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% O 2 ), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O 2 ) 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.

Specific HIF-2α (Hypoxia-Inducible Factor-2) Inhibitor PT2385 Mitigates Placental Dysfunction In Vitro and in a Rat Model of Preeclampsia (RUPP)

“…Because the environment and drug exposure can be tightly controlled, such model is widely used to identify new therapeutic targets and to test medications for placental dysfunction. 44,45 Even if this system is useful to identify drug candidates, animal models remain necessary to evaluate the ultimate clinical outcomes, namely maternal symptoms and fetal growth.…”

Exploring maternal-fetal interface with in vitro placental and trophoblastic models

“…Consequently, the in vitro formation of STB during different developmental processes of trophoblast cells can effectively be studied using human full-term placenta. The isolation of chorionic trophoblast cells has been conducted in multiple laboratories, followed by freezing and storage for various programs (Sagrillo-Fagundes et al, 2016). In the 1980s, a traditional tryptic digestive procedure was established and has been utilized since then.…”