Discontinuous gas exchange in Madagascar hissing cockroaches is not a consequence of hysteresis around a fixed PCO2 threshold.

It has been hypothesised that insects display discontinuous gas-exchange cycles (DGCs) as a result of hysteresis in their ventilatory control, where CO2-sensitive respiratory chemoreceptors respond to changes in haemolymph PCO2 only after some delay. If correct, DGCs would be a manifestation of an unstable feedback loop between chemoreceptors and ventilation, causing PCO2 to oscillate around some fixed threshold value: PCO2 above this ventilatory threshold would stimulate excessive hyperventilation, driving PCO2 below the threshold and causing a subsequent apnoea. This hypothesis was tested by implanting micro-optodes into the haemocoel of Madagascar hissing cockroaches and measuring haemolymph PO2 and PCO2 simultaneously during continuous and discontinuous gas exchange. The mean haemolymph PCO2 of 1.9 kPa measured during continuous gas exchange was assumed to represent the threshold level stimulating ventilation, and this was compared with PCO2 levels recorded during DGCs elicited by decapitation. Cockroaches were also exposed to hypoxic (PO2 10 kPa) and hypercapnic (PCO2 2 kPa) gas mixtures to manipulate haemolymph PO2 and PCO2. Decapitated cockroaches maintained DGCs even when their haemolymph PCO2 was forced above or below the putative ∼2 kPa ventilation threshold, demonstrating that the characteristic oscillation between apnoea and gas exchange is not driven by a lag between changing haemolymph PCO2 and a PCO2 chemoreceptor with a fixed ventilatory threshold. However, it was observed that the gas exchange periods within the DGC were altered to enhance O2 uptake and CO2 release during hypoxia and hypercapnia exposure. This indicates that while respiratory chemoreceptors do modulate ventilatory activity in response to haemolymph gas levels, their role in initiating or terminating the gas exchange periods within the DGC remains unclear.

Automated 3D Microphysiometry Facilitates High-Content and Highly Reproducible Oxygen Measurements within 3D Cell Culture Models.

Microphysiometry is a powerful technique to study metabolic parameters and detect changes to external stimuli. However, applying this technique for automated label-free and real-time measurements within cell-laden three-dimensional (3D) cell culture constructs remains a challenge. Herein, we present an entirely automated microphysiometry setup that combines needle-type microsensors with motorized sample and sensor positioning systems inside a standard tissue-culture incubator. The setup records dissolved oxygen as a metabolic parameter along the z-direction within cell-laden 3D constructs in a minimally invasive manner. The microphysiometry setup was applied to characterize the spatial oxygen distribution within thick cell-laden 3D constructs, study the time-dependent changes on the oxygen tension within 3D breast cancer models following a chemotherapeutic treatment, and identify kinetics and recovery effects after drug exposure over 5 weeks. Our data suggest that the microphysiometry setup enables highly reproducible measurements without human intervention, due to the high degree of automation and positional accuracy. The results demonstrate the applicability of the setup to provide valuable long-term insights into oxygenation within 3D models using minimally invasive, label-free, and entirely automated analysis methods.

A New Non-invasive Technique for Measuring 3D-Oxygen Gradients in Wells During Mammalian Cell Culture.

Oxygen tension plays an important role in overall cell function and fate, regulating gene expression, and cell differentiation. Although there is extensive literature available that supports the previous statement, little information is to be found about accurate O2 measurements during culture. In fact, O2 concentration at the cell layer during culture is commonly assumed to be equal to that of the incubator atmosphere. This assumption does not consider oxygen diffusion properties, cell type, cell density, media composition, time in culture nor height of the cell culture medium column. In this study, we developed a non-invasive, optical sensor foil-based technique suitable for measuring the 3D oxygen gradient that is formed during cell culture as a result of normal cell respiration. For this propose, we created a 3D printed ramp to which surface an oxygen optode sensor foil was attached. The ramps were positioned inside the culture wells of 24 well plate prior cell seeding. This set up in conjunction with the VisiSens TD camera system allows to investigate the oxygen gradient formation during culture. Cultivation was performed with three different initial cell densities of the cell line A549 that were seeded on the plate containing the ramps with the oxygen sensors. The O2 gradient obtained after 96 h of culture showed significantly lower O2 concentrations closer to the bottom of the well in high cell density cultures compared to that of lower cell density cultures. Furthermore, it was very interesting to observe that even with low cell density culture, oxygen concentration near the cell layer was lower than that of the incubator atmosphere. The obtained oxygen gradient after 96 h was used to calculate the oxygen consumption rate (OCR) of the A549 cells, and the obtained value of ~100 fmol/h/cell matches the OCR value already reported in the literature for this cell line. Moreover, we found our set up to be unique in its ability to measure oxygen gradient formation in several wells of a cell culture plate simultaneously and in a non-invasive manner.

