Ultrasound Trap Research Articles

Hematocolpos Secondary to an Unrecognized Diagnosis of Hymenial Imperforation: A Case Report and Review of the Literature

A relatively rare congenital malformation, hematocolpos is the progressive accumulation of menstrual blood in the vaginal cavity at puberty. It is often the consequence of a hymen imperforation. It is clinically manifested by cyclic pelvic pain and primary amenorrhea and, more rarely, by a pelvic mass syndrome. The diagnosis is primarily clinical. Ultrasound and magnetic resonance imaging (MRI) are additional tests to confirm hematolcolpos and exclude other associated genitourinary malformations. Treatment consists of a hymenotomy to drain the hematocolpos. The age of discovery of hematocolpos varies from 13 to 14 years. We report a case of hematocolpos secondary to hymenal imperforation diagnosed in a 17-year-old girl with periodic pelvic pain, primary amenorrhea and a pelvic mass. A first ultrasound trap had evoked a large ovarian cyst but the repeat pelvic ultrasound in our hospital confirmed the diagnosis of hematocolpos.

Gene Expression Analysis of Mouse Embryonic Stem Cells Following Levitation in an Ultrasound Standing Wave Trap

In the present paper, gene expression analysis of mouse embryonic stem (ES) cells levitated in a novel ultrasound standing wave trap (USWT) ( Bazou et al. 2005a) at variable acoustic pressures (0.08–0.85 MPa) and times (5–60 min) was performed. Our results showed that levitation of ES cells at the highest employed acoustic pressure for 60 min does not modify gene expression and cells maintain their pluripotency. Embryoid bodies (EBs) also expressed the early and late neural differentiation markers, which were also unaffected by the acoustic field. Our results suggest that the ultrasound trap microenvironment is minimally invasive as the biologic consequences of ES cell replication and EB differentiation proceed without significantly affecting gene expression. The technique holds great promise in safe cell manipulation techniques for a variety of applications including tissue engineering and regenerative medicine. (E-mail: Bazoud@tcd.ie)

Modelling condensation and the initiation of chondrogenesis in chick wing bud mesenchymal cells levitated in an ultrasound trap

Chick wing bud mesenchymal cell micromass culture allows the study of a variety of developmental mechanisms, ranging from cell adhesion to pattern formation. However, many cells remain in contact with an artificial substratum, which can influence cytoskeletal organisation and differentiation. An ultrasound standing wave trap facilitates the rapid formation of 2-D monolayer cell aggregates with a defined zero time-point, independent from contact with a surface. Aggregates formed rapidly (within 2 min) and intercellular membrane spreading occurred at points of contact. This was associated with an increase in peripheral F-actin within 10 min of cell-cell contact and aggregates had obtained physical integrity by 30 min. The chondrogenic transcription factor Sox9 could be detected in cells in the ultrasound trap within 3 h (ultrasound exposure alone was not responsible for this effect). This approach facilitates the study of the initial cell-cell interactions that occur during condensation formation and demonstrates that a combination of cell shape and cytoskeletal organisation is required for the initiation and maintenance of a differentiated phenotype, which is lost when these phenomena are influenced by contact with an artificial substrate.

Biochemical properties of encapsulated high-density 3-D HepG2 aggregates formed in an ultrasound trap for application in hepatotoxicity studies

This paper describes the alginate encapsulation of preformed high-density 3-D HepG2 cell aggregates that guarantees good maintenance of liver-specific biomarker expression. The process involves forming a high-density (> or =7 x 10(4) cells/aggregate) discoid 3-D cell aggregate in an ultrasound trap, which is subsequently recovered and encapsulated in alginate/CaCl(2) hydrogel. Glucose secretion/consumption, lactate release, detoxifying enzyme capacity, cytokeratin-18 expression as well as hypoxia were characterized in encapsulated 3-D HepG2 aggregates over 10 days in culture. Encapsulated 3-D HepG2 aggregates released glucose into the media, although this ability was exhibited only after 1 day in culture and was subsequently lost over the ensuing 9 days. In contrast, lactate was constantly released into the media. Significantly more lactate was secreted after 3 days in culture indicating a more hypoxic environment and hence a higher rate of anaerobic glycolysis. Aggregates consistently expressed cytokeratin-18. Cytochrome P450-1A1 activity reached a maximum on day 1 of culture followed by a progressive reduction to basal levels, while P450-3A4 activity was up-regulated in a time-dependent manner reaching a peak on day 7 in culture. Glutathione-S-transferase activity, on the other hand, was at more physiological levels and remained constant over the 10-day culture period. The ultrasound trap allowed the rapid (within 5 min) generation of uniformly shaped and sized aggregates. The results reported here suggest that ultrasound-formed 3-D HepG2 aggregates can serve as alternative in vitro models providing a quick outlook on toxicity, in a tissue-mimetic manner, thus offering the future option of a cost-effective screening platform for pharmaceutical development.

