Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.
Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.
Placental development is a crucial step in the establishment of pregnancy, impacting both maternal and fetal health. However, the mechanistic basis by which this process occurs is not fully understood. Cell migration is an important biological process that contributes to the establishment and functions of the placenta during pregnancy. Following blastocyst implantation, the trophectoderm differentiates into villous trophoblasts, which cover the surface of the chorionic villi and are involved in gas and nutrient exchange, and extravillous trophoblasts (EVTs), which migrate out from the villi and invade the maternal decidua and vasculature1. Migration of the EVTs is essential for remodeling maternal spiral arteries and establishing uteroplacental circulation to support fetal growth2. Inadequate trophoblast invasion during pregnancy results in abnormal placental development and may contribute to pregnancy complications such as preeclampsia, gestational hypertension, intra-uterine growth restriction, and preterm birth3,4. Therefore, understanding the factors that affect trophoblast motility is essential to determining the pathways required for normal placentation.
Trophoblast migration is controlled by a complex network of signaling molecules, including growth factors, cytokines, hormones, and angiogenic factors5. Due to the limitations of studying the placenta in vivo, in vitro assays utilizing immortalized human trophoblast cell lines have been crucial to identifying factors that contribute to trophoblast motility. Scratch and invasion assays have been used extensively to quantitatively assess the role of individual molecules on trophoblast cell migration6,7,8. However, while useful in tandem to investigate how changes in migration may be caused by changes in cell invasiveness, these two assays do not account for additional mechanisms that may contribute to altered rates of cell migration. For example, reductions to the rate of cell proliferation may result in fewer cells available to migrate.
Here, we describe quantitative in vitro methods for assessing trophoblast migration using scratch, cell proliferation, and cell invasion assays. In the scratch assay, a uniform wound is created in a cell monolayer, and the migration of cells to fill the gap is measured by automated, time-lapse imaging of the wound size and density of cells within the wound. The cell proliferation assay is based on calculating the ratio of cells at each time point compared to a known starting quantity of cells. In the cell invasion assay, cells are seeded atop an extracellular matrix-coated cell culture insert chamber (e.g. Transwell), and the number of cells that invade through the extracellular matrix in response to chemo-attractant are counted.
The scratch assay is a simple and effective tool that may be used to determine how different environmental conditions affect cell migration. The proliferation and invasion assays may subsequently be used to determine the contribution of cell proliferation and invasiveness to overall changes in cell migration. Collectively, these assays provide robust measurements of biological processes that may contribute to cell motility. Two immortalized first-trimester trophoblast cell lines were used in the described assays, Swan.71 (Sw.71) and HTR-8/SVneo9,10. However, these assays may also be optimized for use in other cell types to identify key modulators of cell migration and invasion.
1. Cell Preparation
2. Scratch Assay
3. Invasion Assay
4. Proliferation Assay
The immortalized human first-trimester trophoblast cell lines Sw.71 and HTR-8/SVneo were used in these experiments to determine the role of glucocorticoids in trophoblast cell migration12. Glucocorticoids were used in these experiments as they have recently been shown to alter trophoblast functions, although alternative treatments could be used12. Both cell lines were treated with vehicle (1x PBS) or 100 nM of the synthetic glucocorticoid dexamethasone (Dex) for all experiments. Wound density and size were measured every 2 h for 72 h using an automated, time-lapse imaging system. Figure 1 shows an example of images and results obtained from the scratch assay at different timepoints. Sample images from 0, 8, and 18 h timepoints for both treatments have been provided (Figure 1A). The wound density and wound size are graphed over time for samples treated with vehicle control (Veh) or 100 nM Dex (Figure 1B). Dex treatment reduced the rate of wound closure as determined by both wound density and wound size, indicating that glucocorticoids inhibit cell migration in first-trimester trophoblast cells.
Figure 2 shows an example of images taken of the cell culture insert following the invasion assay. Cells were treated for 24 h with Veh or 100 nM Dex. Invaded cells were counted from four independent replicates per treatment using four unique fields of view per replicate. Glucocorticoid treatment reduced cell invasiveness, as shown by a 15% reduction in the number of invaded cells.
Figure 3 shows an example of the cell proliferation assay. Cells were counted at 24, 48, and 72 h following treatment with Veh or 100 nM Dex. Glucocorticoid treatment reduced cell proliferation, as indicated by fewer cells at each time point. Overall, these assays demonstrate that glucocorticoid exposure reduces cell motility by inhibiting both cell proliferation and invasion.
Figure 1: Cell Scratch Assay. (A) Representative images of wounds for Veh- and Dex-treated Sw.71 cells at 0, 8, and 18 h are shown. Red lines indicate wound size. (B) Wound density and wound size were measured in Sw.71 and HTR-8/SVneo cells treated with vehicle (Veh) or 100 nM Dex. Pictures were taken every 2 h for 72 h using an automated, time-lapse microscope. Data represents the mean of four independent replicates ± SEM. Please click here to view a larger version of this figure.
Figure 2: Cell Invasion Assay. Invasion assays were conducted with Sw.71 and HTR-8/SVneo cells treated for 24 h with vehicle (Veh) or 100 nM Dex. (A) Representative images from the invasion assay for Veh- and Dex-treated cells after 24 h incubation. Inset image is negative control with no cells added to the upper chamber. Images taken at 200x magnification. Scale bars are 120 µm. (B) Invaded cells were counted and the bar graphs represent the normalized mean of four independent replicates ± SEM. Please click here to view a larger version of this figure.
