## 1 Introduction  
Mixed gas is one of the essential conditions for the development of mammalian cells in vitro, and, normally, the main components of the mixed gas are oxygen (O\({}_{2}\)), carbon dioxide gas (CO\({}_{2}\)), and nitrogen (N\({}_{2}\)). CO\({}_{2}\) plays a key role in regulating and maintaining the pH of the culture medium within the optimal range of 7.2-7.4 [1, 2]. Generally, the standard concentration of CO\({}_{2}\) in mixed gas is 5%. However, different cells have varying demands for oxygen at different stages of development [3, 4]. For instance, embryos grow best under 6% O\({}_{2}\) before the early blastocyst stage but require over 20% O\({}_{2}\) after the gastula stage [5, 6]. Oxygen availability also influences organogenesis and tissue development [7, 8]. The early development of organoids often relies on physiological hypoxia, while in later phases of growth, a sufficient supply of oxygen is required to support proliferation, final differentiation, and maturation [9, 10]. Therefore, it is of great significance to support wide-range oxygen concentration regulation for an excellent cell culture system.  
In cell laboratories, two types of incubators are commonly used for cell culture: CO\({}_{2}\) incubators and tri-gas incubators. CO\({}_{2}\) incubators utilize CO\({}_{2}\) as the sole gas source, while tri-gas incubators use a combination of CO\({}_{2}\), O\({}_{2}\), and N\({}_{2}\). CO\({}_{2}\) incubators typically allow for adjusting the CO\({}_{2}\) concentration from 0-20%, regardless of the O\({}_{2}\) level, suitable for general cell culture requirements. On the other hand, tri-gas incubators offer the flexibility to adjust both CO\({}_{2}\) and O\({}_{2}\) concentrations, ranging from 0 to 20% and 0 to 90%, respectively,catering to the needs of hypoxia and hyperoxia cell culture. Both types of incubators have an internal volume of approximately 180 L, resulting in a gas concentration recovery time of over 60 min after opening and closing the door, leading to gas environment disruption and high gas consumption [11].  
In recent years, with the increasing requirements for specialization and customization in cell culture, multi-channel incubators have become more and more popular [12; 13; 14]. These incubators can consist of either single or multiple small culture chambers, offering superior temperature and gas control capabilities due to the smaller interior volume of each chamber. To maintain the internal gas environment of the chamber, a small flow of gas is usually used for continuous gas supply. One common approach involves releasing mixed gas into the chamber and then venting it into the atmosphere, such as Genea Biomedex Geri [15]. As a result, this method results in significant gas consumption over time. A more efficient alternative is to circulate the gas within the system for reuse, such as ESCO medical Miri TL [16]. Although the circulation method can reduce gas consumption, it imposes higher demands on gas control and still represents relatively high levels.  
To establish an ideal gas environment to meet the hypoxia and hyperoxia requirements of cell culture, we propose a tri-gas supply system and method for the multi-channel incubator. The system allows for gas circulation inside to supply gas to multiple chambers simultaneously and achieves control of gas concentration through a quantitative step-by-step adjustment strategy. This method enables the system to achieve a wide range of gas concentrations with low gas consumption while maintaining high control accuracy. Consequently, we offer an exceptional gas system for cell culture that can accommodate nearly all applications.