The Art of Inducing Hypoxia

Round 1

Reviewer 1 Report

This manuscript compares three different ways to induce hypoxia in cell cultures. The Authors used cells related to osteogenesis, but the results are extensible to many, perhaps all cell lines used to address the effects of hypoxia. Although rather technical, it may be useful for many experimenters. However, a few issues deserve assessment.

Lines 45-54. Not only I fully agree with the Authors, but also encourage them to add the consideration that performing cell cultures at 18% O2 (e.g., in room air, as in almost all the papers published until now) exposes cells to a hyperoxic environment, not certainly a “control” normoxic one, which may bias considerably the results in several instances.

Table 1. In my opinion, increasing the medium height may induce a time-dependent situation where cells are initially exposed to a normoxic situation that progressively becomes hypoxic due to the relatively slow diffusion of oxygen across the medium. This rate of deoxygenation can be calculated from the oxygen diffusion constant, the area of the liquid-air interface and  the rate of oxygen consumption. Indeed, oxygen keeps diffusing from the atmosphere above the medium thereby leading to (probably) a decay curve converging to an asymptote whose value depends not only by the medium height, but also by the number of live breathing cells and their specific oxygen consumption. Therefore, anoxic, probably dying cells breath in a different way thereby altering the oxygen decay in the medium. The Authors are invited to comment on this.

Table 3. Disadvantages of MH. Is “low induction of hypoxia” to be replaced by “slow”? Add horizontal grids to avoid confusion between two successive rows. Add a row entitled “ability to monitor PO2”.

Paragraph 2.6. If the red hypoxia reagent functions as an oxygen sensor, the Authors must provide data on its sensitivity for oxygen and linearity range. In facts, the dye color change may correspond to the oxygen tension only within a range of PO2.

GOX/CAT system. This elegant system may induce anoxia rather than hypoxia because all available oxygen is consumed if enzymes are present in excess. Anoxia is clearly a quite different condition than hypoxia because it is lethal. Also, the most recent Km and Vmax values of catalase (0.2384 mM and 13.3156 s/mM, respectively, Isolation, Fractionation and Characterization of Catalase from Neurospora crassa, Suryani1*, L Ambarsari1 and E Lindawati1 IOP Conf. Series: Earth and Environmental Science 58 (2017) 012068 doi:10.1088/1755-1315/58/1/012068) are not compatible with complete lack of H2O2 in the reaction mixture. Therefore, the GOX/CAT system may induce some ROS release that ultimately conflicts with the observed hypoxia response. Please comment.

Apparently the effects of CoCl2 of HIF-related parameters may seem minor, but they depend on the concentration used. Are the Authors sure that they used a saturating concentration?

Figure legends. The list of the various parameters must be consistent in sequence with the corresponding parameters in the figures.

The discussion is fairly too long and concerns osteogenic cell physiology. Reduce it by half at least and concentrate on the central issue of this manuscript: the art of inducing hypoxia.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Major comments

• The manuscript is missing some discussion/characterisation of HIF-2α. It is mentioned once in the discussion as ‘reacting to more physiological oxygen concentrations’, so why wasn’t HIF-2α expression determined in parallel to HIF-1α?
• What is the positive control for all the experiments? There is no comparison to a known hypoxia-inducing environment (e.g. a hypoxic chamber), so it is impossible to interpret these results.
• Is it possible that the chemical/enzymatic modifications may influence the experimental outcome in a way that is not hypoxia-related? There is no discussion/control groups that interrogate this phenomenon.
• How is the modification of oxygen concentration is never quantified in this study, e.g. by an oxygen sensing probe. Only a ‘hypoxia dye’ is used to confirm hypoxia, but there is no information/discussion supplied on what level of hypoxia this dye is supposed to detect. This is much too vague.
• Information is missing from the methods on the exposure time of the hypoxia modulations, i.e. once adding CoCl2/GOXCAT/increasing medium height, how long does it take to ‘become hypoxic’?

General comments

• Requires a general spelling/grammar check.
• Are cells mycoplasma-tested? Not in methods.
• Line 227 ‘as a second readout cells were stained with hypoxia reagent’. What was the first readout? My understanding is that this hypoxia staining is the first readout, and the HIF western is the second readout. Can you please clarify?
• Line 256 does control without stimulus mean cells under aerobic conditions/in air?
• Line 262 do you mean not significantly by CoCl₂?
• Why is a CoCl₂ group missing from Figure 4A?
• In Figure 5, why use HIF-regulated genes without a comparison to HIF-lα itself?
• Figure 2; Why does CoCl₂ not reduce oxygen levels according to the hypoxia reagent but then HIF is upregulated?
• Figure 2; Why does 5.4 mm of medium reduce oxygen levels according to the hypoxia reagent but this does not correspond with HIF stabilisation?
• Fig 3 chemokine analysis; If hypoxia induces chemokine release, why the variation across chemokines under the different hypoxic conditions (CoCl₂, MH)? It is either hypoxic or not.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

All concerns have been answered.

Author Response

We thank the reviewer for her/his review. 

Reviewer 2 Report

Thank you for addressing the majority of my concerns. There are a few things remaining.

1. Can you please supply details of the mycoplasma test used.
2. Line 168: 'when oxygen concentrations decrease below 5%
   which can be generally considered as hypoxic.' Can you please supply a reference for this? An O₂ partial pressure of 15 mmHg (20 µM concentration in solution, equivalent to 2% O₂ in the gas-phase) constitutes an approximate threshold between pathological hypoxia and physiological hypoxia. In fact, the level of oxygen in normal tissues ranges from 3-7%. 
3. You mention 'The effect of hypoxia chambers was extensively studied elsewhere so we focused on the other induction methods in comparison to these studies.' But I see no comparison in this manuscript to these 'other studies'.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf