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Biologie du développement

Menneskelig Primær trophoblast? Trophoblast Cell Culture modell for å studere den beskyttende effekten av melatonin mot Hypoksi / Reoksygeneringen-indusert Forstyrrelse

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

Gjennom menneskets svangerskapet, placenta cytotrophoblast celler, som er mononukleærerte stamceller, raskt spre seg og differensiere til enten villous eller extravillous cytotrophoblast celler. Extravillous cytotrophoblasts invadere og oppussing spiralarteriene av livmorveggen. Villous cytotrophoblasts, derimot, fortsetter å spre seg, skille og sikring for å danne multinucleated syncytiotrophoblast (den syncytium) 1. Vedlikehold av villøse trophoblast homeostase er viktig for fosterets trivsel og sunn graviditet. Faktisk villous trophoblasts tillate maternal-føtal utveksling av oksygen og næringsstoffer, og produserer viktige hormoner for graviditet. Dessuten er det syncytiotrophoblast den eneste celletype i direkte kontakt med morens blodsirkulasjonen og gir en vesentlig fysisk og immunologisk barriere. Derfor må syncytiotrophoblast gjennomgå apoptose og erstatning for homeostatic vedlikehold og til avoid morkake patologier 2-5.

Teknikken er utviklet av Kliman et al. 6 i 1986 for å isolere primær villous cytotrophoblasts fra menneskelige placentas forårsaket en revolusjon i placenta forskning ved at studiet av de molekylære mekanismene som er involvert i villøse trophoblast differensiering. Dette klassisk teknikk, basert på sekvensielle enzymatiske digestions med trypsin og DNase, etterfulgt av isolasjon i tetthet sentrifuge media (kolloidalt silikapartiklene belagt med polyvinylpyrrolidon, eller Percoll) er nå anerkjent som gullstandarden for å isolere villous cytotrophoblast celler. Teknikken kan optimaliseres ved magnetisk immunorensing, en prosedyre som skiller villous cytotrophoblasts fra ikke-trofoblastiske celler basert på en annerledes ekspresjon av spesifikke antigener på overflaten av disse cellene. Vi valgte human leukocytt antigen ABC (HLA-ABC) på grunn av fravær av dets ekspresjon på trophoblastic celle membrane 7,8.

Morkaken er et organ som gjennomgår dramatiske variasjoner i oksygennivået i løpet av svangerskapet. I det første trimester, er den oksygenforholdet fysiologisk meget lav (2% O 2), men øker til mild grad av oksygenering (8% O 2) i den andre og tredje trimester. Tuuli et al. 9 beskrevet at in vitro reproduksjon av trophoblast miljøet inne i placenta villi er en utfordring og variasjoner i oksygennivået kan også føre til fenotypiske endringer. Det er derfor foreslått å innføre 8% oksygen som normoxia å etterligne oksygen spenning funnet i placenta villi i løpet av tredje trimester av svangerskapet 8,9. Chen et al., 10 grundig studert av flere variable knyttet til oksygen spenning i trophoblast cellekultur og vist betydningen av å bestemme oksygennivået i en pericellulær miljø. Nivåene av oksygen i de villi har en tendens til å økepå grunn av vaskulogenese. Blodstrøm i placenta villi øker jevnt og nivået av hydrogenperoksid (en rikelig reaktive oksygenforbindelser) er et viktig signal som styrer vaskulogenese 11,12. I svangerskapskomplikasjoner, mangel på vaskulogenese genererer hypoksi, og enda viktigere, intermitterende varianter av oksygenering (kalt hypoksi / Reoksygeneringen). Disse forholdene fører til en unormal økning av oksidativt stress, som kompromisser placenta og fosterets levedyktighet 13,14. De endringer som trophoblast celler gjennomgår in vivo under episoder av hypoksi / Reoksygeneringen kan etterlignet in vitro som følger: villous cytotrophoblasts blir opprettholdt under normoksisk forhold (8% O 2) til de differensiere i syncytiotrophoblast. De blir deretter utsatt for hypoksiske betingelser (0,5% O 2) i 4 timer, etterfulgt av ytterligere 18 timer av normoxia (reoksygenering). Ved hjelp av denne hypoksi / Reoksygeneringen tilnærming, trophoblasts exHibit deregulert redox status og økte nivåer av indre apoptose 8, som har blitt observert i enkelte svangerskapskomplikasjoner. Derfor er dette en nyttig in vitro modell for å vurdere nye forebyggende og terapeutiske tilnærminger for å bekjempe graviditet komplikasjoner forbundet med placenta hypoksi / reoksygenering.

Placentale celler produserer melatonin, som har flere viktige funksjoner, for eksempel en evne til å unngå oksidativt stress og placenta dysfunksjon 15. Her presenterer vi de eksperimentelle tilnærming og cellemodeller som brukes til å demonstrere den beskyttende effekten av melatonin i placenta trophoblast celler på molekylært, cellulært og funksjonsnivå 8.

Protocol

Placentas ble innhentet umiddelbart etter spontane vaginale fødsler fra ukompliserte svangerskap på kompis-St-Luc Hospital, Montreal, QC, Canada, med informert pasienten samtykke og godkjennelse av etiske komiteer (kompis-St-Luc Hospital og INRS-Institut Armand-Frappier, Laval, QC, Canada). 1. Isolering og rensing av villøse Cytotrophoblast Cells Løsninger og media Forbered transportmedium ved å supplere Dulbeccos modifiserte Eagle Medium Høy Glukose (DMEM-HG) med 1% vol / vol …

Representative Results

Isolasjon og immunorensing av villous cytotrophoblast celler fra en normal sikt placenta oppnådd ved vaginal fødsel ga 1 x 10 8 levende celler. Placenta veide 350 g, var 19 cm i diameter og 4 cm høy med skiveformet fasong og gjennomsiktige membraner. Ingen cotyledon misdannelse ble oppdaget. Navlestrengen hadde paracentral lokalisering og en lengde på 56 cm. Renheten ble evaluert ved flow cytometri ved hjelp vimentin og cytokeratin-7 markører. Mer enn 98% av cellene var n…

Discussion

I pattedyr er fosterutvikling direkte avhengig av tilstrekkelig placenta funksjon. Utviklings opprinnelse lidelser er basert på en hypotese om at årsaken til sykdommer manifestert senere i livet kan spores tilbake til tidlig utvikling og at morkaken har en mekanistisk rolle i foster programmering 30-32. Morkaken er nøkkelen formidler av fosterets vekst og utvikling: det regulerer næringstransport, beskytter mot skadelige eksponeringer, og har store endokrine funksjoner. Utviklingen av Kliman et al.</e…

Divulgations

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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References

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Tags

Keywords: 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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