# Development of a Perfusing Small Intestine-Liver Microphysiological System Device
## 3 Results and Discussion  
### Configuration of Small Intestine-Liver Device  
The body is composed of multi-organ systems that maintain integrated functions. When conducting safety and pharmacokinetics analyses, it is particularly important to consider the interaction between each organ. We developed an MPS small intestine-liver device that may be useful in evaluating drug bioavailability in this organ-organ system (Figure 1). The small intestine-liver device can be used to evaluate the interaction of tissues (particularly gut and liver tissues) via the perfusion system comprising six-strand pumps. This device model is an open system, which facilitates cell seeding and collection as well as substrate addition and sampling. In addition, the small intestine portion can be easily detached as a cassette. PDMS and polystyrene (PS) are commonly used materials for cell plates. PDMS is useful for biological applications because it is impermeable to water and gases while being non-toxic to cells. The major advantage of PDMS over glass and silicon is the ease  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Gene Names** & **Forward Primer Sequences** & **Reverse Primer Sequences** \\  & **(5’-3’)** & **(5’-3’)** \\ \hline _AhR_ & GCACGAGAGCCTCAGGTATCA & GTGCATTAGACTGGACCGAGTC \\ _CAR_ & TGATCAGCTGCAGAGAGAGAG & TGGATGTGTCGGATTGGGA \\ _CYP1A2_ & CTTTGACAAGAACAGGTCCG & _AGGTCCAGCTCTTCTTGGAT_ \\ _CYP2C9_ & _GACATGAAACCCTCAGCAGCTT & TGCTTGTGTwith which it can be fabricated and bonded to other surfaces. However, there is a risk of potential adsorption of medium components on the device, particularly those made of PDMS. Therefore, we selected tissue culture PS (TCPS) as the plate material. We selected two different tube materials for the model. The silicone tube was used for the driving part of six-strand pumps; owing to its excellent heat resistance, it can be autoclaved, allowing for sterilization. However, silicone may cause drug sorption. To avoid this limitation, we also used PEEK for the remainder of the tubing, as it is suitable for collecting biological samples and exhibits almost no drug adsorption or sorption. In our small intestine-liver device, mediums can be simultaneously placed in six wells to flow one way or circulate from inlet to outlet. In our experiments, we allowed the medium to circulate to evaluate the effects of co-culture with gut and liver cells. Furthermore, we designed planar (without patterning: 2D) and spheroid (with patterning: 3D) cultures in the hepatocyte seeding area. This would also reflect the effect of different hepatocyte culture methods while perfusing. Notably, 3D cultures of hepatocytes with Cell-able(r) plates enable high functional activity and long-term culture compared to 2D culture [10]. Moreover, the 3D culture of hepatocytes co-cultured with feeder cells, swiss-al albino 3T3 cells, yields more robust hepatocyte spheroid formation and higher metabolic activity [12]. We believe that the 3D culturing of hepatocytes using this device would yield sufficiently high functional activity for the evaluation of pharmacokinetics. Hepatocytes Morphology at 0, 2, and 4 Days after Seeding on Small Intestine-Liver Device (2D and 3D Cultures)  
Figure 3 shows the morphological changes in hepatocytes on days 0, 2, and 4 after 2D or 3D culture. In terms of morphology, Hepatocytes on day 4 after 2D culture were large and flat. Unattached hepatocytes tended to be flowed by perfusion for 2 days. Hepatocyte morphology on days 2 and 4 was unchanged. Hepatocytes (HC10-10) had a poor adhesion rate and could not be 3D cultured without Swiss-albino 3T3 cells. Hepatocytes with swiss-albino 3T3 cells exhibited immediate gathering in cell adhesion areas after seeding on day 0 after 3D culture. In addition, hepatocytes after 3D culture were distinguishable in a spheroid of 100 \(\upmu\)m on day 4. Therefore, we illustrated that hepatocytes (HC10-10) with Swiss-albino 3T3 cells could yield more robust spheroid formation than hepatocytes alone (HC10-10) (Figure 3).