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Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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Developmental Biology

Trofoblasto humana primaria cultivo celular modelo para estudiar los efectos protectores de la melatonina contra la hipoxia / reoxigenación interrupción inducida

Published: July 30, 2016
doi:
10.3791/54228
, , , , , , , , ,
1INRS-Institut Armand-Frappier

Summary

This manuscript presents a unique in vitro model of immunopurified human villous cytotrophoblast cells cultured under hypoxia/reoxygenation. This model is suitable to study the protective effects of promising treatments, such as melatonin, on pregnancy complications associated with increased oxidative stress and altered placental function.

Abstract

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation.

Introduction

Durante el embarazo humano, las células citotrofoblastos placentarios, que son células madre mononucleadas, rápidamente proliferan y se diferencian en cualquiera de las células citotrofoblastos vellosos o extravillous. citrofoblastos extravelloso invaden y remodelar las arterias espirales de la pared uterina. Citotrofoblastos vellosos, por el contrario, siguen proliferar, diferenciarse y se fusionan para formar sincitiotrofoblasto multinucleadas (el sincitio) 1. El mantenimiento de la homeostasis del trofoblasto velloso es esencial para el bienestar del feto y el embarazo saludable. De hecho, los trofoblastos vellosidades permiten el intercambio materno-fetal de oxígeno y nutrientes, y producen hormonas esenciales para el embarazo. Por otra parte, el sincitiotrofoblasto es el único tipo de células en contacto directo con la circulación de la sangre materna y proporciona una barrera física e inmunológica esencial. Por lo tanto, el sincitiotrofoblasto debe someterse a la apoptosis y sustitución para el mantenimiento de la homeostasis y para evIdentificación del placentaria patologías 2-5.

La técnica desarrollada por Kliman et al., 6 en 1986 para aislar citrofoblastos vellosidades primarias de placentas humanas provocó una revolución en la investigación de la placenta al permitir el estudio de los mecanismos moleculares implicados en la diferenciación del trofoblasto velloso. Esta técnica clásica, sobre la base de las digestiones enzimáticas secuenciales con tripsina y DNasa, seguido de aislamiento en los medios de densidad de centrifugación (partículas de sílice coloidal recubierto por polivinilpirrolidona, o Percoll) es ahora reconocido como el estándar de oro para el aislamiento de células del citotrofoblasto velloso. La técnica se puede optimizar mediante inmunopurificación magnético, un procedimiento que separa citotrofoblastos vellosos de las células no trofoblásticas en base a la expresión diferencial de antígenos específicos en la superficie de estas células. Elegimos el antígeno leucocitario humano ABC (HLA-ABC) debido a la ausencia de su expresión en la célula trofoblástica membrane 7,8.

La placenta es un órgano que sufre variaciones importantes en los niveles de oxígeno durante el embarazo. En el primer trimestre, la relación de la oxigenación es fisiológicamente muy baja (2% de O2), pero aumenta a niveles leves de oxigenación (8% O 2) en el segundo y tercer trimestre. Tuuli et al. 9 describe que la reproducción in vitro del medio ambiente en el interior del trofoblasto vellosidades placentarias es un desafío y las variaciones en los niveles de oxigenación pueden incluso conducir a cambios fenotípicos. Es, por lo tanto, sugiere adoptar 8% de oxígeno como normoxia para imitar la tensión de oxígeno que se encuentra en vellosidades placentarias durante el tercer trimestre de gestación 8,9. Chen et al. 10 ampliamente estudiado diversas variables relacionadas con la tensión de oxígeno en cultivos de células del trofoblasto y demostró la importancia de la determinación de los niveles de oxígeno en un ambiente pericellular. Los niveles de oxígeno en el vellosidades tienden a aumentardebido a la vasculogénesis. El flujo de sangre en las vellosidades placentarias aumenta constantemente y el nivel de peróxido de hidrógeno (una abundancia de especies reactivas del oxígeno) es una señal importante que controla la vasculogénesis 11,12. En las complicaciones del embarazo, la falta de vasculogénesis genera hipoxia, y lo más importante, las variaciones intermitentes de oxigenación (hipoxia llamados / reoxigenación). Estas condiciones llevan a un aumento anormal en el estrés oxidativo, lo que compromete la placenta y del 13,14 viabilidad fetal. Las alteraciones que se someten las células del trofoblasto in vivo durante los episodios de hipoxia / reoxigenación se pueden imitar in vitro de la siguiente manera: citrofoblastos vellosidades se mantienen en condiciones de normoxia (8% O 2) hasta que se diferencian en sincitiotrofoblasto. A continuación, se someten a condiciones de hipoxia (0,5% de O 2) para 4 horas, seguido de un 18 hr adicional de normoxia (reoxigenación). El uso de este enfoque hipoxia / reoxigenación, trofoblastos exHiBit desregulado estado redox y el aumento de los niveles de apoptosis intrínseca 8, como se ha observado en ciertas complicaciones del embarazo. Por lo tanto, este es un útil modelo in vitro para evaluar nuevos enfoques preventivos y terapéuticos para combatir las complicaciones del embarazo asociados con la placenta hipoxia / reoxigenación.

