets represents 2 µm.

initiation of cell–cell contact. Cells lost their roundedmorphology within 5 min and exhibited membranespreading (increased interfacial length at points ofcontact), which resulted in a reduction in the voidindex. While HepG2 integrin expression is requiredto facilitate cell adhesion, spreading, and loss of around morphology on a substratum (Yin et al., 2003),the present study showed that spreading and morpho-logical changes are also induced by cell–cell contactin suspension.

Actin reorganisation at points of cell–cell contacthas previously been reported in a number of systems(Bazou et al., 2004, 2005a; Chu et al., 2004; Helwaniet al., 2004; Huo et al., 2004; Ivanov et al., 2005;Zeggers et al., 1998; Zhang et al., 2005). Helwaniet al. (2004) demonstrated that E-cadherin colocal-ized with cortactin at points of adhesion betweenMDCK cells, which was required for F-actin recruit-ment to cell junctions. The result that cytoplasmicF-actin adjacent to the membrane had increased by

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CELL ADHESION IN HepG2 AGGREGATES 17

Figure 6. (A) Graphical representation of mean F-actin intensityplot profiles demonstrating a significant buildup of F-actin at thecell–cell boundary in aggregates aged greater than 3 min. A timezero control F-actin distribution across the periphery of a single cellwas shown in mirror image (dotted line). (B) Area under curve andpeak intensity of F-actin from Figure 5 A increase with aggregateage.

2 min (Figure 6B) through cell–cell contact alone iscomparable to the value of 2 min reported for a de-tectable increase in junctional F-actin using the ‘cal-cium switch’ approach to induce cell–cell contactin human keratinocyte cells grown on a monolayerof 3T3 fibroblasts treated with mitomycin-C (Zhanget al., 2005). Ivanov et al. (2005) also employed the‘calcium switch’ approach to study actin reorganiza-tion following the development of cell–cell contacts,namely during the assembly of the epithelial apicaljunctional complex, in T84 intestinal epithelial cells.Nascent E-cadherin-containing junctions developedby 30 min at the cell–cell boundary, which coincided

with an increase in phalloidin-stained junctional F-actin (Ivanov et al., 2005). Peripheral F-actin can bedetected within 4 min when mouse sarcoma cell dou-blets are formed using a pair of pipettes (Chu et al.,2004). By 8 min, F-actin can be detected at cell–celljunctions between neural cells in the USWT (Bazouet al., 2005a), and levels have stabilized between 8min and 15 min of human keratinocyte cell contact(Zhang et al., 2005). Increased peripheral F-actinalso occurs where HepG2 cells grown on quartz glasscoverslips form cell–cell contacts (Huo et al., 2004;Zegers et al., 1998).

Morphological changes preceded the recruitmentof F-actin to regions of cell contact. Membranespreading (increased interfacial length) followed areduction in circularity, but both showed a 50%change in 2.2 min (Table 1). Changes in cell mor-phology preceded F-actin redistribution by 1 minfor actin area and by 2.2 min for actin peak in-tensity at the midpoint between cells (Table 1),as assessed by comparing the time required for a50% change. On the assumption that membranespreading arises from receptor–receptor interactions(Bazou et al., 2005a), these results demonstrate thatcell–cell contact through aggregation leads to mem-brane spreading and a loss of circular morphology,followed by F-actin recruitment to regions of cell–cell contact. These early changes in F-actin distribu-tion appear to be a response to changes in cellularmorphology brought about by contact, rather thanthe morphological changes being driven by the re-distribution of F-actin. A comparable relationshipbetween morphology and cytoskeletal rearrange-ments can be observed in the early developmentand polarisation of embryos. Following cell division,

TABLE 1 Changes in indices of cell shape and F-actin withincreasing aggregate age

IndexChange over

10 min

Time for 50%of total change

over 10 min

Circularity 0.053 2.2Interfacial Length (µm) 3.7 2.2Actin: Area under curve 72 3.2Actin: Peak intensity (µm2) 39.2 4.4

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18 G. O. EDWARDS ET AL.

daughter cells form adhesive junctions displayingmembrane spreading at points of cell–cell contactwhere F-actin (Albertini et al., 1987) and adhesivemolecules such as JAM-1 (Thomas et al., 2004) andF-actin (Albertini et al., 1987) become localized.