Oxygen-distribution within 3-D collagen I hydrogels for bone tissue engineering.

Tissue engineering (TE) approaches typically envisage the structural and functional reconstitution of previously damaged tissue in situ. An adequate three-dimensional environment is therefore of fundamental importance for the designated cells associated to the scaffold material. The sufficient supply with nutrients and oxygen in vitro and in vivo mark thereby critical challenges of TE. In this study, we intended to analyse the level of locally dissolved oxygen within 3-D cell-loaded collagen I gels in vitro. For the analysis of the oxygen levels in situ, we employed an optical fibre-based micro sensor setup, as well as a camera supported non-invasive optical sensor foil based technique. These complementary analytical tools enable the identification, localization, and temporal follow-up investigation of specified regions of interest within TE constructs. Human adipose-derived mesenchymal stem cells (hAdMSCs) cultured in collagen I gels under normoxic conditions were analysed periodically and kinetically up to 70 days - thereby revealing dynamic changes of the level of dissolved oxygen inside the gel constructs. Dependent on the applied cell concentration, the in vitro oxygen concentration (cO2) within the gels reached physiological ranges (7-9%) after 21 days, or 35 days of culture. The minimal cO2 was measured after 35 days in vitro, featuring an oxygen level of 4.8 ±â€¯1.3%. Upon prolonged culture, a plateau-like status of the cO2 around 8-9% established, indicating a change in the physiological activity of the cells under investigation. The expression patterns of BCL2, CASP3 and MCM5 revealed significant differences among the proliferative and apoptotic stages of the cell-loaded samples at the investigated time points of 7 and 70 days in culture. In summary, these data show the temporary dynamic nature of the oxygen distribution in cell-loaded gel constructs. The applied technique is an ideal tool for the evaluation of multiple parameters affecting the oxygen distribution in vitro. We conclude that it takes 5 weeks for establishing an equilibrium of cO2. Levels reached in a 3-D gel construct are comparable with physiological oxygenation ranges in bone-associated tissues.

Changes in hemolymph total CO2 content during the water-to-air respiratory transition of amphibiotic dragonflies.

Dragonflies (Odonata, Anisoptera) are amphibiotic; the nymph is aquatic and breathes water using a rectal gill before metamorphosing into the winged adult, which breathes air through spiracles. While the evolutionary and developmental transition from water breathing to air breathing is known to be associated with a dramatic rise in internal CO2 levels, the changes in blood-gas composition experienced by amphibiotic insects, which represent an ancestral air-to-water transition, are unknown. This study measured total CO2 (TCO2) in hemolymph collected from aquatic nymphs and air-breathing adults of Anax junius, Aeshna multicolor (Aeshnidae), Libellula quadrimaculata and Libellulaforensis (Libellulidae). Hemolymph PCO2  was also measured in vivo in both aeshnid nymphs and marbled crayfish (Procambarus fallax. f. virginalis) using a novel fiber-optic CO2 sensor. The hemolymph TCO2 of the pre- and early-final instar nymphs was found to be significantly lower than that of the air-breathing adults. However, the TCO2 of the late-final instar aeshnid nymphs was not significantly different from that of the air-breathing adults, despite the late-final nymphs still breathing water. TCO2 and PCO2  were also significantly higher in the hemolymph of early-final aeshnid nymphs compared with values for the water-breathing crayfish. Thus, while dragonfly nymphs show an increase in internal CO2 as they transition from water to air, from an evolutionary standpoint, the nymph's ability to breathe water is associated with a comparatively minor decrease in hemolymph TCO2 relative to that of the air-breathing adult.