# Development of a Perfusing Small Intestine-Liver Microphysiological System Device
## 3 Results and Discussion
### Configuration of Small Intestine-Liver Device
The expression of metabolic enzymes and the adhesion rate of hepatocytes vary significantly [13]; therefore, the lot selection of hepatocytes is particularly important. Additionally, co-culturing hepatocytes exhibiting weak adhesion with swiss-albino 3T3 cells when using our device is critical. Figure 3: Hepatocyte morphology 0, 2, and 4 days after seeding on the small intestine–liver device. Morphological images of 2D or 3D hepatocyte cultures. Images were taken on days 0, 2, and 4 after perfusion. White arrows represent large and flat hepatocytes. Scale bar: 200 \(\upmu\)m.  
mRNA Expression of Drug Metabolizing Enzymes and Nuclear Receptors in HiEs or Hepatocytes on Day 4 after Culturing on the Small Intestine-Liver Device (2D and 3D Cultures)  
To examine the effect of perfusion and HiE-Hepatocytes interactions on gene expression levels, we evaluated the mRNA expression levels of each cell type. The mRNA expression levels of CYP1A2 and CYP3A4 in hepatocytes were significantly increased in the 3D culture compared with those in the 2D culture (Figure 4A). Conversely, the mRNA expression levels in the HiEs of 2D or 3D cultured hepatocytes were almost equivalent. The gene expression levels of HiEs were higher than or almost equivalent to the levels of human adult small intestine cells, excluding CYP3A4 (Figure 4B). Figure 4: Expression levels of drug-metabolizing enzymes, transporter in HiEs or hepatocytes on day 4 after perfusion. **(A)** Samples were obtained from hepatocytes in HiEs–Hepatocytes interactions cultured under perfusion and hepatocytes at 0 h. HPRT was used as housekeeping gene. Bars indicate means \(\pm\) SD (\(n=3\)). The graph shows mRNA expression relative to that of hepatocytes at 0 h at considered as 1. * \(p\) \(<\) 0.05, **_p_ \(<\) 0.01 for comparison between 2D and 3D. **(B)** Samples were obtained from HiEs in HiEs–Hepatocytes interactions cultured under perfusion condition and small intestine (SI). HPRT was used as a housekeeping gene. Bars indicate means \(\pm\) SD (\(n=3\)). The graph shows mRNA expression relative to that of SI considered as 1. CYP enzyme induction is regulated by aromatic hydrocarbon receptor (AHR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR) that activate gene transcription [14; 15]. Nuclear receptors should be studied to elucidate the regulation of CYP enzyme induction. The mRNA expression levels of the AhR and PXR in hepatocytes were similar to those in hepatocytes (0 h) (Figure 5A). The mRNA expression levels of CAR in hepatocytes were approximately one-tenth the levels of that in hepatocytes (0 h). The mRNA expression levels of PXR and CAR, excluding AhR, did not significantly differ between 2D or 3D cultures. Conversely, the mRNA expression of CAR in HiEs exceeded the levels in the small intestine (Figure 5B). The mRNA expressions of CAR and PXR in HiEs for 2D or 3D culture did not change. Pleiotrophin, released from Swiss-albino 3T3 cells, is a secreted growth factor with mitogenic properties for fibroblasts, epithelial cells, and endothelial cells [12]. In addition, swiss-albino 3T3 cells produce collagen type I and are involved in the maturation of hepatocytes [13]. Therefore, the function of hepatocytes co-cultured with Swiss-albino 3T3  
Figure 5: Expression levels of nuclear receptors in HiEs or hepatocytes on day 4 after perfusion. (**A**) Samples were obtained from hepatocytes in HiEs–hepatocytes interactions cultured under perfusion and hepatocytes at 0 h. HPRT was used as a housekeeping gene. Bars indicate means \(\pm\) SD (\(n=3\)). The graph shows mRNA expression relative to that of hepatocytes at 0 h considered as 1. * \(p\) < 0.05 vs. between 2D and 3D cultures. (**B**) Samples were obtained from HiEs in HiEs–hepatocytes interactions cultured under perfusion conditions and SI. HPRT was a reference gene. Data are presented as means \(\pm\) SD (\(n=3\)). The graph shows mRNA expression relative to that of SI considered as 1.  
cells may have been enhanced by pleiotrophin and collagen type I, along with perfusion.