HepG2 cells expressing GFP-actin rapidly adhereto fibronectin-treated surfaces, leading to an increasein cell area resulting from membrane spreading,reaching 50% of its steady-state value by 6 min (Fenget al., 2005). In the current cell–cell adhesion study,where membrane spreading was inferred from an in-crease in interfacial length rather than an increase incell area, the duration of contact required to producea 50% increase was shorter, at 2.2 min. Fluorescentstaining demonstrated a 50% increase in F-actin areafollowing 3.2 min and a 50% increase in peak inten-sity by 4.4 min (Table 1).

Changes in morphology and increased F-actinlead to the formation of a stable aggregate. Retentionof aggregate integrity upon sedimentation following15 min levitation demonstrated the acquisition ofmechanical stability, a conclusion strengthened bythe fact that large regions of the aggregate retainedtheir structural integrity during the stress of removalfrom the trap for studying F-actin distribution at timepoints prior to 15 min. Both observations suggestthat aggregates in suspension may be mechanicallyrobust enough to withstand manipulation after 15min. By using pairs of pipettes to hold doublets ofmouse sarcoma cells together, it has been shown thatstrong adhesion develops between 4 min and 30 min,which is associated with an increase in actin at thecell–cell boundary (Chu et al., 2004).

Adhesive cell–cell interactions leading to cy-toskeletal remodelling involve a range of cell-surface receptors, namely cell adhesion molecules(CAMs) and cadherins. It has previously been shownthat wild-type HepG2 cells do not express the CAMsICAM-1 (Qin et al., 2005), hepaCAM (Moh et al.,2005), and LI-CAM (Wong et al., 2003). The ex-pression of E-cadherin in the HepG2 cell line is acontentious issue. On employing RT-PCR and im-munofluorescence, E-cadherin was not detected inthe HepG2 cell line by Yano and Yamasaki (2001).Cui et al. (2006) demonstrated that it could only be

detected when cells were treated with the promoterdemethylating agent 5-aza-2′-deoxycytidine. How-ever, Lin et al. (2006) detected E-cadherin in HepG2cells by means of immunofluorescence and West-ern blotting, a result confirmed by the work of Liuet al. (2006). During the current study, immunofluo-rescent detection of E-cadherin demonstrated a dif-fuse, punctuate cytoplasmic distribution rather thanat cell–cell junctions, thus it was concluded thatthe HepG2 clone employed here did not expressE-cadherin capable of forming functional adherensjunctions (unpublished data).

Exposure to ultrasound is not likely to influencethe properties of cell adhesion molecules. Aggre-gation occurs at a pressure node, where both localacoustic pressure and the axial direct radiation forcethat drives aggregation are zero (Kuznetsova andCoakley, 2004). Calculation of the acoustic interac-tion force between particles at such a pressure nodeare comparable to the Van der Waals force at mem-brane separations of 50 nm (Bazou et al., 2005b)and decreases relative to the van der Waals force atsmaller cell separations. The Van der Waals forceis in turn much smaller than the attractive force in-volved in surface receptor interactions at the mem-brane separations at which receptors engage (Bazouet al., 2006). We conclude that adhesion followingcell–cell contact is due to normal receptor phenom-ena rather than to direct effects of experimental ul-trasound exposure.

The formation of 2-D cell aggregates in the USWTallows the clear temporal optical microscopic ex-amination of adhesion to be compared to detectionof junctional F-actin in recovered aggregates. Theultrasound methodology also has the potential torapidly form 3-D aggregates on a fast timescale. Like3-D culture models, spheroids show enhanced liver-specific behavior in comparison to monolayers, mak-ing them an attractive model for in vitro toxicologicalstudies (Ma et al., 2003). Current technology basedon a rotating plate culture system provides spheroidsfollowing 7 days for studying liver-specific behaviorand drug metabolism (Damelin et al., 2004) and fortoxicological assessment (Ma et al., 2003; Xu et al.,2003). The data presented here provide guidance on

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CELL ADHESION IN HepG2 AGGREGATES 19

the time required to rapidly form a 3-D aggregateof biologically interacting HepG2 cells in an ap-propriately designed ultrasound trap. The extent towhich such ultrasonically generated 3-D aggregatesformed from single-cell HepG2 suspensions developa biochemistry comparable to spheroids will be in-vestigated in future work.

ACKNOWLEDGMENTS

This work was supported by the Biotechnologyand Biological Sciences Research Council, GrantNo. BB/C515220/1.

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