In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Summary

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.

Abstract

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.

Introduction

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. Migr....

Protocol

1. Cell Preparation

1. Maintain cells in T-75 flasks in standard growth media at 37 °C, 5% CO₂, and 95% humidity.
   NOTE: The cells used in these experiments were the immortalized first-trimester trophoblast cell lines Swan.71 (Sw.71) and HTR-8/SVneo^9,10. The Sw.71 cells were maintained in DMEM/F-12 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1.0 mM sodium pyruvate, 10 mM HEPES, 0.10 mM MEM non-essential amino acids, 100 units/mL penicillin, and 100 µg/mL streptomycin. The HTR-8/SVneo cells were maintained in RPMI-1640 supplemented with 5....

Results

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 de.......

Discussion

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,.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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