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Cell culture techniques are used in many areas of biomedical research with significant importance to human health, including cancer biology, stem cell research, cardiovascular biology and neuroscience.
Across these diverse fields, there is a growing appreciation that in vitro cell culture should be performed under conditions that mimic the in vivo physiological environment as closely as possible. This is necessary for the accurate reproduction of in vivo cellular processes because cells react in different and complex ways, metabolically and morphologically, in response to environmental factors.
Within the living tissues of humans and other mammals, cells are typically exposed to oxygen concentrations in the range of 0.5 – 10%, depending on the tissue type – significantly lower than the 21% oxygen present in ambient air. However, limitations in the available equipment for atmospheric control necessitated classical cell culture techniques which included enrichment of atmospheric CO2 while O2 was maintained at its standard atmospheric level. This approach to in vitro culture provides conditions that can be considered “hyperoxic” for most cell types.
Culture of cells under normoxic, “physiologically appropriate” conditions require precise control of O2, CO2, temperature and humidity. One item of equipment developed to fulfil this need is the cell culture incubator (or “tri-gas” incubator), which mixes air with CO2 and N2 gasses to produce the required atmosphere [1]. However, each time the incubator door is opened, air flows in and the atmospheric gas mixture is temporarily disturbed, subsequently having to be re-established. Thus, cells are exposed to inconsistent, variable oxygen concentrations. Some tri-gas incubators have a divided or segmented inner chamber with individual doors to minimize disturbance of atmospheric conditions, but this cannot overcome the changes in gas concentrations and temperature that occur when cells are removed from the incubator for harvesting or periodic replacement of the culture medium.
When cells are exposed to low oxygen concentrations, a group of physiologically important genes such as erythropoietin and vascular endothelial growth factor are induced. These genes are transcriptionally upregulated by hypoxia-inducible factor 1 (HIF-1), a global regulator protein composed of α and β subunits. HIF-1 activity is primarily determined by hypoxia-induced stabilization of HIF-1α, which is rapidly degraded in the presence of oxygen. In recent years, multiple roles of HIF-1 in the pathophysiology of various diseases, including cancer, have been demonstrated. It is a crucial mediator of the hypoxic response and regulator of oxygen metabolism, thus contributing to tumour development and progression [4, 5]. Expression of HIF-1α is upregulated in cancer cells and promotes tumour survival by multiple mechanisms. Several compounds targeting HIF-1-associated processes are now being used to treat different types of cancer [6]. In characterizing the behaviour of tumour cells and their response to these anti-cancer compounds, it is essential to provide uninterrupted hypoxic conditions so that in vivo experimental results can accurately predict clinical efficacy.
Recent studies have also demonstrated that culture under continuous low-oxygen conditions can have beneficial effects on mesenchymal stem cells (MSCs). These stem cells have been evaluated as a potential tool to treat tissue injuries, degenerative diseases and immune disorders. This is due to their multipotent differentiation capacity, immunomodulatory effects and angiogenic / neurogenic properties. MSCs can be isolated from various tissues including bone marrow, adipose tissue, umbilical cord and dental pulp. For both research and clinical applications, in vitro culture of MSCs is required to obtain sufficient cell numbers – but poor growth rate, early senescence, DNA damage and limited survival after transplantation are significant issues in this field. A hypoxic environment can greatly improve growth kinetics and genetic stability during in vitro expansion of MSCs and can improve the success of MSC-based regenerative therapies [7].
The benefits of hypoxic incubation have been studied using dental pulp stem cells (DPSCs) - a unique type of MSC which are promising in many regenerative therapies. In one study [8], the ideal hypoxic culture environment for DPSCs was determined using specially designed culture chambers within a tri-gas incubator. Each chamber comprised a plastic box through which stable mixtures of O2, CO2 and N2 were continuously injected. Using this system to compare DPSC growth in 21% O2, 5% O2 and 3% O2, the authors reported that 5% O2 was optimal for growth, stem cell properties, and secretome trophic effect (the ability of bioactive molecules secreted by DSCs to promote cell growth and regeneration).
The studies described above demonstrate that it is possible to provide a hypoxic environment for the culture and physiological study of tumour cells and stem cells by constructing bespoke systems based on conventional cell culture incubators. However, the development of dedicated hypoxia workstations such as the Whitley Hypoxystation range has enabled more convenient, accessible and precise control of oxygen, carbon dioxide, humidity and temperature, to provide continuous, uninterrupted physiologically relevant conditions for cell culture. In particular, routine manipulations required for the maintenance of cultured cells, together with morphological, metabolic and genomic investigations, can be performed under the same stable conditions used for initial culture. The ability to manipulate cultured cells with no interruption of environmental conditions is an important step in ensuring that experimental results can replicate in vivo cell behaviour as closely as possible.
Written by DWS' Head of Science, Dr Andrew Pridmore.
References:
1. Freshney RI (2015) In: Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications 7th edn, Ch. 8, 125–148 (Wiley Blackwell).
2. Pattappa G et al. (2012) Continuous and uninterrupted oxygen tension influences the colony formation and oxidative metabolism of human mesenchymal stem cells. Tissue Eng Part C Methods 19, 68–79.
3. Basciano L et al. (2011) Long term culture of mesenchymal stem cells in hypoxia promotes a genetic program maintaining their undifferentiated and multipotent status. BMC Cell Biol. 30, 12.
4. Sun J and Zhu S (2023) Identifying the role of hypoxia-related lncRNAs in pancreatic cancer. Genomics 115, 110665.
5. Zhao Y et al. (2024) HIF-1α signaling: Essential roles in tumorigenesis and implications in targeted therapies. Genes Dis. 11, 234–251.
6. Qannita RA et al. (2024) Targeting Hypoxia-Inducible Factor-1 (HIF-1) in Cancer: Emerging Therapeutic Strategies and Pathway Regulation. Pharmaceuticals 17, 195. doi: 10.3390/ph17020195.
7. Haque N et al. (2013) Hypoxic Culture Conditions as a Solution for Mesenchymal Stem Cell Based Regenerative Therapy. Scientific World Journal, Volume 2013, Article ID 632972. doi: 10.1155/2013/632972.
8. Ahmed N et al. (2016) The effects of hypoxia on the stemness properties of human dental pulp stem cells (DPSCs) Scientific Reports 6, Article number: 35476.
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