Cell culture chamber with gas supply for prolonged recording of human neuronal cells on microelectrode array

Abstract

Background: Typically, live cell analyses are performed outside an incubator in an ambient air, where the lack of sufficient CO₂ supply results in a fast change of pH and the high evaporation causes concentration drifts in the culture medium. That limits the experiment time for tens of minutes. In many applications, e.g. in neurotoxicity studies, a prolonged measurement of extracellular activity is, however, essential.

New method: We demonstrate a simple cell culture chamber that enables stable culture conditions during prolonged extracellular recordings on a microelectrode array (MEA) outside an incubator. The proposed chamber consists of a gas permeable silicone structure that enables gas transfer into the chamber.

Results: We show that the culture chamber supports the growth of the human embryonic stem cell (hESC)-derived neurons both inside and outside an incubator. The structure provides very low evaporation, stable pH and osmolarity, and maintains strong signaling of hESC-derived neuronal networks over three-day MEA experiments.

Comparison with existing methods: Existing systems are typically complex including continuous perfusion of medium or relatively large amount of gas to supply. The proposed chamber requires only a supply of very low flow rate (1.5ml/min) of non-humidified 5% CO₂ gas. Utilizing dry gas supply makes the proposed chamber simple to use.

Conclusion: Using the proposed culture structure on top of MEA, we can maintain hESC-derived neural networks over three days outside an incubator. Technically, the structure requires very low flow rate of dry gas supporting, however, low evaporation and maintaining the pH of the culture.

Keywords: Cell culture environment; Human embryonic stem cells; Long-term culture; MEA; Neuronal cells; Osmolarity; PDMS; pH.