Name: Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis (Video) | JoVE Journal
Uploaded: 2024-11-08T12:20:00.000+00:00
Duration: 5 min 30 s
Description: Learn trophoblast cell recovery from angiogenesis-tube formation assays for differentiation marker analysis using HTR-8/SVneo cells. This method enables efficient RNA and protein isolation for studying placental development and preeclampsia mechanisms.

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5 minutes

Funding source FONDECYT 1221362, ANID, for JG. Convocatoria Nacional Subvenci&#243;n a la Instalaci&#243;n en la Academia, ANID, No. SA77210087 for ICW.

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 &#181;M 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.).
<ol>
	<li>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 &#176;C.</li>
	<li>Prepare the basement membrane matrix as follows. Slowly thaw the basement membrane matrix by placing the bottle at 4 &#176;C for 3 to 4 h and cool the MW-6 dish at -20 &#176;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 &#176;C until use. After thawing everything must be worked at 4 &#176;C to avoid premature polymerization of the matrix.</li>
	<li>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 &#176;C.</li>
</ol>
2. Tube formation assay
<ol>
	<li>Add 60 &#956;L/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 &#956;L of basement membrane matrix -70%.</li>
	<li>Transfer the dishes with basement membrane matrix to a cell incubator (5% CO2 and 37 &#176;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.</li>
	<li>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.</li>
	<li>Transfer the seeded cells to the cell incubator and culture for 12 h, 24 h, and 48 h at 5% CO2 and 37 &#176;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.</li>
</ol>
3. Cell recovery from basement membrane matrix
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Remove PBS and add 700 &#956;L 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.</li>
	<li>Gently recover the suspension of the tubular network with a micropipette (p1.000) in a 1.5 mL tube.</li>
	<li>Centrifuge the suspension at 1,000 x g for 10 min at 4 &#176;C. At the end, a white pellet of cells should be visible.</li>
	<li>Eliminate the supernatant by inversion of the tube, trying to leave it as dry as possible.</li>
	<li>Add 1 mL of PBS at 4 &#176;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.</li>
	<li>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 &#181;L for RNA extraction and the remaining 700 &#181;L for protein extraction, etc.</li>
	<li>Centrifuge the tubes at 1,000 x g for 10 min at 4 &#176;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 &#176;C up to a month.</li>
</ol>

