Pericellular oxygen concentration of cultured primary human trophoblasts

Abstract

Introduction Oxygen is pivotal in placental development and function. In vitro culture of human trophoblasts provides a useful model to study this phenomenon, but a hotly debated issue is whether or not the oxygen tension of the culture conditions mimics in vivo conditions. We tested the hypothesis that ambient oxygen tensions in culture reflect the pericellular oxygen levels. Methods We used a microelectrode oxygen sensor to measure the concentration of dissolved oxygen in the culture medium equilibrated with 21%, 8% or <0.5% oxygen. Results The concentration of oxygen in medium without cells resembled that in the ambient atmosphere. The oxygen concentration present in medium bathing trophoblasts was remarkably dependent on the depth within the medium where sampling occurred, and the oxygen concentration within the overlying atmosphere was not reflected in medium immediately adjacent to the cells. Indeed, the pericellular oxygen concentration was in a range that most would consider severe hypoxia, at ≤ 0.6% oxygen or about 4.6 mm Hg, when the overlying atmosphere was 21% oxygen. Conclusions We conclude that culture conditions of 21% oxygen are unable to replicate the pO2 of 40–60 mm Hg commonly attributed to the maternal blood in the intervillous space in the second and third trimesters of pregnancy. We further surmise that oxygen atmospheres in culture conditions between 0.5% and 21% provide different oxygen fluxes in the immediate pericellular environment yet can still yield insights into the responses of human trophoblast to different oxygen conditions.

Limitations of oxygen delivery to cells in culture: An underappreciated problem in basic and translational research

“…This gradient is not stable, but is ever increasing as cell proliferation occurs (Figure 6) [39]. There is essentially an infinite number of gradient conditions that may occur depending on the cell density, OCR, and medium thickness utilized in a specific experimental protocol [10,20,34,39–45]. For example, high OCR cells such as primary hepatocytes or hepatocellular carcinoma cell lines seeded at high density may reach pericellular oxygen concentration near zero within 4 hours and possibly in as little as 30 minutes [6,42].…”

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

“…Normoxia mimics the concentration of oxygen in the intervillous space of the placenta during the second and the third trimesters of pregnancy. H/R mimics in vitro the variation of oxygen in the intervillous space of placentas from patients with pregnancy disorders, such as preeclampsia . If not treated with melatonin, trophoblasts were treated at 72 hour of culture with pyrrolidine dithiocarbamate (PDTC) 25 μmol/L (Tocris, Bristol, UK), rapamycin 300 nmol/L (Tocris), or lipopolysaccharide (LPS; Sigma‐Aldrich) 1 μg/mL, which are, respectively, an NFκB inhibitor, a positive control for autophagy, and a positive control for inflammation.…”

Dissolved oxygen concentration in the medium during cell culture: Defects and improvements

“…Among various physical conditions involved in cell culture, medium oxygen concentration is thought to be one of the most important factors (Metzen et al, 1995). Previous reports have indicated that the oxygen tensions in the culture medium is not only affected by the oxygen concentration within the incubators (Newby et al, 2005;Chen et al, 2013) and the depth from the medium surface (Pettersen et al, 2005;Oze et al, 2012) but also by the medium volume (Oze et al, 2012).…”

“…In these studies, the cells are cultured in tri-gas humidified incubators or hypoxia chambers (Yu and Hales, 2011;Hsiao et al, 2013;Liu et al, 2013), so changes of the culture medium are often performed in normoxia (Xiong et al, 2009;Raymond et al, 2011). It has also been reported that the oxygen tension in the medium depended on the oxygen concentration within the overlying atmosphere (Newby et al, 2005;Chen et al, 2013). Thus, the replacement of the medium in normoxia will induce cellular reoxygenation (Kato et al, 2011;Ibuki et al, 2012;Ren et al, 2013).…”