# Cell Stress Reduction by a Novel Perfusion-Culture System Using Commercial Culture Dish
## 3 Results and Discussion  
### Analysis of Culture Medium Pumping Methods and Evaluation of CD-Adapter Deflection  
Figure 4 shows the result of relationships between pressure on the CD-Adapter and displacement of the center membrane. The position of the center membrane at atmospheric pressure (100 kPa) was defined as 0. The average value of the three-measurement data was plotted as the displacement. As shown in Figure 4, precipitous deformation of the membrane was confirmed between 79 kPa and 120 kPa. This result means that the container volume can be easily changed by the feeding pressure of the culture medium. In contrast, precipitous deformation by positive pressure was prevented by applying the holding jig, as shown in dotted line \"\(a\)\" of Figure 4.  
Evaluation of the Expression of the Stress Marker DDIT3 in Perfusion Culture and Manual Medium Change  
The expression of the stress marker _DDIT3_ by 293T cells cultured under perfusion, manual medium change, and no medium replacement conditions is presented in Figure 5B. The results reveal that, in perfusion culture, the relative expression levels were 1.00 on the inlet side and 1.23 on the outlet side. Similarly, the relative expression levels for manual medium change were 1.94 and 2.40, respectively, and those for no culture replacement conditions were 9.20 and 8.94, respectively. Compared with the results of the perfusion condition, the no culture replacement condition and the manual medium change condition showed an approximate nine-fold and two-fold increase in _DDIT3_ expression, respectively, indicating that perfusion culture reduced the amount of stress on cells compared with conventional manual medium change. Furthermore, we demonstrated that, under all the examined conditions, _DDIT3_ expression was almost equal in cells growing on the inlet and outlet sides. In perfusion culture using the system described in this paper, we showed that the cell culture environment was uniform throughout the culture dish. In addition, cell growth was evaluated for differences in each culture condition. After three days of culture, cells reached confluence, so the cell number after two days was counted. As shown in Figure 6, there was no difference in cell proliferation rate and morphology owing to differences in culture methods. After two days of culture, there was no significant difference in cell number, so there was no device advantage for cell growth at this stage. At 1.4 \(\mu\)L/min set in the experiment, we assume that the pressure was hardly applied because the medium did not leak from the CD-Adapter even when the tube was removed from the CD-Adapter. Overall, these results demonstrate that perfusion cultures using this novel system decreased cell stress and created improved culturing environment for cells compared with conventional manual medium change techniques. Figure 4: Evaluation result of the pressure and the deflection of the center membrane. The deflection is defined as 0 at atmospheric pressure (100 kPa). Dotted line “\(a\)” is the deflection of the membrane with the holding jig. These results indicate that perfusion culture with the system reduced the stress on cells, suggesting that the disorderly medium replacement by the manual method may be the cause of stress. Figure 5: Gene expression analysis results. (**A**) Cell collection area on a culture dish. Cells cultured under each condition were collected from two halves of the culture dish: the inlet side and the outlet side. (**B**) Gene expression values of _DDIT3_ by 293T cells cultured for three days. The three experimental conditions are as follows: perfusion culturing with the system, culturing by manual medium change, and culturing without medium exchange. Each value was obtained by qRT-PCR. CD-Adapters were applied to all dishes to standardize the shape of the culture container. Figure 6: Microscopic image and cell growth rate of cultured 293T cells. (**A**) Results observed at \(\times 40\) magnification.