Analyzing Angiogenesis: In Vitro Assay for Tube Formation on BM Matrix

<ol><li>Cartwright, J. E., Fraser, R., Leslie, K., Wallace, A. E., James, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Remodelling+at+the+maternal-fetal+interface%3A+relevance+to+human+pregnancy+disorders%5BTitle%5D)+AND+%22Reproduction%22%5BJournal%5D)">Remodelling at the maternal-fetal interface: relevance to human pregnancy disorders.</a> Reproduction. 140 (6), 803-813 (2010).</li>
<li>Burton, G. J., Woods, A. W., Jauniaux, E., Kingdom, J. C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rheological+and+Physiological+Consequences+of+Conversion+of+the+Maternal+Spiral+Arteries+for+Uteroplacental+Blood+Flow+during+Human+Pregnancy%5BTitle%5D)+AND+%22Placenta%22%5BJournal%5D)">Rheological and Physiological Consequences of Conversion of the Maternal Spiral Arteries for Uteroplacental Blood Flow during Human Pregnancy.</a> Placenta. 30 (6), 473-482 (2009).</li>
<li>Huppertz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+anatomy+of+the+normal+placenta%5BTitle%5D)+AND+%22J+Clin+Pathol%22%5BJournal%5D)">The anatomy of the normal placenta.</a> J Clin Pathol. 61 (12), 1296-1302 (2008).</li>
<li>Bulmer, J. N., Innes, B. A., Levey, J., Robson, S. C., Lash, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+vascular+smooth+muscle+cell+apoptosis+and+migration+during+uterine+spiral+artery+remodeling+in+normal+human+pregnancy%5BTitle%5D)+AND+%22FASEB+J%22%5BJournal%5D)">The role of vascular smooth muscle cell apoptosis and migration during uterine spiral artery remodeling in normal human pregnancy.</a> FASEB J. 26 (7), 2975-2985 (2012).</li>
<li>Paul, M., Chakraborty, S., Islam, S., Ain, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trans-differentiation+of+trophoblast+stem+cells%3A+implications+in+placental+biology%5BTitle%5D)+AND+%22Life+Sci+Alliance%22%5BJournal%5D)">Trans-differentiation of trophoblast stem cells: implications in placental biology.</a> Life Sci Alliance. 6 (3), 202201583(2023).</li>
<li>Pollheimer, J., Vondra, S., Baltayeva, J., Beristain, A. G., Knöfler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+placental+extravillous+trophoblasts+by+the+maternal+uterine+environment%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">Regulation of placental extravillous trophoblasts by the maternal uterine environment.</a> Front Immunol. 9, 1-18 (2018).</li>
<li>Sato, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endovascular+trophoblast+and+spiral+artery+remodeling%5BTitle%5D)+AND+%22Mol+Cell+Endocrinol%22%5BJournal%5D)">Endovascular trophoblast and spiral artery remodeling.</a> Mol Cell Endocrinol. 503, 110699(2020).</li>
<li>Huppertz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+critical+role+of+abnormal+trophoblast+development+in+the+etiology+of+preeclampsia%5BTitle%5D)+AND+%22Curr+Pharm+Biotechnol%22%5BJournal%5D)">The critical role of abnormal trophoblast development in the etiology of preeclampsia.</a> Curr Pharm Biotechnol. 19 (10), 771-780 (2018).</li>
<li>Khong, T. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sampling+and+definitions+of+placental+lesions+Amsterdam+placental+workshop+group+consensus+statement%5BTitle%5D)+AND+%22Arch+Pathol+Lab+Med%22%5BJournal%5D)">Sampling and definitions of placental lesions Amsterdam placental workshop group consensus statement.</a> Arch Pathol Lab Med. 140 (7), 698-713 (2016).</li>
<li>Kleinman, H. K., Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Matrigel%3A+Basement+membrane+matrix+with+biological+activity%5BTitle%5D)+AND+%22Semin+Can+Biol%22%5BJournal%5D)">Matrigel: Basement membrane matrix with biological activity.</a> Semin Can Biol. 15, 378-386 (2005).</li>
