Placenta
Volume 31, Issue 4 , Pages 347-350, April 2010

New technology for investigating trophoblast function

  • R.J. Keogh

      Affiliations

    • Department of Perinatal Medicine Pregnancy Research Centre, Royal Women's Hospital, Locked Bag 300, Corner Grattan Street and Flemington Road, Parkville 3052 Victoria, Australia
    • University of Melbourne, Department of Obstetrics and Gynaecology, Royal Women's Hospital, Locked Bag 300, Corner Grattan Street and Flemington Road, Parkville 3052 Victoria, Australia
    • Corresponding Author InformationPregnancy Research Centre, Royal Women's Hospital, Locked Bag 300, Corner Grattan Street and Flemington Road, Parkville 3052 Victoria, Australia. Tel.: +61 3 8345 3749; fax: +61 3 8345 3746.

Accepted 11 February 2010. published online 08 March 2010.

Article Outline

Abstract 

Measuring trophoblast function involves performing end-point assays that represent the response at a single time point. New technology from Roche Applied Science enables continuous monitoring of cells in real-time using specialized culture dishes containing micro-electrodes. The xCELLigence System allows continuous measurement and quantification of cell adhesion, proliferation, migration and invasion, thus creating a true picture of trophoblast function. Lag and log growth phases can be determined thus pinpointing optimal times to treat and harvest cells. Use of this system will provide valuable insights into trophoblast functions as well as the behaviour of other cell types found at the maternal–fetal interface.

Keywords: Trophoblast, Migration, Invasion, Proliferation, xCELLigence, Technology

 

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1. Introduction 

The mainstays of assessing extravillous trophoblast function are to measure cell migration, invasion and proliferation. This is achieved through functional assays such as wound-healing assays [1], Boyden chamber experiments [2] and [3H]-thymidine uptake [3], all with single or end-point analysis. There has been no way to quantitatively monitor cell behaviour in real-time. Whilst time-lapse video microscopy does allow continuous monitoring of cells, substantial analysis of the data collected is still required post-experiment [4], [5].

The Roche xCELLigence System is an innovative technology that allows continuous, quantitative monitoring of cells as they adhere, proliferate and migrate by measuring electrical impedance. Gold micro-electrodes are incorporated into the bottom of custom tissue culture dishes. By passing a current through the micro-electrodes at regular intervals, the electrical impedance at a particular time can be measured. The change in impedance caused by cell attachment and spreading is then expressed as the Cell Index (CI). This is an arbitrary measurement defined as (Rn − Rb)/15, where Rb is the background impedance of the well measured with medium alone and Rn is the impedance of the well measured at any time (t) with cells present. The CI is thus a reflection of overall cell number, attachment quality and cell morphology which can change as a function of time.

This report demonstrates the use of the xCELLigence System to measure growth characteristics of two extravillous trophoblast cell lines and the adhesive, proliferative, migratory and invasive responses to treatment with the chemokine CCL2.

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2. Methods 

SGHPL-4 cells were kindly provided by Professor Guy Whitley and Dr Judith Cartwright (St George's, London, UK) and HTR8/SVneo cells were kindly provided by Professor Charles Graham (Queen's University, Kingston, Canada). Cells were cultured in Ham's F-10 (SGHPL-4) or RPMI (HTR8/SVneo) supplemented with 10% or 5% fetal calf serum (FCS) respectively, l-glutamine (2 mmol/L), penicillin (100 IU/ml) and streptomycin (100 μg/ml) and were maintained in 95% air/5% CO2 at 37 °C. All tissue culture reagents were from Invitrogen Corp. (Carlsbad, CA USA) except for FCS, which was from Thermo Fisher Scientific (Sydney, NSW Australia).

