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

Summary

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.

Abstract

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 maternal decidua and uterus toward the uSA³. Once there, EVTs induce the migration and apoptosis of the maternal vascular-associated smooth muscle cells (VSMC) and endothelial cells (EC)⁴. Subsequently, EVTs acquire an endothelial-like phenotype through a mesenchymal to endothelial-like transition (MELT)^5,6, replacing the VSMC and EC of the vessel⁷.

Preeclampsia (PE) is a pregnancy-specific syndrome characterized by the onset of maternal hypertension, accompanied by proteinuria and/or other symptoms of maternal end-organ dysfunction, determined since the 20^th week of gestation⁸. Although the precise etiology of PE is not fully understood, the most accepted hypothesis relates to a defective uSA remodeling⁹, generating a permanent state of insufficient blood flow to the placenta, accompanied by placental damage by ischemia-reperfusion, hypoxia, and oxidative stress, activating the release of placental factors to the maternal circulation, triggering the characteristic maternal symptoms of PE⁹. Thus, it is critical to determine the key cellular and molecular mechanisms involved in the uSA remodelling by trophoblast cells.

The evaluation of the MELT of EVTs is classically achieved by the in vitro basement membrane matrix tube-formation assay¹⁰, by which individual cells migrate and organize themselves in two-dimensional structures that resembles blood vessel. This protocol requires the seeding of EVTs cell lines on a thin layer of polymerized extracellular matrix, usually, referred to as basement membrane matrix, which is a commercially available extracellular matrix composed of a solubilized basement membrane extracted from the Engelbreth-Holm-Swarm mouse sarcoma, enriched by Laminin, as well as Collagen IV, heparan sulfate proteoglycans and entactin/nidogen¹⁰. The analysis of the formed tube-like structures is usually achieved by the identification of established characteristic in photomicrographs (e.g., number of branches or mesh index) and the quantification of these by the ImageJ software (Angiogenesis analyzer plugin)¹¹. Although this method is widely used to describe the formation of the tube-like structures^12,13,14, they are limited to the characterization of the organization of the cells, since it is not possible to collect these structures for biochemical analysis^12,13,14, largely due to the impossibility to separate the cells/tubes from the basement membrane matrix, which contains high amount of different proteins. Thus, it is not possible to determine the real contribution of cell-derived proteins, which is necessary to correctly understand the expression of cellular proteins. Since most of the proteins in these extracts would come from the basement membrane matrix, the representation of the cellular proteins turns out to be very low, so a large amount of proteins must be loaded into an SDS-page gel for analysis by Western blot, affecting the resolution and transfer of the proteins. Making proper analysis almost impossible. This excess of basement membrane matrix derived proteins also profoundly affects the extraction of RNA with the required purity for further analysis.

In this report we describe a simple, fast, and cost-effective method, based on PBS-EDTA treatment, which allows the release of tube-like structures from the basement membrane matrix which could be processed for biochemical analysis of cell extracts. This method yields high amounts of biomolecules, RNA, and proteins, and it is compatible with classical biochemical analysis, qPCR, and Western blot. We propose that this strategy could be applicable to other assays, such as sub-cellular fractionation, determination of epigenetic markers by pyrosequencing or chromatin immune precipitation assays. In brief, the protocol consists of incubation of the tube-like structures in the culture dish with PBS supplemented with 50 mM of EDTA at 4 °C to dissolve the basement membrane matrix. By this simple incubation, the tubular structures remain in suspension, which after a series of washes with PBS to eliminate the EDTA and the excess of diluted basement membrane matrix, can be obtained as a ready-to-use pellet of tubular structures and/or individual cells. The subsequent sections describe the protocols for the MELT assay and the recovery of tube- like structures.

