Pericellular oxygen depletion during ordinary tissue culturing, measured with oxygen microsensors

Abstract

Recent research has found important differences in oxygen tension in proximity to certain mammalian cells when grown in culture. Oxygen has a low diffusion rate through cell culture media, thus, as a result of normal respiration, a decrease in oxygen tension develops close to the cells. Therefore, for the purpose of standardization and optimization, it is important to monitor pericellular oxygen tension and cell oxygen consumption. Here, we describe an integrated oxygen microsensor and recording system that allows measurement of oxygen concentration profiles in vertical transects through a 1.6-mm deep, stagnant, medium layer covering a cell culture. The measurement set-up reveals that, when confluent, a conventional culture of adherent cells, although exposed to the constant oxygen tension of ambient air, may experience pericellular oxygen tensions below the level required to sustain full oxidative metabolism. Depletions reported are even more prominent and potentially aggravating when the cell culture is incubated at reduced oxygen tensions (down to around 4% oxygen). Our results demonstrate that, if the pericellular oxygen tension is not measured, it is impossible to relate in vitro culture results (for example, gene expression to the oxygen tension experienced by the cell), as this concentration may deviate very substantially from the oxygen concentration recorded in the gas phase.

How much medium do you use for cell culture? Medium volume influences mineralization and osteoclastogenesis in vitro

“…Glucose was the principal energy source required for bone degradation (29), and hypoxia-inducible factor 1 alpha was stabilized in osteoclasts, leading to osteoclast activation (30). This suggests that the differentiation of large multinuclear osteoclasts may be influenced by the glucose content, and also by low concentrations of oxygen in the medium (6,7). Notably, the total number of large osteoclasts (≥8 nuclei) was observed to increase in a medium volume-dependent manner.…”

“…Furthermore, the rates of lactate reuptake appear to be highly dependent on the culture medium volume, indicating there may be a volume-induced stimulation of oxidative lactate metabolism (3). In addition, the concentration of oxygen in the medium has been demonstrated to decrease in line with increasing medium depth (6,7). Collectively, this indicates that the medium volume may impact a variety of cell culture factors.…”

“…This result indicates that osteoblast mineralization may be promoted in an autocrine manner, regardless of the glucose content. Previous studies have also demonstrated that oxygen concentrations in the culture media decrease in line with increasing medium depth (6,7). The media volumes of 0.4, 0.6, 0.8, 1.0, 1.5 and 2.0 ml in a 24-well plate (2 cm 2 /well) correspond to media depths of 2, 3, 4, 5, 7.5 and 10 mm, respectively.…”

Pression partielle en oxygène et culture de cellules cancéreuses

“…C'est ainsi que les cellules cultivées dans les conditions « standard » actuelles sont bien soumises à chaque changement de milieu à un apport de milieu de culture hyperoxique, mais cette hyperoxie pourra n'être que transitoire compte tenu de la consommation d'oxygène par les cellules. De ce fait, le milieu atteindra progressivement et transitoirement une normoxie péricellulaire en fonction du métabolisme et de la densité cellulaires, et pourra même devenir hypoxique lorsque le tapis cellulaire sera très confluent [16].…”

“…Par ailleurs, dans les sphéroïdes, d'autres gradients liés au métabolisme cellulaire (pH, lactate, glucose, facteurs de croissance…) vont s'ajouter au gradient d'O 2 . Dans le cas de l'oxygène, l'existence de ce type de gradient n'est pas retrouvée dans les cultures monocouches [16]. Ces conditions hétérogènes et imparfaitement définies au sein d'une même culture pourraient néanmoins avoir l'avantage pour l'expéri-mentateur, à la condition d'en maîtriser les paramètres, de « mimer » in vitro certains aspects retrouvés in vivo dans les masses tumorales mal perfusées.…”