# Cell Stress Reduction by a Novel Perfusion-Culture System Using Commercial Culture Dish  
[PERSON]  
1 Laboratory of Animal Molecular Morphology, Department of Animal Science, Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan; [EMAIL_ADDRESS] (R.Y. ); [EMAIL_ADDRESS] (N.H.)  
[PERSON]  
3 Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tokyo 135-0064, Japan; [EMAIL_ADDRESS]  
[PERSON]  
2 Laboratory of Animal Molecular Morphology, Department of Animal Science, Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan; [EMAIL_ADDRESS] (R.Y. ); [EMAIL_ADDRESS] (N.H.)  
[PERSON]  
2 Laboratory of Animal Molecular Morphology, Department of Animal Science, Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan; [EMAIL_ADDRESS] (R.Y. ); [EMAIL_ADDRESS] (N.H.)  
[PERSON]  
2 Laboratory of Animal Molecular Morphology, Department of Animal Science, Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan; [EMAIL_ADDRESS] (R.Y. ); [EMAIL_ADDRESS] (N.H.)  
[PERSON]  
2 Laboratory of Animal Molecular Morphology, Department of Animal Science, Graduate School of Agricultural Science, Kobe University, Kobe 657-8501, Japan; [EMAIL_ADDRESS] (R.Y. ); [EMAIL_ADDRESS] (N.  
is a method that involves completely removing the culture medium and replacing it with new media. However, in manual medium change, it is easy for waste products to accumulate and for nutrients to be replenished, which may cause cell stress owing to conditions such as acidosis, which lowers the quality of the cells [10]. Additionally, the culture medium is replaced manually within a biosafety cabinet outside the incubator, which exposes the culture to the external environment. In this way, manual medium change involves large changes in the conditions of the culture environment that may differ greatly from those in the in vivo environment in a way that is detrimental to the cells (Figure 1). On the other hand, perfusion culture, which involves continuous delivery and discharge of culture medium, easily overcomes the issues of unstable medium conditions due to the manual medium change. The most important components of the perfusion culture are specific enclosed containers for cell culture, such as microfluidic devices or flow chambers. Although the superiority has not been sufficiently assessed, perfusion culture is expected to cause lower stress levels in cells compared with manual medium change. Perfusion culture can be performed easily by setting-up those containers and the related apparatus in an incubator [11; 12]. However, in the laboratory, for retaining cells and allowing them to proliferate, commercial disposable culture dishes are widely used as containers [13]. The size of the culture dish varies for each manufacturing company. For example, the inner diameter of 35 mm culture dish from IWAKI is 35 mm, but that from Nunc is 33.6 mm. Researchers hesitate in changing the cell culturing condition formed in each dish because they have a knowledge of the cell culturing process for each culture dish. Using a different culturing container requires additional basic study for bridging the gap between conventional and new protocols. Hence, a new perfusion culturing system applicable to commercially available culture dishes is required to utilize existing resources and knowledge. To this end, several perfusion-culture systems employing commercial culture dishes have been reported in the literature, but these systems still serve as research platforms specialized for specific applications [14; 15]. RC-37 Series (Warner Instruments) and Hiper-Go (LUMITOS AG) are commercially available; however, it is not easy to change the configuration flexibly to adjust to various experimental conditions. In this study, we developed a versatile system for perfusion culture that can be easily equipped with various commercial 35 mm culture dishes.