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A summary of published physioxic cell culture conditions

Technical Note

Physiological Oxygen

Using physiologic oxygen levels for cell culture is crucial because it more accurately replicates the natural environment of most cell types, leading to healthier and more functional cells. Culturing cells at atmospheric oxygen (21%) can cause oxidative stress and alter gene expression, skewing experimental outcomes. In contrast, physiologic oxygen levels (typically 1-8%) reduce cellular stress and align gene expression with in vivo conditions, enhancing cell viability, proper differentiation, and metabolic activity. This approach results in more biologically relevant data, improving the reliability of studies in areas such as drug development, stem cell research, and cancer biology.

Published Data

Numerous studies have identified differences in the behavior and function of many cell types when incubated at 21% oxygen compared to more physioxic levels. The table below summarizes published data on various cell lines and primary cell types, showing the tested concentrations of oxygen used in the study and the experiment results. The publications listed are but a small handful of all the publications on the topic and is by no means comprehensive. It is important to note in some cases, published results may contradict each other, so careful consideration and further evaluation may be required.


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Cell Line/Primary Cell Oxygen Levels Results Citation
201B7 2.5%, 5% ↑ colonies of uniformly smaller size were observed at 2.5% and 5% O2 than at 20% O2. Western blot analysis showed that the iPS cells cultured at 2.5% and 5% O2 expressed significantly more Nanog but less 53BP1 than those cultured at 20% O2. Some genes involved in cellular and metabolic processes were amplified in the low oxygen culture, particularly at 2.5% O2. 1
253G1 2.5%, 5% ↑ colonies of uniformly smaller size were observed at 2.5% and 5% O2 than at 20% O2. Western blot analysis showed that the iPS cells cultured at 2.5% and 5% O2 expressed significantly more Nanog but less 53BP1 than those cultured at 20% O2. Some genes involved in cellular and metabolic processes were amplified in the low oxygen culture, particularly at 2.5% O2. 1
ASC (adipose tissue-derived stromal cells) 2% 2% O2 ↑ proliferation with an approximately six- to sevenfold higher ASC expansion over 6 weeks. ↑ expression of Oct3/4 and Nanog (stem-cell marker) and secreted growth factors, promoting the maintenance of stem cells. 2
bEnd3 5% Attenuated reoxygenation-induced ROS production at 18% O2 in response to sulforaphane but not at 5% O2 3
C2C12 5% Differential effects of Resveratrol on H2O2 production, proliferation, and mitochondrial network dynamics 4
C2C12 5% Differential H2O2 production, metabolism, and mitochondrial network dynamics 5
C3A 5% Hyperoxia increases hepatocyte differentiation in HepaRG and C3A cells. In contrast, hypoxia (5%) maintains stem cell characteristics and inhibits hepatic differentiation of HepaRG cells. 6
Clonal primitive neural stem cells (pNSC) 4% Low (4%) oxygen ↑ survival of pNSCs by inhibiting apoptosis-inducing factor (AIF)-dependent cell death, whereas pNSCs undergo both AIF- and caspase-mediated cell death in 20% oxygen. Low (4%) oxygen had 10-fold greater colonies and colony size was 48.7% larger than at normoxic conditions. 7
HaCaT 2% Differential regulation of skin barrier and dermal network-related genes induced by glycolic acid, gluconolactone, and salicylic acid 8
HCT116 5% ↓ apoptosis induced by doxorubicin 9
HEK293 3% Rounder mitochondria at 18% O2 10
HepaRG, C3A 5% Hyperoxia increases hepatocyte differentiation in HepaRG and C3A cells. In contrast, hypoxia (5%) maintains stem cell characteristics and inhibits hepatic differentiation of HepaRG cells. 6
HepG2 3%, 8% ↑ hepatotoxicity , differential regulation of phase I and II enzymes induced by acetaminophen, ↑ hepatotoxicity induced by cyclophosphamide and aflatoxin B 11
Hs68 2% Differential regulation of skin barrier and dermal network-related genes induced by glycolic acid, gluconolactone, and salicylic acid 8
HT1080 0.2% ↓ invasive ability under low oxygen conditions 12
Huh-7 5% ↑ mitochondrial footprint at 18% O2, ↓ basal and maximal OCR at 18% O2 13
Human adult cardiac stem cells 5% Low 5% O2 culture doubled the cell production and markedly diminished the frequency of aneuploidy. Cells expanded in 5% O2 showed lower intracellular levels of reactive oxygen species, less cell senescence, and higher resistance to oxidative stress than those grown in 20% O2, although the expression of stem cell antigens and adhesion molecules was comparable between groups, as was the paracrine secretion of growth factors into conditioned media. In vivo, the implantation of 5% O2 cells into infarcted hearts of mice resulted in greater cell engraftment and better functional recovery than with conventionally cultured cells. 14
