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Abstract
Introduction
Protocol
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Developmental Biology

Trophoblast primário humano Cultura celular modelo para estudar os efeitos protetores da melatonina contra a hipóxia / Disruption induzida pela reoxigenação

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 a gravidez humana, as células citotrofoblasto da placenta, que são células mononucleadas de haste, proliferam rapidamente e se diferenciam em células quer citotrofoblasto vilosos ou extravilosas. citotrofoblastos extravilosas invadir e remodelar as artérias em espiral da parede uterina. Citotrofoblastos viloso, por outro lado, continuar a proliferar, diferenciar-se e fundem para formar trofoblasto multinucleadas (a sincício) 1. A manutenção da homeostase trofoblasto viloso é essencial para o bem-estar fetal e gravidez saudável. Na verdade, trofoblastos vilosidades permitir a troca materno-fetal de oxigênio e nutrientes, e produzir hormônios essenciais para a gravidez. Além disso, o trofoblasto é o único tipo de célula em contacto directo com a circulação do sangue materno e proporciona uma barreira física e imunológica essencial. Portanto, o sinciciotrofoblasto deve sofrer apoptose e substituição para a manutenção da homeostase e avoid placentária patologias 2-5.

A técnica desenvolvida pela Kliman et ai. 6, em 1986, para isolar citotrofoblastos vilosidades primárias a partir de placentas humanas causou uma revolução no domínio da investigação de placenta, permitindo que o estudo dos mecanismos moleculares envolvidos na diferenciação trofoblasto viloso. Esta técnica clássica, com base em digestões enzimáticas sequenciais com tripsina e DNase, seguido pelo isolamento em meio de centrifugação de densidade (partículas de sílica coloidal revestido por polivinilpirrolidona, ou Percoll) é hoje reconhecida como o padrão de ouro para o isolamento de células citotrofoblasto vilosidades. A técnica pode ser optimizado por imunopurificação magnética, um processo que separa citotrofoblastos vilosidades a partir de células não-trofoblásticas com base na expressão diferencial de antigénios específicos nas superfícies destas células. Nós escolhemos o antigénio de leucócitos humanos ABC (HLA-ABC), devido à ausência da sua expressão na célula Membran trofoblásticae 7,8.

A placenta é um órgão que sofre variações dramáticas em níveis de oxigénio durante a gravidez. No primeiro trimestre, a relação de oxigenação é fisiologicamente muito baixa (2% de O 2), mas aumenta para níveis moderados de oxigenação (8% de O2) no segundo e terceiro trimestre. Tuuli et al. 9 descrito que a reprodução in vitro do meio ambiente trophoblast dentro das vilosidades da placenta é um desafio e variações nos níveis de oxigenação pode até levar a mudanças fenotípicas. É, por conseguinte, sugerido para adoptar 8% de oxigénio como normóxia para imitar a tensão de oxigénio encontradas em vilosidades da placenta durante o terceiro trimestre da gravidez 8,9. Chen et al. 10 extensivamente estudadas diversas variáveis ​​relacionadas com a tensão de oxigênio em cultura de células trofoblasto e demonstrou a importância de determinar os níveis de oxigênio em um ambiente pericelular. Os níveis de oxigénio no vilosidades tendem a aumentardevido à vasculogénese. O fluxo de sangue no vilosidades aumentos placentários constantemente e o nível de peróxido de hidrogênio (uma espécie abundante reativas de oxigênio) é um sinal importante que controla vasculog�ese 11,12. Em complicações na gravidez, a falta de vasculog�ese gera hipóxia, e mais importante, as variações intermitentes de oxigenação (chamada hipóxia / reoxigenação). Estas condições levam a um aumento anormal de estresse oxidativo, o que compromete placentária e 13,14 viabilidade fetal. As alterações que se submetem a células trofoblásticas in vivo durante episódios de hipoxia / reoxigenação pode ser mimetizado in vitro, como segue: citotrofoblastos vilosidades são mantidas sob condições de normóxia (8% O 2) até que se diferenciam em trofoblasto. Elas são então submetidas a condições de hipóxia (0,5% de O2) durante 4 horas, seguido de um adicional de 18 h de normóxia (reoxigenação). Usando essa abordagem hipóxia / reoxigenação, trofoblastos exHiBit desregulada estado redox e níveis aumentados de apoptose intrínseca 8, como tem sido observada em certas complicações na gravidez. Assim, este é um útil modelo in vitro para avaliar novas abordagens terapêuticas e preventivas para combater a complicações na gravidez associados com placentária hipoxia / reoxigenação.

Células da placenta produzir melatonina, que tem várias funções importantes, tais como a capacidade de evitar o estresse oxidativo e disfunção placentária 15. Aqui, apresentamos os modelos de abordagem e de células experimentais utilizados para demonstrar os efeitos protectores da melatonina em células trofoblásticas da placenta, a nível molecular, celular e funcional 8.

Protocol

Placentas foram obtidas imediatamente após partos vaginais espontâneos de gestações sem complicações no CHUM-St-Luc Hospital, Montreal, QC, Canadá, com o consentimento do paciente informado e aprovação dos comitês de ética (CHUM-St-Luc Hospital e INRS-Institut Armand-Frappier, Laval, Quebec, Canadá). 1. Isolamento e purificação de células das vilosidades citotrofoblasto Soluções e meios de comunicação Preparar por um meio de transporte complementar do meio modificado por Du…

Representative Results

Isolamento e imunopurifica�o de células citotrofoblasto vilosidades da placenta normais prazo obtidos por parto vaginal rendeu 1 x 10 8 células viáveis. A placenta pesava 350 g, foi de 19 cm de diâmetro, 4 cm de altura com forma discóide e membranas transparentes. Nenhuma malformação cotilédones foi detectada. O cordão umbilical teve localização paracentral e um comprimento de 56 cm. A pureza foi avaliada por citometria de fluxo usando-vimentina e citoqueratina…

Discussion

Nos mamíferos, o desenvolvimento fetal é diretamente dependente da função placentária adequada. As origens do desenvolvimento de distúrbios de saúde baseiam-se na hipótese de que a causa de doenças que se manifestam mais tarde na vida pode ser rastreada até ao início do desenvolvimento e que a placenta tem um papel mecanicista na programação fetal 30-32. A placenta é o mediador chave do crescimento e desenvolvimento fetal: regula a transferência de nutrientes, protege contra as exposições pre…

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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