Rapid and effective modulation of dissolved oxygen levels
in cell culture media for improved hypoxia studies
Param Mohan, Tina P. Dale, Saniya Gupta, Alasdair G. Kay, and Nicholas R Forsyth
Guy Hilton Research Centre, Institute of Science and Technology in Medicine, Keele University, UK, ST4 7QB.
[email protected]
Cell-based hypoxia studies are reliant on close control of O2 concentration in the surrounding environment.
Exposure to ambient O2 (21%) concentrations for short or sustained periods of time can result in
confounding results due to oxygen toxicity. Reduced O2 cell culture incubators and workstations are
examples of adopted measures designed to reduce this oxygen toxicity and to promote consistency of
observation. We have identified a further layer of complexity to this scenario – culture media. With a starting
O2 concentration of approximately 10% O2, a high salt content, and high operating temperature (37oC) it can
take culture media approximately 3 hrs to reduce to 2% O2 in the presence of cells and essentially never in
the absence of cells and in large volumes.
To overcome this deficiency we have developed a system for the rapid and effective modulation of dissolved
oxygen levels in cell culture media. We have engineered a simple agitation device within a refrigerated unit
with both O2 and CO2 gas level control. The system will reproducibly deoxygenate 500 ml bottled volumes of
culture media with sterility retained through vented cap incorporation. Deoxygenation of large medium
volumes to defined set-points (0.5%, 1%, 2%, etc) is readily achievable within 90 minutes.
Controlled experimentation to evaluate human mesenchymal stem cell (hMSC) recovery from bone marrow
aspirate in a 2% O2 workstation revealed a 50% improvement in both colony-forming unit-fibroblasts (CFU-F)
and in absolute cell numbers when pre-deoxygenated (2% O2) medium was used as compared to medium
stored in 2% O2 workstation prior to its use. The dramatic improvement in cell number was accompanied by
widespread global transcriptional alterations at both transcript and exon levels as identified through Exon
1.0ST microarray analysis. This included 167 and 71 unique upregulated and down-regulated transcripts as
compared to 2% O2 workstation-alone recovered hMSC when compared to 21% O2 recovered hMSC.
In summary we have developed a system for the rapid and effective modulation of dissolved oxygen levels
in cell culture media. The application of this technology resulted in improved hMSC CFU-F and cell number
recovery which was accompanied by widespread global transcriptional changes.
HypOxyCool prototype
SCI-tive and dissolved oxygen sensor
Rapid deoxygenation to desired use point in HypOxyCool prototype
140
15
14
13
12
11
10
9
8
120
Cell culture media has variable dissolved O2 levels
100
DMEM
450
IMEM
40
350
20
300
250
0
0
200
1
2
3
4
5
6
7
8
9
MEM
IMEM
7
6
5
4
3
2
1
0
60
MEM
400
Media volume (ml)
RPM
80
500
10
0
Dissolved Oxygen Concentration after 3 Hours
1
2
150
100
0
2
4
6
8
10
12
14
16
18
3
Time (hours)
4
5
6
HypOxyCool unit set at 1% O2, variable rpm, vented cap, 500ml media bottle.
Measurements performed inside SCI-tive operating at 37oC, 2% O2
50
0
DMEM
Dissolved Oxygen Concentration
(%)
120 rpm after 2 hours
HypOxyCool deoxygenation curve
20
Dissolved oxygen (% )
Pre-deoxygenated media enhances hMSC recovery and stabilises transcriptome
120
37oC
4oC
500
6 Hours
400
400
350
350
300
250
200
150
0
0
16
18
400
400
350
350
300
250
200
150
0
0
18
20
3500000
3000000
*
60
40
* p=0.04
2500000
2000000
1500000
1000000
** p=0.03
500000
0
2% O2
65 genes
20
0 hrs
6 hrs
hMSC recovered from bone
marrow - CFU-F quantified and
transcriptome determined at P0.
2%WS O2
65 genes
41
6
10
8
39
10
Genes Upregulated in 2% O2 = Downregulated in 21% O2
2% O2
144 genes
2%WS O2
160 genes
17
44
54
29
56
33
63
138
2%PG O2
91 genes
2%PG O2
254 genes
150
50
6
8
10
12
14
16
Dissolved Oxygen Concentration (% )
2% O2 SciTive
200
50
4
18
250
100
2
6
8
10
12
14
16
Dissolved Oxygen Concentration (% )
300
100
0
4
450
Media Volume (ml)
AGITATED
Media Volume (ml)
450
2
500
6 hrs
**
Genes Upregulated in 21% O2 = Downregulated in 2% O2
0
20
0 Hrs
500
21% O2
0
150
50
6
8
10
12
14
Dissolved Oxygen Concentration (%)
2% O2 SCI-tive + 2% O2
HypOxyCool Media
4000000
200
50
4
80
2% O2 SCI-tive
20
250
100
2
6 hrs
300
100
0
0 hrs
450
Media Volume (ml)
Media Volume (ml)
450
STATIC
500
0 Hours
CFU per T75 Flask
Media will equilibrate to reduced O2 after 6 hours at 4oC only
100
21% O2
2% O2 SCI-tive + 2% O2
HypOxyCool Media
Cells per T75 Flask
All dissolved O2 measurements were performed with the HI 2400
Dissolved Oxygen Bench Meter with salinity and temperature correction.
0
2
4
6
8
10
12
14
16
Dissolved Oxygen Concentration (% )
18
20
Media bottle placed into SCI-tive workstation operating at 2% O2,
opened, measured immediately and again after 6 hours.
•Optimised frequency of CFU isolation
•Optimised cell numbers (per flask)
•Greater CFU recovery with controlled rate of expansion
•Reduced transcriptional alteration
•Maintenance of in vivo profile?