Human Primary Trofoblast Cell Cultuur aan de beschermende effecten van melatonine tegen hypoxie Studie / reoxygenation-geïnduceerde Disruption
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
Gedurende de zwangerschap, de placenta cytotrofoblastcellen, die mononucleaire stamcellen zijn, zich snel vermenigvuldigen en differentiëren in ofwel villous of extravilleuze cytotrofoblastcellen. Extravilleuze cytotrofoblasten binnen te vallen en de vorm van de spiraal arteriën van de baarmoederwand. Villous cytotrofoblasten, anderzijds blijven prolifereren, differentiëren en zekering multinucleated syncytiotrofoblast (de syncytium) 1 vormen. Het onderhoud van villi trofoblast homeostase is essentieel voor foetale welzijn en gezonde zwangerschap. In feite, villous trofoblasten mogelijk maternale-foetale uitwisseling van zuurstof en voedingsstoffen en hormonen produceren essentieel voor de zwangerschap. Bovendien is het syncytiotrofoblast het enige celtype in direct contact met de maternale bloedsomloop en een essentiële fysische en immunologische barrière. Daarom moet de syncytiotrofoblast apoptose en vervanging ondergaan homeostatische onderhoud en AVOid placenta pathologie 2-5.
De door Kliman et al. 6 ontwikkeld in 1986 tot primaire villous cytotrofoblasten isoleren van menselijke placenta's techniek zorgde voor een revolutie in de placenta onderzoek doordat de studie van de moleculaire mechanismen die betrokken zijn bij villous trofoblast differentiatie. Deze klassieke techniek, waarbij in de enzymatische digesties met trypsine en DNase, gevolgd door isolatie van dichtheidscentrifugatie media (colloïdale silicadeeltjes bekleed met polyvinylpyrrolidon of Percoll) wordt nu erkend als de gouden standaard voor het isoleren villeus cytotrofoblastcellen. De techniek kan worden geoptimaliseerd door magnetische immunozuivering, een procedure die villous cytotrofoblasten scheidt van niet-trofoblastcellen gebaseerd op de differentiële expressie van specifieke antigenen op het oppervlak van deze cellen. We kozen voor het menselijke leukocyt antigen ABC (HLA-ABC) vanwege de afwezigheid van de expressie op het trofoblastische cel membraame 7,8.
De placenta is een orgaan dat dramatische verschillen in zuurstofgehalte ondergaat tijdens de zwangerschap. In het eerste trimester is de oxygenatie verhouding fysiologisch zeer laag (2% O 2) maar neemt milde niveaus van oxygenatie (8% O 2) in het tweede en derde trimester. Tuuli et al. 9 beschreven dat het in vitro reproduceren van de trofoblast omgeving in de placenta villi is een uitdaging en variaties oxygenatie niveau kan zelfs leiden tot fenotypische veranderingen. Het is daarom voorgesteld 8% zuurstof vast die normoxia de zuurstofspanning in placenta villi in het derde trimester van de zwangerschap 8,9 nabootsen. Chen et al. 10 uitgebreid bestudeerd verscheidene variabelen in verband met zuurstofspanning in trofoblast celkweek en toonde het belang van het bepalen van het zuurstofgehalte in een pericellulaire omgeving. De niveaus van zuurstof in de villi neiging te stijgendoor vasculogenese. De bloedstroom in placenta villi voortdurend toeneemt en het niveau van waterstofperoxide (een overvloedige reactieve zuurstofspecies) een belangrijk signaal dat vasculogenese 11,12 regelt. In de zwangerschap complicaties, een gebrek aan vasculogenese genereert hypoxie, en nog belangrijker, intermitterende variaties van oxygenatie (genaamd hypoxie / reoxygenatie). Deze omstandigheden leiden tot een abnormale verhoging van de oxidatieve stress, die de placenta en de levensvatbaarheid van de foetus 13,14 compromissen. De veranderingen die trofoblast cellen ondergaan in vivo tijdens episodes van hypoxie / reoxygenation kan worden nagebootst in vitro als volgt: villous cytotrofoblasten onder normoxische omstandigheden (8% O 2) totdat ze differentiëren tot syncytiotrofoblast worden gehandhaafd. Zij worden vervolgens onderworpen aan hypoxische omstandigheden (0,5% O2) gedurende 4 uur, gevolgd door nog 18 h normoxia (reoxygenatie). Met behulp van deze hypoxie / reoxygenation benadering, trofoblasten exHiBit gedereguleerd redox status en verhoogde intrinsieke apoptose 8, zoals in sommige zwangerschap complicaties waargenomen. Daarom is het zinvol om in vitro model om nieuwe preventieve en therapeutische mogelijkheden te evalueren bestrijding zwangerschap complicaties van placenta hypoxie / reoxygenatie.
Placenta cellen produceren melatonine, dat een aantal belangrijke functies, zoals het vermogen om oxidatieve stress en placentale dysfunctie 15 overbodig is. Hier presenteren we de experimentele aanpak en mobiele modellen die worden gebruikt om de beschermende effecten van melatonine in de placenta trofoblast cellen vertonen op moleculair, cellulair en functioneel niveau 8.
Protocol
Representative Results
Discussion
In zoogdieren, ontwikkeling van de foetus is direct afhankelijk van een adequate placenta-functie. De oorsprong van ontwikkeling stoornissen zijn gebaseerd op de hypothese dat de oorzaak van ziekten manifesteren op latere leeftijd naar vroege ontwikkeling en dat de placenta een mechanistische rol bij de foetale 30-32 kunnen worden getraceerd. De placenta is de sleutel mediator van de foetale groei en ontwikkeling: het reguleert nutriëntentransfers, beschermt tegen schadelijke blootstelling, en heeft grote en…
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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