Introduction Mimicking physiological conditions for hypoxic in vitro studies requires special consideration regarding the preconditioning of culture media with respect to dissolved oxygen level (DO). The proliferation and phenotype of highly differentiated cells from tissues such as tendons, ligaments and bone marrow, can alter when exposed to different oxygen levels than in their normal niche.¹ Establishment of hypoxic culture conditions within media requires specific time frames depending on final level of hypoxia desired.² In this study the equilibration time required for DO level reaching the set point was investigated. We show that DO equilibrium is not attained for several hours and may have implications on readouts in early sampling of experimental tissues such as ex vivo tendon explants.

Methods Four liquid media were tested: distilled de-ionised water; Dulbecco’s phosphate buffer saline; Dulbecco’s modified eagle medium containing 10% foetal bovine serum (FBS) (D10) and DMEM containing 15% FBS (D15) (all initially equilibrated at ambient atmospheric gas composition) were exposed to 8% O₂ (balance 5% CO₂, 87% N₂) in a humidified incubator at 37 ^oC and DO level measured up to 8 h with a Jenway 970 portable DO₂metre (Jenway, UK). Media was also stirred (S) using a magnetic bar to study the effect of mixing on oxygen dissolving rate. Real-time monitoring of the temperature was measured.

Results There was a gradual reduction of DO toward the set point (8%) although the set point was not reached until 8 h in all media (Figure 1). There was no significant different between the media in the rate of DO reduction. Stirring facilitated faster oxygen removal in the first 2 h period however this initial advantage was not observed at later time points. The presence of cells cultured in D10 did not influence the DO reduction pattern compared to S and non-stirring (NS) conditions.

Discussion In both S and NS group a minimum of 8 h pre-conditioning was required to reach the designated oxygen set point. These results show that preconditioning of culture media in hypoxic experiments and optimisation of this period is essential. This pre-conditioning period is critical as the gradual decrease of DO towards the set point can constantly alter the cellular and molecular profile of the experiment and may impact early sampling.

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Abstract 96 Figure 1

DO level in D10 media in non-stirring (NS) and stirring (S) conditions. Bone marrow mesenchymal stem cells (BM-MSCs) cultured in D10 were assayed in NS conditions only. The horizontal line denotes the 8% oxygen set point. Data is shown as mean ± SEM.

References

1. Chung DJ, et al. Res Vet Sci. 2012;92(1):66–75

2. Newby D. et al. Placenta. 2005;26(4):353–357