# Development of a Perfusing Small Intestine-Liver Microphysiological System Device
## 2 Materials and Methods
### Small Intestine-Liver Device  
A small intestine-liver device model was developed in our laboratory to serve as a multi-organ MPS (Figure 1) and common evaluation system for pharmacokinetics and safety studies.  
The small intestinal cells are seeded on cell culture inserts, which are widely used for absorption evaluation. Hepatocytes require direct seeding on the device. The device material was made of tissue culture PS (TCPS). In addition, the hepatocyte seeding area is designed to allow flat (no patterning: 2D) or spheroid-forming (with patterning: 3D) culture. The 3D plate is aligned regularly at 100 \(\upmu\)m intervals on the bottom of the device. The cell culture area is located inside the circle coated with collagen type I. Non-adherent areas of the cells are covered with super-hydrophilic poly (ethyleneglycol) molecules. The 3D plate on the device is similar to the Cell-able(r) plate system [10].  
Differentiation of Human iPS Cell-Derived Small Intestinal Epithelial Cells and Culture of Cryopreserved Hepatocytes on Small Intestine-Liver Device  
The iPS cells were differentiated over 26 days, as previously reported [11]. The small intestine-liver device was produced by Shinko Chemical Co., Ltd. (Kanazawa, Japan). At least 24 h before hepatocytes were thawed and seeded, 2D plates were coated with 0.01% collagen solution (Research Institute for the Functional Peptides, Yamagata, Japan) and washed with PBS solution. 3.0 \(\times\) 10\({}^{5}\) cells/lane of hepatocytes were seeded on the 2D plate using OptiThaw Hepatocyte Media contained in OptiThaw Hepatocyte Kit K800 (Sekisui Xenotech). The feeder cells, Swiss-albino 3T3 cells, were cultured in D-MEM (high glucose) with phenol red containing 10% FBS, 1% L-glutamine solution (Biological Industries, Beit-Haemek, Israel), 1% PS, and 1% NEMA. The small intestine-liver device plate was designed in a spheroid (with patterning: 3D) culture in the hepatocyte seeding area by Toyo Gosei Co., Ltd. (Tokyo, Japan). When swiss-albino 3T3 cells were confluent to approximately 80%, cells were detached using 0.5 \(w\)/\(v\)% trypsin-EDTA and seeded at 3.84 \(\times\) 10\({}^{4}\) cells/lane in the hepatocyte seeding area of the 3D plate. The following day, hepatocytes were seeded at 1.92 \(\times\) 10\({}^{5}\) cells/lane using OptiThaw Hepatocyte Media. Immediately after seeding, the cells were placed on a clean bench for approximately 10 min to facilitate adherence. The cells were incubated at 37 \({}^{\circ}\)C for 4 h and then replaced with OptiPlate Hepatocyte Media K8200 (Sekisui Xenotech). The cells on the 2D plate were incubated at 37 \({}^{\circ}\)C for 20 h before co-culturing with HiEs that had been differentiated for 24 d. Swiss-albino 3T3 cells were not seeded in 2D plates. The cells on the 3D plate were incubated at 37 \({}^{\circ}\)C for 72 h before co-culturing with HiEs that had been differentiated for 26 d (Figure 2).  
Figure 1: Architecture of the small intestine–liver device. The perfusion culture device consisted of a small intestine–liver-connected device plate, six-strand pump, and holder. The perfusing small intestine–liver-connected device plate contained a six-strand well. Adapters are available for the device as various types of cell culture inserts can be set in the small intestine culture section of the six-strand well plate. To prevent cells from drying, there is a groove in the gap between each well where sterile water can be added. A cassette-type set is inserted in the small intestine culture section of the small intestine–liver device body. The hepatocyte culture section comprised two culture types, with or without patterning. The plate with patterning allows for the combination of co-culture with other cells and 3D culture. The small intestine–liver device was perfused with culture medium at a flow rate of 172 \(\mu\)L/h. The holder was designed to ease mobility and prevent contamination.