<li>Carpentier, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Angiogenesis+analyzer+for+ImageJ+-+A+comparative+morphometric+analysis+of+%26%2334%3BEndothelial+Tube+Formation+Assay%26%2334%3B+and+%26%2334%3BFibrin+Bead+Assay.%26%2334%3B%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Angiogenesis analyzer for ImageJ - A comparative morphometric analysis of &#34;Endothelial Tube Formation Assay&#34; and &#34;Fibrin Bead Assay.&#34;.</a> Sci Rep. 10 (1), 1-13 (2020).</li>
<li>Arderiu, G., Peña, E., Aledo, R., Badimon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+factor-Akt+signaling+triggers+microvessel+formation%5BTitle%5D)+AND+%22J+Thrombosis+Haemostasis%22%5BJournal%5D)">Tissue factor-Akt signaling triggers microvessel formation.</a> J Thrombosis Haemostasis. 10 (9), 1895-1905 (2012).</li>
<li>Leung, K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ginsenoside+Rb1+inhibits+tube-like+structure+formation+of+endothelial+cells+by+regulating+pigment+epithelium-derived+factor+through+the+oestrogen+%CE%B2+receptor%5BTitle%5D)+AND+%22British+J+Pharmacol%22%5BJournal%5D)">Ginsenoside Rb1 inhibits tube-like structure formation of endothelial cells by regulating pigment epithelium-derived factor through the oestrogen β receptor.</a> British J Pharmacol. 152 (2), 207-215 (2007).</li>
<li>Xue, L., Greisler, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Angiogenic+effect+of+fibroblast+growth+factor-1+and+vascular+endothelial+growth+factor+and+their+synergism+in+a+novel+in+vitro+quantitative+fibrin-based+3-dimensional+angiogenesis+system%5BTitle%5D)+AND+%22Surgery%22%5BJournal%5D)">Angiogenic effect of fibroblast growth factor-1 and vascular endothelial growth factor and their synergism in a novel in vitro quantitative fibrin-based 3-dimensional angiogenesis system.</a> Surgery. 132 (2), 259-267 (2002).</li>
<li>Ji, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Placental+trophoblast+cell+differentiation%3A+Physiological+regulation+and+pathological+relevance+to+preeclampsia%5BTitle%5D)+AND+%22Mol+Asp+Med%22%5BJournal%5D)">Placental trophoblast cell differentiation: Physiological regulation and pathological relevance to preeclampsia.</a> Mol Asp Med. 34 (5), 981-1023 (2013).</li>
<li>Liu, C. Y., Lin, H. H., Tang, M. J., Wang, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vimentin+contributes+to+epithelial-mesenchymal+transition+ancer+cell+mechanics+by+mediating+cytoskeletal+organization+and+focal+adhesion+maturation%5BTitle%5D)+AND+%22Oncotarget%22%5BJournal%5D)">Vimentin contributes to epithelial-mesenchymal transition ancer cell mechanics by mediating cytoskeletal organization and focal adhesion maturation.</a> Oncotarget. 6 (18), 15966-15983 (2015).</li>
<li>Kolesnichenko, O. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelial+progenitor+cells+derived+from+embryonic+stem+cells+prevent+alveolar+simplification+in+a+murine+model+of+bronchopulmonary+dysplasia%5BTitle%5D)+AND+%22Front+Cell+Dev+Biol%22%5BJournal%5D)">Endothelial progenitor cells derived from embryonic stem cells prevent alveolar simplification in a murine model of bronchopulmonary dysplasia.</a> Front Cell Dev Biol. 11, 1209518(2023).</li>
<li>Gutiérrez, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preeclampsia+associates+with+RECK-dependent+decrease+in+human+trophoblasts+migration+and+invasion%5BTitle%5D)+AND+%22Placenta%22%5BJournal%5D)">Preeclampsia associates with RECK-dependent decrease in human trophoblasts migration and invasion.</a> Placenta. 59, 19-29 (2017).</li>
<li>Ucuzian, A. A., Greisler, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+models+of+angiogenesis%5BTitle%5D)+AND+%22World+J+Surg%22%5BJournal%5D)">In vitro models of angiogenesis.</a> World J Surg. 31 (4), 654-663 (2007).</li>
<li>Rand, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium+depletion+dissociates+and+activates+heterodimeric+Notch+receptors%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">Calcium depletion dissociates and activates heterodimeric Notch receptors.</a> Mol Cell Biol. 20 (5), 1825-1835 (2000).</li>
</ol>