Figure 3: Cell Proliferation Assay. Sw.71 and HTR-8/SVneo cells treated with vehicle (Veh) or 100 nM Dex were counted every 24 h using an automated cell counter. Cell counts are graphed as the mean ± SEM of at least 11 independent replicates. Please click here to view a larger version of this figure.
This procedure builds on the use of migration and invasion assays to include the rate of cell proliferation as a potential contributor to measured differences in trophoblast cell movement. Used together, these in vitro assays are simple and effective methods that can be used to identify the cellular pathways that regulate trophoblast migration. As described above, trophoblast cells may be treated with hormones, cytokines, growth factors, or other molecules to determine their impact on trophoblast movement. Alternatively, target proteins may be depleted from cells to elucidate their functional role in trophoblast motility. Identifying key modulators of trophoblast migration may provide a better understanding of the basic mechanisms underlying complications of pregnancy related to placental defects, including preeclampsia, intrauterine growth restriction, and preterm birth.
There are several critical steps in this protocol that are required to obtain accurate results. Because phenol-red has estrogenic activity at concentrations used in cell culture media13, it is important that phenol-red free media is used for experimental endpoints to prevent undesired activation of the estrogen receptor in cells. During the transwell invasion assay, it is important to ensure that the extracellular matrix is homogenous, level, and that cold supplies are used to prevent premature polymerization of the matrix. When conducting the cell proliferation and invasion assays, it is important to count samples immediately to prevent re-adherence of the cells to tissue culture plates. It should also be taken under consideration that cell death due to experimental treatment may influence the results of all three assays. Therefore, markers of cell death (e.g., lactate dehydrogenase release, phosphatidylserine externalization, or DNA degradation) in response to treatment should be evaluated in the experimental cell line prior to evaluating migration, invasion, and proliferation14. Some limitations exist for these assays, for example, the process of creating a wound in a cell monolayer induces a cell injury response than may confound the cell migration process. Furthermore, manual scraping may introduce interexperiment variability, although utilizing a wound-maker tool that creates uniform wound widths can partially mitigate this limitation. The disadvantage of the invasion assay is that it is an endpoint assay, and the kinetics of movement are not easily attained. Despite these limitations, the assays described here provide reproducible, quantitative data at relatively low-cost.
The assays described here can also be modified to measure cell migration in other cell types, although these assays would not be suitable for non-adherent cell types. In the scratch assay, the imaging intervals for time-lapse microscopy may be adjusted to sufficiently capture differences in the rate of cell migration. Automated systems for live cell imaging can capture wound size images as frequently as every 15 min. Images taken at least every 30 min can be stitched together to create a movie of wound healing. In the invasion assay, the concentration of extracellular matrix, the total number of cells seeded, and the length of incubation prior to fixing and counting cells may be adjusted to account for the specific rate of invasion in different cell types. In the cell proliferation assay, the number of cells seeded onto the plates and the time points for cell counting may be adjusted to account for different rates of cell growth. Overall, these assays are flexible and highly accessible tools that can be used in a wide range of cell types to identify the mechanisms underlying cell migration.
The authors have nothing to disclose.
The authors thank Dr. Gil Mor for providing the Sw.71 cells. This research was supported by an Albert McKern Scholar Award to S.W.
0.4% Trypan blue stain | Thermo Fisher Scientific | 15250-061 | |
0.5% Trypsin-EDTA | Life Technologies | 15400-054 | |
1.0 M HEPES | AmericanBio | AB06021-00100 | |
10 mM MEM non-essential amino acids | Life Technologies | 11140-050 | |
100 mM sodium pyruvate | Life Technologies | 11360-070 | |
24-well plate transwell inserts | Corning | 3422 | Contain polycarbonate filters with an 8 μm pore size |
Charcoal dextran-stripped FBS | Gemini Bio-Products | 100-119 | |
Countess II Automated Cell Counter | Thermo Fisher Scientific | AMQAX1000 | |
Crystal Violet | Sigma Aldrich | C0775 | |
Cytoseal 60 | Thermo Fisher Scientific | 8310-4 | |
Dexamethasone (Dex; 1, 4-pregnadien-9α-fluoro-16α-methyl-11β, 17, 21-triol-3, 20-dione; ≥98% TLC) | Steraloids | P0500-000 | |
DMEM/Ham’s F12 | Life Technologies | 11330-032 | |
Fetal Bovine Serum | Sigma Aldrich | F2442 | |
IncuCyte 24-well ImageLock Plates | Essen BioScience | 4365 | If using the IncuCyte Zoom imaging system, seed cells onto IncuCyte ImageLock Plates. These plates have fiducial markers on the bottom of the plate that are used as a reference for repeat imaging in a constant field of view. |
IncuCyte software | Essen BioScience | 2010A Rev2 | |
IncuCyte ZOOM system | Essen BioScience | N/A | Scratch assay images were captured and analyzed using the preset “Scratch Wound” parameters on the IncuCyte ZOOM system. Other time-lapse microscopes may also be used. |
Matrigel Membrane Matrix | Becton Dickinson | 356231 | Growth factor reduced, phenol-red free |
Micro Slides | VWR International, LLC | 48311-7003 | |
Microscope cover glass | Fisher Scientific | 12-545-E | |
Olympus IX71 inverted microscope | Olympus | IX71 | |
Penicillin (10,000 units/mL)/streptomycin (10,000 µg/mL) | Life Technologies | 151-40-122 | |
Phenol-red free DMEM/Ham's F12 | Life Technologies | 11039-021 | |
Semi-automatic wound maker tool | Essen BioScience | N/A |