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Method Article
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.
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 alphav-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.
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 20th 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 structures12,13,14, they are limited to the characterization of the organization of the cells, since it is not possible to collect these structures for biochemical analysis12,13,14, 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.
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.).
2. Tube formation assay
3. Cell recovery from basement membrane matrix
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...
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...
The authors declare that they have no conflicts of interest.
Funding source FONDECYT 1221362, ANID, for JG. Convocatoria Nacional Subvención a la Instalación en la Academia, ANID, No. SA77210087 for ICW.
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 |
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