Células de la placenta producen melatonina, que tiene varias funciones importantes, tales como la capacidad de evitar el estrés oxidativo y la disfunción placentaria 15. A continuación, presentamos los modelos de aproximación y de células experimentales utilizados para demostrar el efecto protector de la melatonina en las células trofoblásticas de la placenta a nivel molecular, celular y funcional 8.

Protocol

Placentas fueron obtenidos inmediatamente después de los partos vaginales espontáneos de embarazos sin complicaciones en el CHUM St-Luc del Hospital, Montreal, QC, Canadá, con el consentimiento del paciente informado y la aprobación de los comités éticos (CHUM-St-Luc hospital y INRS-Institut Armand-Frappier, Laval, Quebec, Canadá). 1. Aislamiento y purificación de células citotrofoblastos velloso Soluciones y medios Preparar los medios de transporte, completándolo Dulbecco modificad…

Representative Results

El aislamiento y la inmunopurificación de las células del citotrofoblasto velloso de la placenta normal plazo obtenidos por parto vaginal produjo 1 x 10 8 células viables. La placenta pesa 350 g, fue de 19 cm de diámetro, 4 cm de altura con forma discoidal y las membranas transparentes. No se detectó cotiledón malformación. El cordón umbilical tenía localización paracentral y una longitud de 56 cm. La pureza se evaluó por citometría de flujo utilizando vimentina y …

Discussion

En los mamíferos, el desarrollo fetal depende directamente de la función placentaria adecuada. Los orígenes del desarrollo de trastornos de la salud se basan en la hipótesis de que la causa de las enfermedades que se manifiestan más tarde en la vida se remonta al desarrollo temprano y que la placenta tiene una función mecánica en la programación fetal 30-32. La placenta es el mediador clave de crecimiento y desarrollo del feto: regula la transferencia de nutrientes, protege contra la exposición dañi…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) (no. 262011-2009) to CV and March of Dimes Social and Behavioral Sciences Research grant (#12-FY12-179) to CV and JTS; by studentships to LSF from the Ministère de l’éducation, de l’Enseignement supérieurs et de la recherche (MEESR)-Fonds de recherche du Québec (FRQ)-Nature et technologies (NT) and the Fondation Universitaire Armand-Frappier INRS, to HC from the Réseau Québécois en Reproduction-NSERC-CREATE, to AAHT from the Canadian Institutes of Health Research (CIHR) and FRQ-Santé, and to JBP from NSERC; by a fellowship to EMAS from the Conselho Nacional de Desenvolvimento Cientìfico e Tecnològico (CNPq) and the Programme de bourses d’excellence pour étudiants étrangers MEESR-FRQNT.