Long-term viability and proliferation of alginate-encapsulated 3-D HepG2 aggregates formed in an ultrasound trap

We report proof of principle here of a gel encapsulation technique that departs from the minimum surface area to volume restriction of spherical microcapsules and allows gelation of preformed high-density (>or=2x10(4) cells/aggregate) 3-D HepG2 cell aggregates. The process involves forming a discoid 3-D cell aggregate in an ultrasound standing wave trap (USWT), which is subsequently recovered and encapsulated in alginate/CaCl2 hydrogel. The size of the ultrasound-formed aggregates was dependent upon the initial cell concentration, and was in the range of 0.4-2.6 mm in diameter (for cell concentrations ranging between 10(4) and 5x10(6)/ml). At low cell concentrations (<or=5x10(5)/ml), aggregates were 2-D, while at concentrations of >or=10(6)/ml, 3-D aggregates were generated. Cells in non- and encapsulated 3-D HepG2 aggregates remained 70-80% viable over 10 days in culture. The proliferative activity of the aggregates resulted in the doubling of the aggregate cell number and a subsequent increase in the aggregate thickness, while albumin secretion levels in encapsulated aggregates was 4.5 times higher compared to non-encapsulated, control aggregates. The results reported here suggest that the ultrasound trap can provide an alternative, novel approach of hydrogel cell encapsulation and thus rapidly (within 5 min) produce in vitro models for hepatocyte functional studies (for example, toxicity studies particularly if primary hepatocytes are used) in a tissue-mimetic manner.

NCAM and PSA-NCAM dependent membrane spreading and F-actin reorganization in suspended adhering neural cells

Mediation of synchronous cell-cell interactions by NCAM and PSA-NCAM is examined here in aggregates (monolayers) of C6 polysialylated embryonic neural cells, formed rapidly (within 30 s) in suspension in an ultrasound trap. These cells express all three main isoforms of neural cell adhesion molecule (NCAM). The rate of extension of perimeter contact (i.e., membrane spreading) between closely adjacent cells and the temporal reinforcement of the Filamentous (F)-actin cytoskeleton at those regions were measured. Enzymatic removal of the cell-cell repelling polysialic acid (PSA) increases the rate of NCAM-induced membrane spreading, while removal of NCAM-120 had no detectable effect. Competitive peptide inhibition of the third immunoglobulin domain of NCAM significantly reduced the rate of membrane spreading, while NCAM siRNA transfected cells lost their ability to spread. It is argued that NCAM induced contact is the initial requirement for membrane spreading and facilitates conditions for subsequent cytoskeletal reorganization in these neural cells.

Functional three‐dimensional HepG2 aggregate cultures generated from an ultrasound trap: Comparison with HepG2 spheroids

Three-dimensional culture systems are an ideal in vitro model being capable of sustaining cell functionalities in a manner that resembles the in vivo conditions. In the present study, we developed an ultrasound trap-based technique to rapidly produce HepG2 hepatocarcinoma cell aggregates within 30 min. Enhanced junctional F-actin was observed at the points of cell-cell contact throughout the aggregates. HepG2 aggregates prepared by the ultrasound trap can be maintained in culture on a P-HEMA-coated surface for up to 3 weeks. The cells in these aggregates proliferated during the initial 3 days and cell number was consistent during the following maintenance period. Albumin secretion from these HepG2 aggregates recovered after 3 days of aggregate formation and remained relatively stable for the following 12 days. Cytochrome P450-1A1 activity was significantly enhanced after 6 days with maximal enzyme activity observed between 9 and 18 days. In addition, comparison experiments demonstrated that HepG2 aggregates generated by the ultrasound trap had comparable functional characterizations with HepG2 spheroids formed by a traditional gyrotatory-mediated method. Our results suggest that HepG2 aggregate cultures prepared through the ultrasound trap-based technique may provide a novel approach to produce in vitro models for hepatocyte functional studies.

