A method for the long-term cultivation of mammalian cells in the absence of oxygen: Characterization of cell replication, hypoxia-inducible factor expression and reactive oxygen species production

Differential expression of cytokines and receptor expression during anoxic growth

“…As previously reported, we found no differences in HIF1α expression measured, i.e., at day 3, cells were HIF1α positive and at day 10, cells were negative for HIF expression [ 3 ]. ROS production at day 10 anoxic cultivation was also confirmed using the cell permeant reagent 2′,7′-dichlorofluorescein diacetate (DCFDA) according to manufacturer’s specifications (Abcam), as previously described [ 3 ]. Chemokine levels were measured using Milliplex ® MAP Human Cytokine/Chemokine Magnetic Bead Panel and Bio-Plex MAGPIX Multiplex Reader according to manufacturer’s protocol.…”

“…The IL-6/IL-8 mediated pathway activations result in cell phenotypic alteration with display of dendritic protrusions by monolayer cells and accompanying increased cell migration. In our initial study detailing cell characteristics upon long-term growth (17 days) in the absence of oxygen, we observed cell morphologic changes similar to that described for human fibrosarcoma (HT1080) cells and triple negative metastatic breast cancer (MDA-MB-231) cells in high cell density three dimensional (3D) cultures [ 1 – 3 ]. In addition, these studies indicate that the secretome profiles of HT1080 cells and MDA-MB-231 are altered during the metastatic process [ 1 , 2 , 4 – 6 ].…”

“…To determine whether HeLa 229 cells can engage in anaerobic synthetic metabolism to produce members of its secretome, and if that production differs from normoxic culture expression, culture supernatants (n = 19) from cells were tested after 3 and 10 days’ incubation in the absence of oxygen. To confirm metabolic status, hypoxia inducible factors 1α ( HIF1 α) mRNA and protein expression at day 3 anoxic cell incubation (glycolytic fermentation, HIF1α positive), and day 10 anoxic cell incubation (anaerobic respiration, HIF1α negative) were performed and confirmed, as previously described [ 3 ]. As previously reported, we found no differences in HIF1α expression measured, i.e., at day 3, cells were HIF1α positive and at day 10, cells were negative for HIF expression [ 3 ].…”

“…To confirm metabolic status, hypoxia inducible factors 1α ( HIF1 α) mRNA and protein expression at day 3 anoxic cell incubation (glycolytic fermentation, HIF1α positive), and day 10 anoxic cell incubation (anaerobic respiration, HIF1α negative) were performed and confirmed, as previously described [ 3 ]. As previously reported, we found no differences in HIF1α expression measured, i.e., at day 3, cells were HIF1α positive and at day 10, cells were negative for HIF expression [ 3 ]. ROS production at day 10 anoxic cultivation was also confirmed using the cell permeant reagent 2′,7′-dichlorofluorescein diacetate (DCFDA) according to manufacturer’s specifications (Abcam), as previously described [ 3 ].…”

“…Chemokine levels were measured using Milliplex ® MAP Human Cytokine/Chemokine Magnetic Bead Panel and Bio-Plex MAGPIX Multiplex Reader according to manufacturer’s protocol. HeLa 229 cells were prepared and cultured in the absence of oxygen, as previously described [ 3 ]. Briefly, HeLa 229 cells that were seeded normoxically (5% CO 2 in air) for 24 h in growth media (DMEM: 4.5 g/L glucose, 10% FBS, 50 µg/mL gentamicin; 1.68 × 10 5 cells/well of a 24 well plate) were then placed in an anaerobic chamber (Whitley A35), and medium was immediately replaced with de-gassed PS-74656 medium.…”