Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis

Résumé

The present protocol allows the recovery of tube-like structures formed in the classical in vitro angiogenesis-tube formation assay where cells, such as trophoblast cells, organize in capillary-like structures over a layer of extracellular basement membrane matrix. The recovered structures are used for RNA and protein isolation for various biochemical analyses.

Résumé

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of mesenchymal to endothelial-like transition. Traditionally, this process is evaluated by an in vitro tube-formation assay, where the cells organize themselves into tube-like structures when seeded over a polymerized basement membrane preparation. Although several structural features can be measured in photomicrographs of the structures, to assess the real commitment of EVT to the endothelial-type phenotype, biochemical analysis of cell extracts is required. Scraping the cells from the culture dish to obtain RNA and/or protein extracts is not an alternative since the tube-like structures are severely contaminated by the bulk of proteins from the polymerized basement membrane. Thus, a strategy to separate the cells from the basement membrane proteins prior to the preparation of cell extracts is needed. Here, a simple, fast, and cost-effective method to recover the tube-like structures from the in vitro tube-formation assay and the subsequent analysis by biochemical techniques is presented. Tube-like structures formed by HTR8/SVneo cells, an EVT cell line, were liberated from the polymerized basement membrane by a short incubation with PBS supplemented with ethylenediaminetetraacetic acid (EDTA). After serial washes, a ready-to-use pellet of purified tube-like structures can be obtained. This pellet can be subsequently processed to obtain RNA and protein extracts. qPCR analysis evidenced the induced expression of VE-cadherin and alpha_v-integrin, two endothelial cell markers, in EVT-derived tube-like structures compared to control cells, which was consistent with the induction of the endothelial cell marker, CD31, evaluated by immunofluorescence. Western blot analysis of the tube-like structures' protein extracts revealed the overexpression of RECK in transfected HTR8/SVneo cells. Thus, this simple method allows to obtain cell extracts from the in vitro tube-formation assay for the subsequent analysis of RNAs and protein expression.

Introduction

The success of a pregnancy depends on the proper development of the placenta and the establishment of placental circulation. One of the key events associated with this process is the remodeling of the uterine spiral arteries (uSA)¹, from a high-resistance and low-flow to low-resistance and high-flow blood vessel, increasing the perfusion of maternal blood into the placenta².

The remodeling of uSA is achieved by specialized trophoblast cells derived from the blastocyst. After the implantation of the embryo, fetal extravillous trophoblast (EVTs) migrate from the implantation site through the mat....

Protocole

1. Preparation for the assay

NOTE: All steps must be conducted under sterile conditions. All the plastic materials must be sterile and brand new. Glass bottles must be sterilized by autoclaving. All the materials must be sterilized with 70% ethanol solution before their use in the laminar flow cabinet. Minimum cell culture medium (RPMI) must be sterilized by filtration with a 0.22 µM filter before supplementation with fetal bovine serum (FBS). Always wear personal protective equipment when working with biohazardous waste (lab coat, gloves, long hair tied back, etc.).

1. Prepare RPMI-1640 media (5% FBS complete....

Résultats

To evaluate the protocol described in this report, first we generated the tube-like structures. As shown in Figure 1A, HTR8/SVneo cells (EVT) generated tube-like structures at 12 h, 24 h and 48 h of incubation over the basement membrane matrix. No tube-like structures were observed in the control dishes where HTR8/SVneo were seeded on plastic at the assayed time interval. After photomicrograph acquisition, several parameters of the tube structures were observed and quantified by ImageJ softw.......

Discussion

Tube-like formation assay is a widely used tool to evaluate the interaction between cells in angiogenic processes¹⁹. Here we depict the angiogenic properties of non-Endothelial cells, such as the EVTs, in the remodeling of vascular uterine vessels, a critical event during placentation⁶. Although tube-like formation is very informative about the interaction between cells, deeper analysis at the molecular, epigenetic and protein level during the angiogenic events is needed. T.......

matériels

Name	Company	Catalog Number	Comments
Cell Culture Dishes laboratory (100mm)	Nest	704201
Cell Culure 6-Plate	Nest	0917A
Cell Culure Microscope	Olympus	CKX53SF
Centrifuge	Thermo Scientific	Mega Fuge 8R
Circular Analog Magnetic Hotplate Stirrer	SCI Logex	MS-H-S
CO2 Incubator	Thermo Scientific	HERA cell Vios 160i
Cultrex PathClear Basement Membrane Extract (2 x 5 mL)	R&D SYSTEMS	RD.3432-010-01
Dry Heat Incubators	HES	MK 200-1
Ethylenediaminetetraacetic Acid (EDTA)	Winkler	BM-0680
Fetal Bovine Serum Heat Inactivated	Capricorn Scientific	FBS-HI-12A
Freezer Vertical	DEAWOO	FF-211VSM
HTR8/SVneo	ATCC	CRL-3271
Laboratory bench rocker	TCL	S2025
Laminar flow cabinet	BIOBASE	BSC-1300
Luminescent Image Analyzer	Health Care	Image Quant LAS 500
Matrigel Matrix Growth Factor Reduced, Phenol Red-Free 10 ml	R&D SYSTEMS	356231
Micro Centrifuge	SCI Logex	D1008
Micropipete Lambda Plus, P10	Corning	4071
Micropipete Lambda Plus, P1000	Corning	4075
Micropipete Lambda Plus, P2	Corning	4070
Micropipete Lambda Plus, P20	Corning	4072
Micropipete Lambda Plus, P200	Corning	4074
Microscope Camera-Set	MOTIC	Moticam 2300
Pen-strep-amphot b/Antim	Biological Industries	03-029-1B
pHmeter	HANNA	HI2221
Phophate Buffer Saline 10X (PBS10X)	Corning	46-013-CM
Pippete micro tip with filter, sterile, P10	Jetbiofil	PMT231010
Pippete micro tip with filter, sterile, P1000	Jetbiofil	PMT252000
Pippete micro tip with filter, sterile, P200	Jetbiofil	PMT231200
PowerPac	BIO-RAD	E0203
Radwag	TCL	WTB 200
Remote water Purification System	Millipore	Direct-Q 5 UV
RPMI 1640 Medium, powder	Gibco	31800-022
Thermal Cycler	Bioer Technology	TC-96/G/H(b)C
Thermoregulated bath	Thermo Scientific	TSGP10
Trypsin EDTA 10X	Capricorn Scientific	TRY-1B10
Ultrasonic Processors	SONICS	VCX130PB
Vacuum Pump	HES	ROCKER 300
Vortex Mixer	Thermo Scientific	LP Vortex Mixer