Experiments were carried out using the RTCA DP instrument (Roche Diagnostics GmbH, Germany) which was placed in a humidified incubator maintained at 37 °C with 95% air/5% CO2. Growth curves were constructed using 16-well plates (E-plate 16, Roche Diagnostics GmbH). Briefly, cells were seeded at 1250–40,000 cells/well in medium containing FCS. Cells were monitored once every 2 min for 40 min and then once every hour. For adhesion and proliferation, cells were seeded in E-plates 16 at 40,000 cells/well in FCS-free medium, rhCCL2 (R&D Systems; Minneapolis, MN USA) was added at 20 ng/ml, and the plate was then monitored once every 30 s for 4 h, then once every hour. Cell migration and invasion were assessed using specially designed 16-well plates (CIM-plate 16, Roche Diagnostics GmbH) with 8 μm pores. These plates are similar to conventional transwells with the micro-electrodes located on the underside of the membrane of the upper chamber. Wells were coated on the upper surface of the transwell with Matrigel (BD BioSciences, Bedford, MA USA; 1:10 dilution) for measurement of invasion (wells can also be coated on the lower (electrode) surface with a thin layer of a matrix component such as fibronectin, or diluted Matrigel to aid attachment). CCL2 (20 ng/ml) was added to FCS-free medium in the lower chamber and cells were seeded into the upper chamber at 40,000/well. The CIM-plate 16 was monitored every 10 s for 40 min then once every hour. Data analysis was carried out using RTCA Software 1.2 supplied with the instrument.

HTR8/SVneo functions were also assessed using conventional assays. Proliferation was measured with the CellTiter 96® AQueous One Proliferation Assay (Promega Corporation, Madison, WI USA) according to the manufacturer's instructions. Migration was measured by a wound-healing assay with minor modifications [6]. Briefly, cells were grown to confluency in 6-well plates, a vertical scratch was made using a sterile 200 μl pipette tip and photographs were then taken at t = 0, 3, 24, 27 and 48 h. Images were processed with ImageJ [7] and the extent of cell migration (% wound closure) calculated using Cell Profiler [8]. Matrix metalloprotease (MMP) activity was measured in the medium collected from wound-healing assays by gelatin zymography using methodology as described [9]. The intensity of the band corresponding to MMP9 was measured and analysed with ImageQuant (GE Healthcare Amersham, UK) as an indicator of the invasive potential of the cells.

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3. Results and discussion 

Growth curves generated using the xCELLigence System are characteristic for each cell type. In the HTR8/SVneo growth curves (Fig. 1A) the cells are seen to (i) rapidly attach over the first 2 h, (ii) enter a lag phase of approximately 4 h before (iii) entering log growth. SGHPL-4s show an initial sharp increase in CI due to fast and strong attachment (Fig. 1B). The cells then move into a cytostatic state where the decline in CI is stable at the higher densities, an indication of contact inhibition, whilst the lower densities continue to proliferate. By monitoring cells in real-time and gathering this kinetic data, it is possible to determine the recovery time for cells post plating and the optimal time for addition of treatments. This data also shows the importance of seeding density and how it alters the duration of the lag and log growth phases.

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  • Fig. 1 

    Trophoblast growth curves. Growth curves for A) HTR8/SVneo and B) SGHPL-4 generated by plating cells at densities from 1250 to 40,000 cells/well as indicated and monitoring over 72 h. Annotations represent (i) cell attachment, (ii) lag phase and (iii) log phase.

Experiments were performed to assess the effect of the chemokine CCL2 on HTR8/SVneo proliferation, adhesion, migration and invasion. In each case, 10% FCS was included as a positive control. CCL2 had no effect on HTR8/SVneo proliferation (Fig. 2A) but did stimulate adhesion (Fig. 2B), migration (Fig. 2C) and invasion (Fig. 2D). Values for individual time points can be extracted from the data as well as rates of change by calculating the slope or area under the curve (Fig. 3D–F). An advantage of the xCELLigence System is that adhesion and proliferation data can be obtained from the same well (Fig. 2A and B). Monitoring of the cells over the first 4 h at 30 s intervals provides adhesion data, whilst subsequent monitoring every hour up to 60 h generates proliferation data.

