Is 8% O 2 more normoxic than 21% O 2 for long-term in vitro cultures of human primary term cytotrophoblasts?

Abstract

STUDY QUESTION: Is 8% O 2 a better percentage of atmospheric oxygen for long-term cultures of human primary term cytotrophoblasts than the conventional 21% O 2 traditionally used in cell culture? SUMMARY ANSWER: Human primary term cytotrophoblasts are able to differentiate into syncytiotrophoblasts under both atmospheric oxygen levels. WHAT IS KNOWN ALREADY: Cell culture is traditionally done under 21% O 2 , which is equal to a pO 2 of ~160mmHg. Based on the pO 2 measured after instauration of the blood circulation within the placenta, it has been proposed that cytotrophoblasts culture should be under 8% O 2 , which is equivalent to 60mmHg, and that this percentage should be considered as the physiological normoxia for cytotrophoblasts. STUDY, DESIGN, SIZE, DURATION: Cytotrophoblasts were isolated and purified from human term placentas (n > 4). Cells were cultured under 21% O 2 and 8% O 2 for 12 days. Several cellular parameters were assessed on Days 2, 4, 8 and 12. PARTICIPANTS/MATERIALS, SETTING, METHODS: Placentas were obtained after vaginal or elective cesarean delivery from uncomplicated pregnancies at term (n ≥ 4). Cell viability was measured by a luminescent assay based on quantitation of the ATP content of living cells. Cell fusion was assessed by quantification of syncytin and e-cadherin mRNA expression by real-time PCR and determination of the fusion index by immunofluorescent microscopy. Trophoblast differentiation was assessed by measuring the expression levels of hCGβ, inhibin α subunit (InhA) and placental growth factor (PlGF) by real-time PCR and ELISA. Finally, the effect of the two oxygen levels on apoptosis and cellular oxidative stress was also investigated by quantifying caspase 3/7 activation, superoxide dismutase 1 (SOD-1) mRNA expression and H 2 O 2 generation. MAIN RESULTS AND THE ROLE OF CHANCE: There was no difference between 21% O 2 and 8% O 2 on cell viability. Cell fusion seemed to be enhanced during the first 4 days when the cells were cultured under 21% O 2 compared to 8% O 2 . The expression level of hCGβ was equivalent in both oxygen conditions, indicating that there was no difference in trophoblast differentiation. Interestingly, InhA expression was higher under 8% O 2 , while PlGF expression was inhibited compared to 21% O 2 . This latter result indicates that 8% O 2 may be more hypoxic than normoxic for in vitro culture of primary term cytotrophoblast. This is further corroborated by the fact that 21% O 2 did not significantly increase caspase 3/7 activities and the oxidative stress (SOD-1 mRNA expression and H 2 O 2 generation) in our cell cultures. LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: The in vitro culture of cytotrophoblasts is artificial and does not reflect the in vivo situation. The cell population is nearly 100% pure, cultured as a monolayer, and the cells bath in a chemically defined culture medium deprived of any oxygen carrier. The oxygen molecules available to the cells are passively dissolved in the medium. The gas dissolution properties of the medium and the cellular consumption rate of oxygen may allow the cells to sustain a wide range of oxygen percentages from 8% to 21%. WIDER IMPLICATIONS OF THE FINDINGS: It is possible to culture human primary term cytotrophoblasts for at least 12 days. The O 2 percentage of the air does not negatively affect in vitro cytotrophoblast differentiation. For in vitro culture of cytotrophoblasts, it is not necessary to lower the percentage of atmospheric oxygen to 8%. STUDY FUNDING/COMPETING INTEREST(S): This work was fully supported by 'Fetus for Life' charity. The authors state that there is no conflict of interest to declare regarding the publication of this paper.