Fisher 2020 Placenta
|Fisher J, McKeating D, Pennell E, Cuffe J, Holland O, Perkins A (2020) Mitochondrial isolation, cryopreservation and preliminary biochemical characterisation from placental cytotrophoblast and syncytiotrophoblast. Placenta 82:1-4.|
Abstract: Placental function and homeostasis is very much dependent upon trophoblast mitochondria. Mitochondria are responsible for a myriad of functions including energy production, endocrine functions, nutrient and oxygen transfer as well as protecting the placenta from oxidative insult and cell stress. The materno-fetal interface is comprised of 2 related cell lineages, cytotrophoblast cells which fuse to form the overlaying syncytium. Mitochondrial morphology and function in these 2 lineages is different and, in this study, we describe methods for mitochondrial isolation, cryopreservation and preliminary biochemical characterisation of these discrete forms of mitochondria.
Villous tissue was collected from normal term placentae following vaginal delivery (n=7). Differential centrifugation was used to isolate 2 fractions, larger mitochondria predominantly from the cytotrophoblast cells and smaller mitochondria from the syncytiotrophoblast. Freshly isolated mitochondria were assessed for Complex I and Complex II respiration and maximal respiratory capacity and these were compared to equivalent isolates that had been frozen, stored at -80oC and revived. Other important functional parameters of mitochondrial activity were also assessed post cryopreservation including membrane potential, ATP production, progesterone biosynthesis and anti-oxidant expression.
The methods described resulted in 2 mitochondrial populations which match the types of mitochondria observed in situ with election microscopy. Both forms of mitochondria were metabolically active and recovered well from cryopreservation. There were important functional differences between cyto-mitochondria and syncytio-mitochondria including decreased respiration, decreased oxidative phosphorylation, decreased anti-oxidant expression but increased expression of the enzyme CYP11A1 and increased progesterone production. This study highlights the need to consider different types of trophoblast mitochondria in placental studies and provides methods to facilitate this.
Labels: MiParea: Respiration, Instruments;methods, Developmental biology
Stress:Cryopreservation Organism: Human Tissue;cell: Genital Preparation: Isolated mitochondria
Coupling state: LEAK, OXPHOS, ET Pathway: N, NS, ROX HRR: Oxygraph-2k