Protocol

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 growth medium) by adding sterilized RPMI 1640 and FBS in a sterile glass bottle in the laminar flow cabinet. For 5% FBS solution, add 190 mL of RPMI-1640 and 10 mL of FBS. Warm the RPMI-1640 Media-5% FBS in a temperature-controlled water bath at 37 °C.
2. Prepare the basement membrane matrix as follows. Slowly thaw the basement membrane matrix by placing the bottle at 4 °C for 3 to 4 h and cool the MW-6 dish at -20 °C, for 1 h prior to use. Once thawed, maintain the basement membrane matrix on ice. In the laminar flow cabinet dilute the basement membrane matrix to 70% with ice cold- RPMI1640 without FBS.
   NOTE: Basement membrane matrix should be stored at -80 °C until use. After thawing everything must be worked at 4 °C to avoid premature polymerization of the matrix.
3. Prepare PBS-EDTA by dissolving the appropriate amount of EDTA in PBS to reach a final concentration of 50 mM of EDTA. Maintain the PBS-EDTA at 4 °C.

2. Tube formation assay

1. Add 60 μL/cm² of the diluted basement membrane matrix as indicated in step 1.2. to the dishes. Avoid bubbles, which interfere with the microphotograph needed for quantification of the tubular structures.
   NOTE: In the case of a multiwell-6 (MW6, with an area of 9.6 cm²) the volume is 580 μL of basement membrane matrix -70%.
2. Transfer the dishes with basement membrane matrix to a cell incubator (5% CO₂ and 37 °C) for 2 h to promote basement membrane matrix polymerization.
   NOTE: During the last 1 h of polymerization, prepare the cell suspension of the cells that will be used for the assay, with the aim of completing the 2 h of polymerization when the cells are ready to be seeded.
3. Seed 80,000 cells/cm² of HTR8/SVneo (EVTs cell line) diluted in 2 mL of RPMI-1640- 5% FBS over the polymerized basement membrane matrix and also over the uncoated dishes as control. Carefully seed the cells along the wall of the well to avoid destroying the basement membrane matrix.
   NOTE: Procure a homogeneous cell distribution over the Matrigel, otherwise tubes will not form in areas where there are fewer cells or where the cells are overcrowded.
4. Transfer the seeded cells to the cell incubator and culture for 12 h, 24 h, and 48 h at 5% CO₂ and 37 °C.
   NOTE: Usually, 24 h is sufficient to analyze the tubular structure, however if kinetic protein or RNA expression during this process is needed, incubate the cells for: 3 h, 6 h, 12 h, 24 h and 48 h.

3. Cell recovery from basement membrane matrix

1. Once the selected incubation time has reached (for example, 12 h, 24 h, 48 h), recover the conditioned medium to perform the measurement of choice on it.
2. Gently wash the cells with ice-cold 1x PBS, at least 3x to eliminate cell debris. If microphotographs are needed, pour 1 mL of cold PBS in each well and take pictures within 10 min.
3. Remove PBS and add 700 μL of PBS-EDTA to each well and incubate the plate on ice for 3-5 min.
   NOTE: PBS-EDTA will dissolve the basement membrane matrix, so the entire cell network will remain in suspension.
4. Gently recover the suspension of the tubular network with a micropipette (p1.000) in a 1.5 mL tube.
5. Centrifuge the suspension at 1,000 x g for 10 min at 4 °C. At the end, a white pellet of cells should be visible.
6. Eliminate the supernatant by inversion of the tube, trying to leave it as dry as possible.
7. Add 1 mL of PBS at 4 °C and gently resuspend the cell pellet with a p.1000 micropipette to wash the cells and remove the dissolved basement membrane matrix and EDTA. Repeat at least 2x.
   NOTE: These washing steps are critical. The remaining basement membrane matrix could be observed as a viscous layer over the cell pellet. Repeat the washes until most of the basement membrane matrix layer is eliminated.
8. After the final wash, use the cell pellet directly for protein or RNA extraction. Alternatively, resuspend the pellet in 1 mL of the PBS and distribute into different tubes, depending on the requirements: for example, 300 µL for RNA extraction and the remaining 700 µL for protein extraction, etc.
9. Centrifuge the tubes at 1,000 x g for 10 min at 4 °C to obtain the cell pellet.
   NOTE: The division of the cell suspension in PBS can be in the ratio desired, for example, 1:1, 1:2 or 1:3 to RNA:protein. Here a ratio of 1:2 RNA:proteins is used. At this point, store the cell pellets if not immediately needed, as dry as possible, at -80 °C up to a month.

Results

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...

Materials

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