The authors declare that they have no conflicts of interest.

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. These biochemical approaches require the preparation of pure extracts of cell-derived RNA, DNA and proteins, which is challenging when the cells must be separated from the basement membrane matrix, which could be considered as contaminants in those techniques.
The protocol presented here enriches the analysis related to angiogenesis assay, and is simple, quick, and cost-effective. Because the tube-formation assays presented here were made with basement membrane matrix, we consider that the present protocol is not affected by the basement membrane matrix nature (i.e., no reproducibility of the results). This protocol yields high amounts of biomolecules with low levels of basement membrane matrix proteins or inhibitors that could affect further biochemical analysis.
One of the main considerations of this protocol is that the reagent preparation and sample incubation must be done at 4 &#176;C, since it reduces the activation of signaling pathways such as Notch20. Additionally, the incubation with PBS-EDTA must be brief, considering that the tubular network detaches from the basement membrane matrix quickly, so exceeding the incubation time could only cause over exposure to EDTA, a calcium chelator20. 
A limitation of this method is that in the washing phase to remove the PBS-EDTA and the basement membrane matrix, traces of matrix may remain in the sample, which could overestimate the cellular protein quantification when performing a Western blot. This limitation can be solved by assessing a complete resuspension of basement membrane matrix during the washing steps.
The method proposed is simple, fast, and cost-effective, becoming affordable for use in biochemical techniques to measure the molecular and cellular mechanisms involved in the commitment of an endothelial phenotype. Also, this method allows to evaluate the role of specific proteins or pathways in the angiogenic processes using manipulated cells (e.g., overexpression or knockdown of proteins) in the tube-like formation assay followed by further biochemical analysis.

