# Cell Stress Reduction by a Novel Perfusion-Culture System Using Commercial Culture Dish
## Materials and Methods
### Evaluation of The Stress Marker DDIT3 Expression in Perfusion Culture and Manual Medium Change  
To demonstrate that stable cell culture is possible using the developed perfusion-culture system, we evaluated cell stress by measuring the expression of the cellular stress marker _DDIT3_, which is also known as CHOP (C/EBP homologous protein). CHOP is related to endoplasmic reticulum stress [17], and is involved in promoting apoptosis, ultimately causing cell death [18].  
As a preparation for the evaluation, human embryonic kidney epithelial cells (293T cells), a general culture cell line, were seeded at a density of \(2\times 10^{5}\) cells/mL in 35 mm culture plates and cultured for one day at 37 \({}^{\circ}\)C in a 5% CO\({}_{2}\) environment. Thereafter, 2 mL Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum and used as the culture medium, was added to the culture dish. This evaluation experiment was conducted under three culture conditions, including perfusion culture, manual medium change (replacing the culture medium once per day), and culturing without replacing the culture medium as a negative control. To standardize the shape of the culture container, CD-Adapters were applied to all dishes. The flow rate for perfusion culture was 1.4 uL/min, as this is the flow rate required for exchanging 2 mL of culture medium in one day. After culturing the cells for one and three days under each condition, the cells were collected by trypsin using trypsin-ethylenediaminetetraacetic acid (EDTA), and the cells cultured for two days were counted. After cell collection, total RNA was extracted by RNeasy Plus Mini Kit (74134, QIAGEN, Hilden, Germany) and cDNA was synthesized for 120 min at 37 \({}^{\circ}\)C, and the enzyme was inactivated at 85 \({}^{\circ}\)C for 5 s by High-Capacity cDNA Reverse Transcription Kit (4368814, Thermo Fisher scientific, Waltham, MA, USA) according to the protocol. _DDIT3_ expression was measured using reverse transcription polymerase chain reaction (qRT-PCR; \(n=3\)). According to the protocol, for qRT-PCR, denaturing was performed at 95 \({}^{\circ}\)C for 23 s, as well as annealing and extending at 60 \({}^{\circ}\)C for 30 s, and this was performed for 40 cycles. The primer sequences used for qRT-PCR are presented in Table 1. \(\beta\)-Actin was used as an internal standard. Cell deviation and qRT-PCR results of _DDIT3_ were calculated as standard deviation.  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Gene** & **Primer-Forward** & **Primer-Reverse** \\ \hline \(\beta\)-Actin & 5\({}^{\prime}\)-TGGATCAGCAAGCAGAGAGATG-3\({}^{\prime}\) & 5\({}^{\prime}\)-GCATTGCGGTGAGAGAT-3\({}^{\prime}\) \\ \hline DDIT3 & 5\({}^{\prime}\)-TCAGAGCTGGAACCTGAGGA-3\({}^{\prime}\) & 5\({}^{\prime}\)-CTGCCATCTGCAGTTGGA-3\({}^{\prime}\) \\ \hline \hline \end{tabular}
\end{table}
Table 1: Primers used for reverse transcription polymerase chain reaction (qRT-PCR).  
Figure 3: Deflection of the center membrane owing to liquid pumping. When pumping liquid into the culture dish, the center membrane swells, preventing the culture medium from being maintained at a constant volume in the dish. Additionally, discharging the culture medium causes the center membrane to become concave and may cause the membrane to touch the bottom of the culture dish. Thus, a holding jig was inserted into the CD-Adapter while pumping liquid into the culture dish to prevent physical swelling of the center membrane.