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

Abstract

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 O 2 measurements during culture. In fact, O 2 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 O 2 gradient obtained after 96 h of culture showed significantly lower O 2 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.

Expansion and differentiation of ex vivo cultured erythroblasts in scalable stirred bioreactors

“…Erythroid cells are commonly cultured in static dishes or flasks, in which spatial inhomogeneities in nutrient concentrations occur due to settling of cells to the bottom surface. Moreover, this cultivation system is susceptible to mass transfer limitations as transfer of oxygen to the culture is fully diffusion‐dependent (Peniche Silva et al, 2020 ; Place et al, 2017 ; Sugiura et al, 2011 ). This type of culture systems would culminate in impractical numbers of dishes/flasks (currently about 9000–12,000 T175 flasks for a single transfusion unit) (Timmins & Nielsen, 2009 , 2011 ).…”

Editors’ Choice—Luminescent Oxygen Sensors: Valuable Tools for Spatiotemporal Exploration of Metabolism in In Vitro Systems

“…43 Another approach used an O 2 optode adhered to a hydrogel "ramp," allowing researchers to define a gradient from the bottom of a culture flask to the media-air interface-giving a proxy for metabolic activity relative to O 2 diffusion into the sample. 44 For the user that needs similar measurements to the Clark electrode, but prefers an optical readout, fiber optic sensors are an appropriate tool. 45 Fiber optics largely benefit those working with microvolumes by not consuming the analyte while allowing for a spatial resolution of ∼50 μm.…”

O2-sensitive microcavity arrays: A new platform for oxygen measurements in 3D cell cultures

“…Systems using both optical ( Eggert et al, 2021 ) and electrochemical ( Kagawa et al, 2015 ) needle-based sensors have been described for this purpose. Peniche Silva et al (2020) described a system where they used an oxygen-sensitive film fixed on a ramp in order to measure oxygen gradients and profiles above a monolayer of cells. However, most systems are not really suited for the use in 3D cell culture.…”

“…Also, the parallel investigation of several spheroids is not possible. The system of Peniche Silva et al (2020) would also allow for the introduction of 3D cell cultures, however the system would not allow measurements in the immediate microenvironment of the cells.…”