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

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.

Melatonin modulates autophagy and inflammation protecting human placental trophoblast from hypoxia/reoxygenation

“…Term placentas were obtained immediately after spontaneous vaginal deliveries from uncomplicated pregnancies with informed patient consent. Villous cytotrophoblasts were isolated, based on the classic trypsin‐DNase/Percoll method and modified in our laboratory . Briefly, the placenta was manually minced, weighed, and exposed to four consecutive digestion baths containing trypsin and DNAse.…”

“…The pool of pellets was added to a Percoll column and centrifuged. The layer containing 40‐50% of the density, which contained mainly trophoblasts, was collected . Following the isolation, mononuclear villous trophoblasts were purified by immunomagnetic labeling using autoMACS ™ (Myltenyi Biotec, Santa Barbara, CA, USA), as described previously.…”

Maternal Transient Receptor Potential Vanilloid 6 (Trpv6) Is Involved In Offspring Bone Development

“…Next, we isolated cells from Trpv6 +/+ , Trpv6 +/-, and Trpv6 -/placentas. Staining of the trophoblast-specific marker proteins dysferlin, cytokeratin 7, and GCM1 (28,(30)(31)(32)(33) showed that >90% of the isolated cells were indeed trophoblasts ( Fig. 2B).…”

Current trends in the treatment of chronic placental insufficiency

“…Было установлено, что снижение уровня мелатонина связано с развитием преэклампсии, при этом циркулирующие уровни мелатонина, по-видимому, не подвержены сезонным изменениям во время беременности [19].…”