Mini-incubator For Prolonged Cell Culture And Hypoxia Studies Outside An Incubator
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1
Tampere University of Technology, Department of Automation Science and Engineering, Finland
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2
University of Turku, Department of Medical Biochemistry and Genetics Institute of Biomedicine, Finland
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3
Tampere University of Technology, Department of Automation Science and Engineering, Finland
Motivation
To mimic normal human body conditions in vitro, cells should be cultured in a biomimetic and controlled environment. Typically, living cells are cultured inside an incubator with 5% CO2 and atmospheric O2 concentration (~20%). However, this concentration is not physiologically relevant for most of the human tissues in vivo. For example, for the brain tissue, the normoxia (normal O2 concentration for this specific tissue) is from 0.5% to 7%, for eye from 1 to 5%, and for cancer cells it is near 0%. Moreover, it has been shown that low oxygen conditions can promote growth and influence differentiation of stem cells in vitro. Furthermore, constant culture conditions are difficult to maintain outside an incubator. For example, during the prolonged extracellular recordings with microelectrode arrays (MEA) altering gas concentrations, temperature variations, and evaporation are typical issues. That leads to the variation of pH and molecular concentrations of media. Therefore, to maintain the good culture conditions cells should be cultured in a biomimetic and controlled environment.
We have developed a cell culture platform (See Fig. 1 A and B)) that provides controlled environment for prolonged hypoxia/normoxia studies outside a traditional incubator, e.g. during the MEA recordings on MEA amplifier (Fig. 1C) or on a glass plate on hot plate. The platform maintains the pH, temperature and constant gas concentration, for both 5% CO2 and hypoxia conditions. Cell culture chamber material enables gas exchange but keeps water vapour inside the chamber.
Material and Methods
We have developed a customized mini-incubator platform (See Fig.1) where we can study different cells in controlled truly hypoxic (low oxygen) conditions. Platform is based on the earlier studies were we demonstrated the enhanced differentiation of the human based neuronal cells in a similar structure [1]. Platform consist of (1) a culture substrate (e.g. MEA, Petri dish, or glass plate), (2) a 1-well cell culture chamber, (3) a plastic lid to seal the chamber from contaminations, and (4) a plastic cover to created mini environment around the chamber (See Fig 1A). Analysing the cells outside an incubator, only connection required is a supply of very low flow rate (5 ml/min) of non-humidified (dry) gas to maintain the gas environment of the culture.
In this study we demonstrated our mini-incubator in hypoxic conditions using HeLa carcinoma cells in low oxygen (1% O2, 5% CO2, 94% N2) environment. Hypoxic cancer cells have acquired genetic changes that enable them to proliferate under these conditions. The most often used marker for hypoxia is hypoxia inducible factor 1 alpha (HIF-1a). Under normal oxygen concentration (21% O2) HIF-1a is constantly degraded but in reduced O2 conditions it is stabilized and thus can act as a transcription promoter. We compared the cancer cell cultures in normal CO2 incubator, hypoxia chamber (Baker, Invivo2 400), and our mini-incubator platform.
We also measured the oxygen concentration in the cell culture chamber (without cells) utilizing in-house made a non-invasive optical sensing method for dissolved oxygen. The technology is based on the oxygen dependent fluorescence lifetime of PtOEPK dyes embedded in a dilute concentration (<1%) in a thin (<1 µm) polystyrene film deposited on culturing glass plates. This study was done supplying non-humidified pure N2 gas and air to the mini-incubator with flow rate of 5ml/min.
Results
We showed that we can maintain the pH, temperature, and create truly hypoxic condition for 24h experiments with HeLa cells. HIF-1a induction (as proof of hypoxia) and actin blot as loading control can be seen in western blots in Figure 1D. This demonstrates that in our mini-incubator platform (Fig. 1D, 6) the hypoxic conditions are equal to hypoxia chamber (Fig. 1D, 1 and 2) conditions. In comparison, HIF-1a induction is not expressed in the same structures in standard CO2 incubator (Fig. 1D, 4) and outside incubator without gas supply (Fig. 1D, 3 and 5). pH was also maintained at 7.21 in our platform as it was 7.48 in the CO2 incubator and 7.20 for the fresh medium.
We also showed that we can create truly hypoxic conditions in the cell culture chamber (See Fig 1E) utilizing in-house made non-invasive optical sensing method. This demonstrates the efficiency of the platform. Target level of gas concentration can be reached and time constant for step response from air saturation to pure N2 is 1.7 hours. The dynamics is approximately same compared to commercial hypoxia chamber (measured from water, data not shown).
Conclusions
Low oxygen concentration is a normal condition for most of the cells in vivo. Therefore, it is important to develop tools to study cells in these biomimetic conditions. Here we demonstrate the functionality of our mini-incubator for hypoxia studies. Truly hypoxic conditions can be maintained similarly as inside a commercial hypoxia chamber which was shown with HIF-1a induction of cells. The same system can also be utilized for prolonged MEA and microscopy experiments either in hypoxia conditions or on normal 5% CO2 conditions.
References
[1] J. Kreutzer, L. Ylä-Outinen, P. Kärnä, T. Kaarela, J. Mikkonen, H. Skottman, S. Narkilahti, and P. Kallio, “Structured PDMS Chambers for Enhanced Human Neuronal Cell Activity on MEA Platforms,” J. Bionic Eng., vol. 9, no. 1, pp. 1–10, Mar. 2012.
Figure Legend
Figure 1: A-C) Customized cell culture platforms for prolonged experiments outside the incubator. D) Western plots of HIF-1a (as proof of hypoxia) and actin (as loading control) from cultures in (1-2) hypoxia camber, (3,5) our platforms outside incubator without gas, (4) culture inside normal CO2 incubator, and (6) our platform outside incubator with gas (1% O2) supply. E) Oxygen measurement from cell area in our mini-incubator. Time constant is 1.7h.
Keywords:
pH,
temperature,
hypoxia,
HIF-1α,
Osmolarity,
HeLa,
Normoxia,
Gas supply,
carcinoma cells
Conference:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016.
Presentation Type:
Poster Presentation
Topic:
MEA Meeting 2016
Citation:
Kreutzer
J,
Rantanen
K,
Välimäki
H,
Lekkala
J,
Jaakkola
P and
Kallio
P
(2016). Mini-incubator For Prolonged Cell Culture And Hypoxia Studies Outside An Incubator.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00043
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Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
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Correspondence:
Dr. Joose Kreutzer, Tampere University of Technology, Department of Automation Science and Engineering, Tampere, Finland, joose.kreutzer@tuni.fi