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  • Fig. 2 

    Trophoblast proliferation, adhesion, migration and invasion in response to CCL2 measured with the xCELLigence System. HTR8/SVneo cells were plated at 40,000/well and monitored as described over 60 h. Shown are the Cell Index curves for A) proliferation, B) adhesion, C) migration and D) invasion. Negative control (open circles), CCL2 (20 ng/ml; closed circles), positive control (10% FCS; open squares). Data shown is the mean ± SEM from n ≥ 3 independent experiments with n ≥ 2 replicates in each.

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  • Fig. 3 

    Comparison of conventional assays to the xCELLigence System. HTR8/SVneo cells were stimulated with CCL2 (20 ng/ml) for the times indicated, and measurements made as described using either conventional assays (A–C) or the xCELLigence System (D–F). A and D) proliferation, B) migration, C) invasive potential (MMP 9 activity), E) rate of migration F) rate of invasion. Negative control (open bars), CCL2 (20 ng/ml; grey bars), positive control (10% FCS; hatched bars). The dotted line in (A) shows cell number at t = 0 h. Data shown is the mean ± SEM from n ≥ 3 independent experiments, *p < 0.05.

The data generated with the xCELLigence System (Fig. 3D–F) is in agreement with data collected using conventional techniques (Fig. 3A–C). Treatment of HTR8/SVneo cells with CCL2 for 24 h caused no change in cell number compared to control (Fig. 3A), but stimulated both migration (Fig. 3B) and invasive potential (Fig. 3C) over 48 h. Invasion was not measured directly but invasive potential was assessed by quantifying MMP9 activity with gelatin zymography. The equivalent data produced with the xCELLigence system demonstrates no change in cell number in response to CCL2 (Fig. 3D) and a significant increase in both the rates of migration and invasion determined by calculating the slope of the CI curves (Fig. 3E and F respectively). The limitations of the conventional data are evident, most notably the ability to measure only single time points compared to the continuous measurement facilitated by the xCELLigence System which thus enables rates of migration and invasion to be determined. Furthermore, there are significant time-savings from the collection and analysis of data in real-time which eliminates the need for further assays post-experiment to determine the outcomes.

This technology enables accurate real-time monitoring of cell behaviour without any need to manipulate the cells by removing them from the plate or addition of any labels which may alter cell function. Cells can be fluorescently labelled during the experiment and images captured following completion or they can be stained post experiment. It is also possible to harvest the cells from the plates for subsequent analysis by conventional assays. The technology can be used for a range of other applications including testing cell and compound cytotoxicity [10], receptor-mediated signalling and cytopathogenicity. This is an easy to use system which has the advantage of producing quantitative data in real time. An added advantage is the ability to alter the protocol once the system is running thus enabling users to adjust their output based on the data that they are collecting.

The xCELLigence System is a powerful tool for investigating cellular function. The real-time analysis that it facilitates generates insight into the active nature of cell responses. Application of this technology will undoubtedly provide new understanding of the dynamics of trophoblast behaviour.

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Declaration 

The author and author's institution did not receive any incentive or inducement, financial or otherwise, from Roche Diagnostics GmbH for the use of the xCELLigence System or the preparation of this manuscript. The data in the manuscript was generated by the author in the author's laboratory and was not edited by Roche Diagnostics GmbH.

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Acknowledgements 

The author would like to thank Ms May Wong, Ms Emma Carr and Mr Bruce Mason-Jones for their assistance with this work and critical reading of this manuscript. This work was supported by funding from the Royal Women's Hospital, Parkville, Australia.

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References 

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PII: S0143-4004(10)00087-1

doi:10.1016/j.placenta.2010.02.008

Placenta
Volume 31, Issue 4 , Pages 347-350, April 2010

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