Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced 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

Throughout human pregnancy, the placental cytotrophoblast cells, which are mononucleated stem cells, rapidly proliferate and differentiate into either villous or extravillous cytotrophoblast cells. Extravillous cytotrophoblasts invade and remodel the spiral arteries of the uterine wall. Villous cytotrophoblasts, on the other hand, continue to proliferate, differentiate and fuse to form multinucleated syncytiotrophoblast (the syncytium)¹. The maintenance of villous trophoblast homeostasis is essential for fetal well-being and healthy pregnancy. In fact, villous trophoblasts allow maternal-fetal exchange of oxygen and nutrients, and produce essential hormones....

Protocol

Placentas were obtained immediately after spontaneous vaginal deliveries from uncomplicated pregnancies at the CHUM-St-Luc Hospital, Montreal, QC, Canada, with informed patient consent and approval of ethical committees (CHUM-St-Luc Hospital and INRS-Institut Armand-Frappier, Laval, QC, Canada).

1. Isolation and Purification of Villous Cytotrophoblast Cells

1. Solutions and media
   1. Prepare transport media by supplementing Dulbecco's Modified Eagle's Medium High-Glucose (DMEM-HG) with 1% vol/vol antibiotic (10,000 units/ml penicillin G, 100 mg/ml streptomycin sulphate) and store at 4 °C.
   2. Prepare primary culture....

Results

Isolation and immunopurification of villous cytotrophoblast cells from a normal term placenta obtained by vaginal delivery yielded 1 x 10⁸ viable cells. The placenta weighed 350 g, was 19 cm in diameter, 4 cm tall with discoid shape and transparent membranes. No cotyledon malformation was detected. The umbilical cord had paracentral localization and a length of 56 cm. The purity was evaluated by flow cytometry using vimentin and cytokeratin-7 markers. More than 98% of the cells.......

Discussion

In mammals, fetal development is directly dependent on adequate placental function. The developmental origins of health disorders are based on the hypothesis that the cause of diseases manifested later in life can be traced back to early development and that the placenta has a mechanistic role in fetal programming^30-32. The placenta is the key mediator of fetal growth and development: it regulates nutrient transfer, protects against harmful exposures, and has major endocrine functions. The development by Klima.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Curved Metzenbaum Scissors	Shandon	9212	surgical equipment (cell isolation) (2 units)
Splinter Forceps Fine 41/2 in	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 10 cm x 10 cm	Cardinal Health	361020733
Oblong Glass Baking Dish	Pyrex	1105397	Glassware (2.8 L)
Funnel Buchner	Coorstek Inc	10-356E	Glassware (114 mm 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
10 ml Serological Pipet	Corning	11415038
Cell Strainer 100 μm 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 1 L of digestion solution
Hydroxypiperazineethansulphonic Acid (Hepes)	Life Technologies	15630-080	25 ml (1 M) for 1 L of digestion solution
Trypsin Type I	Sigma-Aldrich	T8003	9,888 U
Deoxyribonuclease Type Iv	Roche	10-104-159-001	402,000 U
Calcium Chloride	Sigma-Aldrich	C4901	100 mM
Magnesium Sulfate	Baker	2500-01	800 mM
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: 36 ml
Isopropanol	Acros	42383-0010	50 ml
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 purified 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