Mini-incubator for prolonged hypoxia studies on MEA: Effect of hypoxia for IPSC-derived cardiomyocytes

Motivation
Cells are normally cultured inside an incubator where 5% from atmospheric air is replaced by CO2. That leads to oxygen concentration around 20% (19.95%) which is not physiologically relevant environment for majority of the cells and tissues. To mimic physiological conditions in vitro, cells should be cultured in a biomimetic and controlled environment. For example, for a muscle, the physioxia (normal O2 partial pressure for this specific tissue) is from 3% to 5%, for brain tissue from 0.5% to 7%, and inside tumors it could be near 0%. It is also known that exposing cells to low oxygen atmosphere, near their natural environment in vivo (0-10% O2), the behavior of cells is drastically different. 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 challenges. That leads to the variation of pH and molecular concentrations of media due to the evaporation. Therefore, to maintain the good culture conditions cells should be cultured in a biomimetic and controlled environment.
We have developed a cell culture chamber that provides controlled environment for prolonged hypoxia, physioxia, and standard-like 5% CO2 “normoxia” studies during the MEA recordings on MEA amplifier. The chamber maintains the pH, temperature and constant gas concentration, for 5% CO2, physioxia and hypoxia conditions.
Material and Methods
A customized culture chamber is based on the earlier studies [1] and provides platform for studies in controlled truly hypoxic (low oxygen) conditions. This mini-incubator platform consist of (1) a culture substrate (e.g. MEA, Petri dish, or glass plate), (2) a 1-well cell culture chamber, (3) a lid to seal the chamber from contaminations, (4) a lid lock to seal the lid water tightly and (5) a cover to create environment around the chamber. A supply of very low flow rate (5 ml/min) of non-humidified (dry) gas is required to maintain the gas environment for the culture.
In this study we demonstrate the functionality of our mini-incubator platform. We measure the oxygen concentration in the cell culture chamber (without cells) utilizing in-house made a non-invasive optical sensing method for dissolved oxygen. We characterize the dynamics of the culture chamber for response of four different gas concentrations (0%, 1%, 5%, and 10%) of oxygen. We also characterize the evaporation and pH from the single mini-incubator chambers after three days.
We also demonstrate the functionality of the mini-incubator platform for long terms experiments and recordings on MEA using cardiomyocytes. We compare the beating rate of cardiomyocytes in hypoxic conditions (1% O2, 5% CO2, 94% N2) to “normoxic” conditions (19% O2, 5% CO2, 76% N2).
Results
We show that we can maintain the pH (~7.3) and evaporation (1 – 3 µl/h) inside the mini-incubator. Compared to standard incubator these parameters are much better maintained in our platform. pH could rise above 7.5 inside an incubator which might partly be due to the evaporation that could be also relatively high (5-7 µl/h). Especially in busy labs where incubator is frequently opened.
We also show that we can create truly hypoxic conditions in the cell culture chamber utilizing in-house made non-invasive optical sensing method. This demonstrates the efficiency of the platform. Target levels of gas concentration can be reached and rise time for step response from air saturation to different oxygen concentrations is ~1 hours.
We also demonstrate that we can maintain the cardiomyocytes on MEA amplifier for five days using 19% O2 + 5% CO2 gas supply and record stable beat rate during the period of experiment. We further show that exposing the cells to reduced oxygen concentration decrease the cardiac beating rate but recover back to normal when reoxygenated back to 19% O2. This will demonstrate that the mini-incubator platform is useful tool for prolonged experiments on MEA but also can be utilized in other heavily used lab tools like microscopy to maintain physiological environment.
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 and physioxia studies. Truly hypoxic conditions can be maintained on prolonged time on MEA.