Hypoxia-Reoxygenation Protocol for Cell Culture

Hypoxia and reoxygenation were performed using OxyGenie mini-incubator (Baker, USA), which is based on our previous studies^{26 ,48 (link),49 (link)}. The portable cell culture incubator includes a battery-operated temperature controller with a heat plate, two prefilled and replaceable gas cylinders and a flow divider to hold six individual 1-well chamber assemblies on a glass plate (Fig. 6a). The 1-well assembly on an MEA plate fits to a MultiChannel Systems signal amplifier allowing long-term recordings outside an incubator. Portability of the OxyGenie and the individual 1-well chambers enable sample preparation and collection one at a time, minimizing the cell culture exposure to ambient air.Figure 6

(a) Portable cell culture instrument include battery operated temperature controller and heat plate, two gas cylinders and flow divider allowing six individual 1-well assemblies. 1-well assembly includes 1-well chamber on plate (glass or MEA), the lid and the lid lock to seal the cell culture and to avoid evaporation, the cover and the cover lock to create and maintain the gas environment around gas permeable 1-well chamber. 1-well assembly on MEA plate can be fitted also to MCS signal amplifier and thus use it for long-term recordings outside the incubator. (b) Hypoxia and hypoxia-reoxygenation protocol.

Day before exposing cells to hypoxia, serum- and glucose-free EB medium (glucose-free DMEM (Gibco) containing 1% MEM NEAA, 1% GlutaMAX and 0.5% Penicillin/streptomycin) was changed. On the following day, the samples were loaded into pre-warmed (37 °C) OxyGenie mini-incubator and hypoxia was initiated using 0% O₂ and 5% CO₂ (hypoxic) gas. The used time periods included 6, 8, 10, 12 and 24 h. Control samples were cultured in serum- and glucose-free EB medium in a standard 5% CO₂ incubator without the lids and covers for the same time periods.
Hypoxia-reoxygenation experiments were performed similarly to the hypoxia experiments, but after 8 or 24 h hypoxia, the gas was exchanged to 19% O₂ and 5% CO₂ (normoxic) gas for 24 h. The control cells were kept in the 1-well chambers on the glass inside the incubator for the whole experiment. Samples were collected after the hypoxia or hypoxia-reoxygenation one by one to minimize the exposure to ambient air. pH was measured from cell culture medium during sample collection using Sentron SI600 pH meter with Sentron MicroFET pH probe.
With MEA plates, hypoxia-reoxygenation was performed so that first the samples were connected to normoxic gas to measure a baseline overnight, after which, hypoxia was initiated using the hypoxic gas for 24 h. After the hypoxia, reoxygenation was performed by connecting the samples again to the normoxic gas for another 24 h. Hypoxia and hypoxia-reoxygenation protocols are presented in Fig. 6b.