Development of charged nanobubble-containing media and their effects on iPSC-derived cell cultures

Nanobubbles (NBs) are gas-filled, spherical structures less than 1 m in diameter that exhibit unique physicochemical properties, including high surface charge and long-term stability. These features distinguish NBs from larger bubbles and enable diverse applications in fields such as environmental engineering and medicine. However, their use in biological contexts, particularly under neutral pH conditions such as those found in cell culture media, has been limited due to challenges in generating stable NBs and controlling their surface charge. In this study, we successfully generated both positively and negatively charged bulk NBs, using air as the gas type, with high zeta potentials in cell culture media (pH 7.4) for human induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) and neurons, and demonstrated the stability of NBs in the media for over a month. We quantitatively assessed their biological effects in the NPCs and neurons by evaluating the number of live cells using fluorescence microscopy. Our findings reveal that positively charged NBs have a significantly greater effect on cell viability more than negatively charged NBs, which may be attributed to electrostatic interactions with negatively charged cell membranes. This work provides the first evidence for the stable incorporation of charged NBs into iPSC-derived neural cell culture systems and establishes a framework that enables systematic investigation of their molecular and cellular functions, paving the way for their application in regenerative medicine.