Materials

Curved Metzenbaum Scissors Shandon 9212 surgical equipment (cell isolation) (2 units)
Splinter Forceps Fine 41/2in Fisherbrand 13-812-42 surgical equipment (cell isolation) (2 units)
Scissors 4.5 Str Dissection Fisherbrand 08-940 surgical equipment (cell isolation) (2 units)
Gauze Sponge 10cm X 10cm Cardinal Health 361020733
Oblong Glass Baking Dish Pyrex 1105397 Glassware (2.8L)
Funnel Buchner  Coorstek Inc 10-356E Glassware (114MM DIAMeter)
Watch Glass  pyrex 9985100EMD Glassware
Formalin solution, neutral buffered, 10% Sigma-Aldrich HT501128-4L histological tissue fixative solution
Trypsinizing Flasks Wheaton 355395 Glassware (1 unit)
Disposable Culture Tubes Kimble 73750-13100 Glassware
Borosilicate Glass Pasteur Pipet (22.8 Cm)  Fisherbrand K63B1367820C Glassware
250 Ml Glass Beakers  Fisherbrand KFS14005250 Glassware
Glass Media Bottles With Cap Fisherbrand KFS14395250 Glassware (8 units)
50 Ml Corex Tube  Corning 8422-A (1 unit)
15 Ml Polystyrene Centrifuge Tube Corning 430791
50 Ml Polystyrene Centrifuge Tube Corning 430829
10ml Serological Pipet Corning 11415038
Cell Strainer 100μm Nylon Corning 431752
Absorbant Liner Scienceware 1199918
500 Ml Bottles Top Filter  Corning Pore: 0,22 µm / medium and HBSS preparation
2 Ml Criogenic Vials Corning 430488
Freezing Container, Nalgene Mr. Frosty Sigma-Aldrich C1562-1EA
Peristaltic Pump Pharmacia Fine Chemicals P3 model
Shaking Water Bath Fisher Model 127
Vacuum Pump ABM 4EKFS6CX-4
Sodium Chloride Fisherbrand EC231-598-3 Saline solution 0.9%
Hank’s Buffered Salt Solution (Hbss) Sigma-Aldrich H2387 Quantity: 9.25 (one vial) for 1L of digestion solution
Hydroxypiperazineethansulphonic Acid (Hepes) Life Technologies 15630-080 25mL (1M) for 1L of digestion solution
Trypsin Type I Sigma-Aldrich T8003 9,888U
Deoxyribonuclease Type Iv Roche 10-104-159-001 402,000U
Calcium Chloride Sigma-Aldrich C4901 100mM
Magnesium Sulfate Baker 2500-01 800mM
Dulbecco’s Modified Eagle Medium High Glucose (Dmem) Life Technologies 10564-045
Penicillin/Streptomycin Sulphate Hyclone SV30010
Fetal Bovine Serum Corning 35-010-CV
Percoll Sigma-Aldrich P1644  Density centrifugation media gradient. Volume: 36mL
Isopropanol Acros 42383-0010 50mL
Dimethyl Sulfoxide Sigma-Aldrich 472301
Automacs Magnetic Separator  Miltenyi Biotec Model 003
Automacs Columns  Miltenyi Biotec 130-021-101
Automacs Running Buffer  Miltenyi Biotec 130-091-221 http://www.miltenyibiotec.com/~/media/Images/Products/Import/0001100/IM0001131.ashx?force=1
Automacs Rinsing Solution  Miltenyi Biotec 130-091-222 http://www.miltenyibiotec.com/en/products-and-services/macs-cell-separation/cell-separation-buffers/automacs-rinsing-solution.aspx
Anti-Human Hla Abc Purified Clone W6/32 Affymetrix eBioscience 14-9983-82 anti-mouse antibody
Anti Mouse Igg Microbeads Miltenyi Biotec 130048401
Multiple Well Plate -  6 Well With Lid Corning 3335 Cell Bind surface
Multiple Well Plate -  24 Well With Lid Corning 3337 Cell Bind surface
Multiple Well Plate -  96 Well With Lid Corning 3300 Cell Bind surface
Modular Incubator Chamber  Billups-Rothenberg MIC-101 A set of two is necessary for simultaneous to generate normoxia and hypoxia/reoxygenation conditions
Single Flow Meter Billups-Rothenberg SFM3001
50 Mm In-Line Filter  Whatman 6721-5010 PTFE, pore: 1.0 µm
Gas Regulator Pro Star PRS301233 A set of two is necessary for simultaneous to generate normoxia and hypoxia/reoxygenation conditions
Gas Hose Class Vi Clear 5/16  Parker 100-05070102 3 pieces with ~ 0.5 m
17 Mm Adjustable Gas Hose Clamp Tiewraps THCSS-16
Normoxia Gas Cylinder  Praxair NI CDOXR1U-K Size K (3rd trimester‘s composition: 5% CO2, 8% O2, Bal. N2)
Normoxia Gas Cylinder  Praxair NI CDOXR1U-K Size K (3rd trimester‘s composition: 5% CO2, 0.5% O2, Bal. N2)
Oxygen Microelectrode Mi-730 Microelectrodes INC 84477
Oxygen Adapter Microelectrodes INC 3572
ROS Detection Reagent: CM-H2DCFDA  Invitrogen C-400
β-hCG ELISA kit  DRG internatinal EIA-4115
Anti-Vimentin ourified antibody eBioscience 14-9897 Host: mouse
Anti-Cytokeratin 7 (FITC) antibody  Abcam ab119697 Host: mouse
Alexa Fluor 488 Goat Anti-mousse IgG H&L antibody Life Technologies A-11029
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Tags

Primary Trophoblast Cell CultureHypoxia/reoxygenationMelatoninPregnancy ComplicationsPlacentaOxidative StressVillous Cytotrophoblast IsolationDensity CentrifugationTrophoblast CultureFirst Trimester Placenta

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Sagrillo-Fagundes, L., Clabault, H., Laurent, L., Hudon-Thibeault, A., Salustiano, E. M. A., Fortier, M., Bienvenue-Pariseault, J., Wong Yen, P., Sanderson, J. T., Vaillancourt, C. Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption. J. Vis. Exp. (113), e54228, doi:10.3791/54228 (2016).
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