Stability of 2-D Colloidal Particle Aggregates Held against Flow Stress in an Ultrasound Trap

The formation of a two-dimensional aggregate of 25 microm latex particles in a 1.5 MHz ultrasound standing wave (USW) field and its disintegration in a flow were studied. The aggregate was held in the pressure node plane, which allowed continuous microscope observation and video recording of the processes. The trajectories and velocities of the particles approaching the formation site were analyzed by particle image velocimetry (PIV). Since the direct radiation force on the particles dominated the drag due to acoustic streaming, the acoustic pressure profile in the vicinity of the aggregate was quantifiable. The drag coefficients D(coef) for 2- to 485-particle aggregates were estimated from the balance of the drag force FD and the buoyancy-corrected gravitational force during sedimentation on termination of the ultrasound when the long axis of the aggregate was in the vertical plane. D(coef) were calculated from FD as proportional to the aggregate velocity. Experiments on particle detachment by flow (in-plane velocity measured by PIV) from horizontal aggregates suspended in deionized water and CaCl2 solution of different concentrations showed that the mechanical strength of the aggregates depended on the acoustic pressure amplitude P0 and ionic strength of the solution. In deionized water the flow velocity required to detach the first single particle from an aggregate increased from 1 mm s-1 at P0 = 0.6 MPa to 4.2 mm s-1 at P0 = 1.4 MPa. The balance of forces acting on particles in a USW trap is discussed. The magnitude of the shear stress employed ( approximately 0.05 Pa) and separation forces suggests that this technique can be applied to studying the mechanical responses of cell aggregates to hydrodynamic flow, where cell-cell interaction can be separated from the effects of solid substrata.

Cell Adhesion Dynamics and Actin Cytoskeleton Reorganization in HepG2 Cell Aggregates

The temporal dependence of cytoskeletal remodelling on cell-cell contact in HepG2 cells has been established here. Cell-cell contact occurred in an ultrasound standing wave trap designed to 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 and change in cell circularity reached 50% of their final values within 2.2 min of contact. Junctional F-actin increased at the interface but lagged behind membrane spreading, reaching 50% of its final value in 4.4 min. Aggregates had good mechanical stability after 15 min in the trap. The implication of this temporal dependence on the sequential progress of adhesion processes is discussed. These results provide insight into how biomimetic cell aggregates with some liver cell functions might be assembled in a systematic, controlled manner in a 3-D ultrasound trap.

Gap junctional intercellular communication and cytoskeletal organization in chondrocytes in suspension in an ultrasound trap

Particles or cells suspended in an appropriately designed ultrasound standing wave field can be aggregated at a node to form a single monolayer in a plane that can be interrogated microscopically. The approach is applied here to investigate the temporal development of F-actin and Cx43 distribution and of gap junctional intercellular communication in 2-D chondrocyte aggregates (monolayers) rapidly and synchronously formed and held in suspension in an ultrasound trap. Development of the F-actin cytoskeleton in the confluent single layer of 'cuboidal' cells forming the aggregate was completed within 1 h. Chondrocytes levitated in the trap synchronously formed functional gap junctions (as assessed by CMFDA dye transfer assays) in less than 1 h of initiation of cell-cell contact in the trap. It was shown that Cx43 gene expression was retained in isolated chondrocytes in suspension. Preincubation of cells with the protein synthesis inhibitor cycloheximide caused a six-fold decrease in Cx43 accumulation (as assessed by immunofluorescence) at the interfaces of chondrocytes in the aggregate. It is shown that the ultrasound trap provides an approach to studying the early stages of cytoskeletal and gap junction development as cells progress from physical aggregation, through molecular adhesion, to display the intracellular consequences of receptor interactions.

Rapid Molecular and Morphological Responses of Prostate Cell Lines to Cell–Cell Contact

Cell-cell adhesion in 2-D PZ-HPV-7 prostate epithelial and DU-145 prostate cancer cell aggregates (monolayers), synchronously and rapidly (within 30 s) formed in suspension in an ultrasound trap has been examined over 60 min. The intracellular distributions of the cadherin/catenin complex components for both cell lines were time-dependent and were clearly identifiable as early as 150 s following cell-cell contact in the trap, while equilibrium positions were reached within 60 min following cell-cell contact. The accumulation of E-cadherin at the cell-cell interface was greater for PZ-HPV-7 than for DU-145 cells over 60 min in the trap, with the apparent formation of adherens junctions over that time scale in PZ-HPV-7 but not in DU-145 cells. The amounts of F-actin, alpha-, beta-, and gamma-catenins recruited to the cell-cell interface of PZ-HPV-7 cells were on average 2.4 times higher than those of DU-145 cells. The ability of different cell types to spread along neighboring cells was 1.5-fold greater for the PZ-HPV-7 than for the DU-145 cells. These results, discussed also in the context of earlier studies of cell adhesion in an ultrasound trap, characterize a reduced adhesiveness of DU-145 cells compared to PZ-HPV-7 cells.