Human chondrocytes and Mesenchymal Stem Cells 5% Chondrocytes senesced after 25-30 PD when they were cultured under standard conditions, however, the same cells cultured in 5% O2 grew to 60 population doublings. High O2 conditions were similarly detrimental to the growth of MSC populations. MSCs cultured in high O2 barely exceeded 10 PD before the cells senesced, but the same populations cultured in low O2 grew to approximately 20 PD. 15
Human corneal endothelial cells 2.5% ↑ ECAR at 2.5% O2 16
Human Mesenchymal Stem cells 3% ↑ Oxygen consumption rate (OCR)/extracellular acidification rate (ECAR) ratio at 18% O2, ↑ expression HIF targets involved in glucose metabolism at 3% O2, growth of hMSC at 20% O2 ↓ lifespan, ↑ oxidative stress and the rate of telomere shortening 17
human neonatal foreskin fibroblasts 4% ↓ Glutathione (GSH) depletion and loss of type I cells at 18% O2 but not at 4% O2 in response to Quercetin 18
Human Pluripotent Stem cells 5% ↑ OCR/ECAR ratio at 18% O2, ↑ glycolytic intermediates at 3% O2, ↑ expression HIF targets involved in glucose metabolism at 3% O2 19
IMR90 5% ↓ apoptosis induced by doxorubicin 9
iTreg (induced CD4+ CD25+ Foxp3+ regulatory T cells) 5% Low oxygenation (5%)↑ the number of generated iTreg cells and relative abundance. 20
LNCaP 5% ↓ mitochondrial footprint at 18% O2, ↓ mean network size 18% O2, ↓ basal OCR at 18% O2, ↑ maximal OCR at 18% O2 13
MDA MB231 5% ↑ invasion at 5% oxygen conditions 12
MCF7 5% ↓ apoptosis induced by doxorubicin 9
MCF7 3% Elongated mitochondria at 18% O2 10
MCF7 3-8% ↓ metabolic activity at 18% O2, compared to 8% O2 10
MCF7 5% ↓ basal and maximal OCR at 18% O2 13
Mouse Hepatocytes 5%, 10% ↓ hepatotoxicity, ↓ mROS and RNS production induced by acetaminophen 21
Mouse Mammary Tumors 3-5% ↓ Cytotoxicity at low O2 with paclitaxel, alpelisib, and erlotinib 22
Neutrophils 3% Hypoxia causes a profound but reversible inhibition of neutrophil apoptosis. This effect was specific for hypoxia and mediated by a ferroprotein-sensing mechanism characteristic of PHD-regulated events in other cell types. 23
Patient-derived Melanoma 6% ↑ Ki-67-positive cells at low O2, reduction of VEGF, PCG-1α, and SLC7A11 levels at low O2 induced by vemurafenib and trametinib 24
PC-3 5% Differential effects of Resveratrol on H2O2 production, proliferation, and mitochondrial network dynamics 4
Rat hippocampal neurons 9% After 3 days in culture, neurons with processes in 9% oxygen were more than double those in normal oxygen. 25
Rat oligodendrial precursor cells (OPC) 5% Low oxygen (5%) ↑ cell survival and morphological complexity compared with 21% oxygen. High oxygen ↓ cell proliferation and the expression of transcription factors important for the regulation of oligodendroglial development.  26
OLN93 5% Low oxygen (5%) ↑ cell survival and morphological complexity compared with 21% oxygen. High oxygen ↓ cell proliferation and the expression of transcription factors important for the regulation of oligodendroglial development.  26
Rat primary cortical neurons 5% ↑ glucose uptake at 5% O2, ↓ glucose oxidation at 5% O2, ↑ lactate levels at 5% O2 27
Rat primary neurons 2%, 5% Globular-shaped mitochondria at 18% O2 (versus elongated at 2% and 5% O2), ↓ mitochondrial network size, mitochondrial fraction, and mitochondrial perimeter at 18% O2 28
RAW 264.7 5% ↓ production of inflammatory mediators at low O2 in response to LPS, ↓ expression of Nrf2 targets and antioxidant response at low O2 in cells treated with dimethyl fumarate 29
SaOS2 5% ↑ mitochondrial footprint at 18% O2, ↓ mean network size 18% O2, ↓ maximal OCR at 18% O2 13
SH-SY5Y 5% ↑ cytotoxicity at low O2, inhibition of ATP synthesis with rotenone at low O2 30
SW480 10% ↑ proapoptotic effect at low O2, ↑ antiproliferative effect at low O2 with teriflunomide, ↑ antiproliferative effect at low O2 with oxaliplatin 31
SW620 10% ↑ proapoptotic effect at low O2, ↑ antiproliferative effect at low O2 with teriflunomide, ↑ antiproliferative effect at low O2 with oxaliplatin 31
T cells 5% ↑ proliferation rates for T cells stimulated and grown under 5% O2 and a mean of 5.8-fold greater total expansion, 2.9-fold ↓ in apoptotic cells. 32
T cells 5%, 10% ↓ T cell proliferation in response to CD3/CD28 crosslinking at low oxygen (5% or 10%). ↓ response to Con A stimulation at 5% or 10% oxygen, whereas the response to a second well known mitogen, PHA, is comparable at all oxygen levels. 33
THP-1 5% Low 5% O2 ↑ the rate of phorbol ester-induced differentiation of THP-1 cells into macrophage-like cells as well as the metabolic activity of both undifferentiated and PMA-differentiated THP-1 cells. 34
Tubular Endothelial Cells 3% Low oxygen ↑ growth by cell proliferation and induces tubulogenesis, endothelial cell differentiation, and vasculogenesis in metanephric kidneys in culture.  35
U2OS 5% ↓ apoptosis induced by doxorubicin 9
U87MG 9% ↓ cytotoxicity at low O2 induced by camptothecin 36
U87MG 3% Rounder mitochondria at 18% O2 10
U87MG 3-8% ↓ metabolic activity at 18% O2, compared to 8–3% O2 10

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Citations

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