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 &#176;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.

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

trophoblast cell recovery from angiogenesis-tube formation assay for differentiation marker expression analysis

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

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.

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 software (angiogenesis analyzer plugin) as shown in Figure 1B. We found statistical differences in every variable measured except for total master segment length, total mesh area, total segment length and mesh index, at 48 h of tube-like formation compared with 12 h and/or 24 h of tube-like formation.
To evaluate the protocol described here to obtain RNA preparations, compatible with qPCR analysis, we obtained total RNA extracts from cells incubated for 0 h, 5 h, and 24 h. The cell pellets were resuspended in the lysis buffer provided in the total RNA kit. The same kit was used for RNA extraction according to the manufacturer's protocol. The concentration of RNA was determined by spectrophotometric absorbance at 260 nm and 280 nm. We obtained ≈10 µg total RNA, at a concentration of ≈190 ng/µL with a 260/280 absorbance ratio ≈2.0 on average, indicating a high yield of pure RNA. Total RNA weighing 0.5 µg was reverse transcribed with the reverse transcriptase kit. The relative quantity of VE-cadherin and alphav-integrin15 mRNAs were determined by qPCR analysis using pre-designed TaqMan primer and probes, for VE-Cadherin and alphav-integrin; beta-tubulin levels were used as housekeeping control15. PCR reactions were prepared with PCR master mix according to manufacturer's protocol and amplified in a thermocycler. Both VE-cadherin and alphav-integrin relative mRNAs levels were increased at 5 h and 24 h, compared to control (0 h). To corroborate the mesenchymal to endothelial like transition (MELT) of EVT, tube-like structures formed after 24 h were fixed directly on basement membrane matrix with 4% paraformaldehyde in PBS (pH 7.4) and immunoassayed for CD31 and Vimentin, classical markers of endothelial and mesenchymal cells, respectively16,17. The nuclei were counterstained with DAPI. The immunofluorescence shown in Figure 3, revealed that the control cells (seeded on plastic) expressed undetectable levels of CD31 which were strongly stained in the tube-like structures. Vimentin presences were evident in both control and tube-like structures. The latter indicates that HTR8/SVneo can acquire an endothelial-like phenotype without losing its cell linage.
To evaluate the expression of proteins by western blot from extracts obtained from the tubular like structures, HTR8/SVneo previously transfected to overexpress RECK18, were subjected to tube-like formation15. After 24 h the structures were photomicrographed and then liberated from the basement membrane matrix following the protocol described here. Total proteins were isolated and quantified. We found that the method yields high levels of proteins, 245 µg total amount with a concentration of 2.5 µg/µL on average considering all samples. By Western blot, we evaluated the accumulation of RECK and β-Tubulin18 in each condition. Using Image J software, we calculated the ratio of RECK/β-Tubulin pixel intensity (Figure 4A). The results showed that the induction of tube-like formation with cells overexpressing RECK leads to a diminishing of the number of isolated segments and total isolated branches length (Figure 4B). A satisfactory western blot, with high quality proteins (Figure 4C) was made following the protocol here, which evidenced that the overexpression of RECK is maintained during tube-like formation (Figure 4D).
<img alt="Cell growth comparison; EVT/enEVT microscopy images at 12, 24, 48h; bar graph shows data analysis." class="xfigimg" src="/files/ftp_upload/66454/66454fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Temporal kinetics of mesenchymal to endothelial-like transition. Human trophoblast cells line was induced to MELT in basement membrane matrix (enEVTs) or cultured in plastic (EVTs). (A) Structures by 12 h, 24 h and 48 h. (B) Analysis of different parameters of the tubular network by ImageJ software with Angiogenesis Analyzer plugin for each time point. Values are Mean ± S.D. α and β indicate p &lt; 0.05 for 12 h and 24 h timepoints, respectively using one-way ANOVA. n= 3. Abbreviations: Nb extrem = number of extremities in the analyzed area; Nb nodes = number of nodes in the analyzed area; Nb Junctions = number of junctions in the analyzed area; Nb master junction = number of master junctions in the analyzed area; Nb master segments = number of master segments in the analyzed area; Tot. master segments length = sum of the length of the detected master segments in the analyzed area; Nb meshes = number of meshes in the analyzed area; Tot.meshes area = sum of mesh areas detected in the analyzed area; Nb pieces = sum of number of segments, isolated elements and branches detected in the analyzed area; Nb segments = number of segments in the analyzed area; Nb branches = number of branches in the analyzed area; Nb isol. seg = number of isolated elements in the analyzed area; Tot. length = sum of length of segments, isolated elements, and branches in the analyzed area; Tot. branching length = sum of length of the trees composed from segments and branches in the analyzed area; Tot. segments length = sum of length of the segments in the analyzed area; Tot. branches length = sum of length of the branches in the analyzed area; Tot. isol. branches length = sum of length of the isolated elements in the analyzed area. Branching interval = mean distance separating two branches in the trees in the analyzed area. [Tot. segments length / Nb branches]; Mesh index = mean distance separating two master junctions in the trees in the analyzed area [Tot. master segments length / Nb master segments]; Mean Mesh Size = mean mesh size in the analyzed area). Scale bar = 400 µm. <a href="https://www.jove.com/files/ftp_upload/66454/66454fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cell migration study; microscopy images at 0h, 5h, 24h; bar graphs show VE-cadherin, AlphaV expression." class="xfigimg" src="/files/ftp_upload/66454/66454fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Endothelial markers in MELT. HTR8/SVneo cells were seeded at a density of 80,000 cells/cm2 on plastic or basement membrane matrix. (A) Representative microphotographs of cells at 0 h (cells in plastic), 5 h and 24 h on basement membrane matrix. (B) After cell recovery using the protocol with PBS-EDTA, we used the pellet to evaluate the relative expression of VE-cadherin and alpha V-integrin by qPCR. Values are mean ± S.E.M *p &lt; 0.05, compared to 0 h using one-way ANOVA. Scale bar = 400 µm. <a href="https://www.jove.com/files/ftp_upload/66454/66454fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="EVTs vs enEVTs fluorescence microscopy; DAPI, CD31, Vimentin staining; cell differentiation analysis." class="xfigimg" src="/files/ftp_upload/66454/66454fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: CD31 is expressed on HTR-8/SVneo cells after 24 h of differentiation with endothelial-like characteristics. CD31 was assessed by immunofluorescence on HTR-8/SVneo cells at 24 h in undifferentiated cells, EVTs (upper panels) and differentiated cells, enEVTs (lower panels). Anti-CD31 (green), anti-vimentin (red) and DAPI (blue) were used. Merge: overlap of CD31, vimentin and DAPI. Representative images with 40x objective in a confocal microscope, n= 3. This image shows how the CD31 increases, a classic endothelial marker, and how vimentin, used as a mesenchymal marker, decreases slightly upon MELT induction in HTR8/SVneo. <a href="https://www.jove.com/files/ftp_upload/66454/66454fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Static equilibrium analysis; microscopy and gel electrophoresis results; EVT, enEVT; RECK expression." class="xfigimg" src="/files/ftp_upload/66454/66454fig04.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: Protein-cell extracts from HTR8/SVneo after MELT assay for Western blot analysis. (A) HTR8/SVneo cell line was infected with a lenti-virus to overexpress human RECK (RECK) or a lentivirus control, which commands the expression of EGFP (Control). At 48 h after transduction, the cells were cultured at 80,000 cell/cm2 in plastic (EVTs) or basement membrane matrix for 24 h to induce MELT (enEVT). (B) Expression of EGFP is observed in control cells by epifluorescence. (C) Ponceau staining of proteins obtained from this assay after SDS-page and transfer to PDVF membrane. (D) Representative Western blot for RECK in cells, using β-tubulin as housekeeping. <a href="https://www.jove.com/files/ftp_upload/66454/66454fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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JoVE Journal

Developmental Biology

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