Molecular adhesion development in a neural cell monolayer forming in an ultrasound trap

A 2-dimensional aggregate of C6 neural cells was formed rapidly (within 30 s) in suspension in a recently developed 1.5 MHz ultrasound standing wave trap. A typical 1 mm diameter aggregate contained about 3,500 cells. Spreading of membrane occurred between the aggregated cells. The rate of spreading of the tangentially developing intercellular contact area was 0.19 microm/min. The form of the suspended aggregate changed from one of a hexagonal arrangement of cells to one of a cell-monolayer-like continuous sheet of mostly quadrilateral and pentagonal cells as in a cell monolayer on a solid substratum. A range of fluorescent indicators showed that the >99% viability of the cells did not change during 1 h exposures; therefore cell viability was not compromised during the monolayer development. The average integral intensities from stained actin filaments at the spreading cell-cell interfaces after 1, 8 and 30 min were 14, 25 and 46 microm(2) respectively. The cells in this work progressed from physical aggregation, through molecular adhesion, to displaying the intracellular consequences of receptor interactions. The ability to form mechanically strong confluent monolayer structures that can be monitored in situ or harvested from the trap provides a technique with general potential for monitoring the synchronous development of cell responses to receptor-triggered adhesion.

Manipulation of cells in ultrasound standing waves

The acoustic radiation force operating on a cell in a 0.5-MPa pressure amplitude 3-MHz standing wave field equals that experienced in a 250-g gravitational field. Consequently, cells may be driven rapidly to preferred points in the sound field. Acoustic radiation pressure can drive bacteria, flowing by an acoustic reflector (prepared as an immunosensor surface), onto that surface where they are then captured. Animal cells have been driven onto the fabric of wide-gauge filters to form, a basis for a supported bioreactor. Real standing wave fields do not have the ideal plane-wave form, particularly when the transducer diameter is much larger than a wavelength (typical transducer diameter of 20 mm, wavelength of 0.5 mm at 3 MHz in water). Pressure variations within a node plane then give rise to regions towards which cells migrate and may be held against flow in potential traps. These traps are exploited commercially to retain cells in fermentors in the pharmaceutical industry. Most recently, ultrasound traps have been employed to concentrate animal cells in the focal plane of a fluorescence microscope so that cell interactions and their consequences at the molecular level can be monitored.

Characterisation of the morphology of 2-D particle aggregates in different electrolyte concentrations in an ultrasound trap

An ultrasound standing wave trap in which growth of two-dimensional aggregates of suspended particles can be optically monitored has recently been developed. The technique is employed here to study the influence of [CaCl 2] in the range 2–20 mM on growth of aggregates of 25 μm latex spheres. The perimeter fractal dimension for closely-packed hexagonally ordered aggregates formed by weakly interacting particles in deionised water was 1.14. The fractal dimension increased with increasing salt concentration to reach 1.58 in 20 mM CaCl 2 where the aggregate resulting from high collision efficiency particle interactions was highly dendritic. The [CaCl 2] at which the aggregate morphology ‘crossed over’ from being essentially closely packed to showing dendritic organisation was about 6 mM. Two analysis techniques, fast Fourier transform (FFT) and void analysis, that are routinely employed for characterising order or ‘openness’ of 2-D structures were applied to the aggregate images. The Fast Fourier Transformation method allowed the detection of the first signs of loss of the internal aggregate order at low salt molarities (2 and 4 mM). An increase in void area with increasing salt concentration was suggested at concentrations (8–20 mM) where the sensitivity of the fractal dimension to change was low.

Cell–cell contact and membrane spreading in an ultrasound trap

An ultrasonic standing wave trap [Langmuir 19 (2003) 3635] in which the morphologies of 2-D latex–microparticle aggregates, forming a pressure node plane, were characterised has been applied here to different cell suspensions with increasing order of specificity of cross-linking molecule, i.e. polylysine with chondrocytes; wheat germ agglutinin (WGA) with erythrocytes and surface receptors on neural cells. The outcome of initial cell–cell contact, i.e. whether the cells stuck at the point of contact (collision efficiency=1) or rolled around each other (collision efficiency=0), was monitored in situ by video-microscopy. The perimeter fractal dimensions (FD) of 2-D hexagonally symmetric, closely packed aggregates of control erythrocytes and chondrocytes were 1.16 and 1.18, respectively while those for the dendrititc aggregates formed initially by erythrocytes in 0.5 μg/ml WGA and chondrocytes in 20 μg/ml polylysine were 1.49 and 1.66. The FDs for control and molecularly cross-linked cells were typical of reaction-limited aggregation (RLA) and transport diffusion-limited aggregation (DLA), respectively. The FDs of the aggregates of cross-linked cells decreased with time to give more closely packed aggregates without clear hexagonal symmetry. Suspensions of neural cells formed dendritic aggregates. Spreading of inter-cellular membrane contact area occurred over 15 min for both erythrocyte and neural cell dendritic aggregates. The potential of the technique to characterise and control the progression of cell adhesion in suspension away from solid substrata is discussed.