In vitro triple cell model in a 3D printed platform for assessing drug absorption and metabolism

Morten Leth Jepsen, Andreas Willumsen, Line Hagner Nielsen, Anja Boisen, Martin DufvaThe Danish National Research Foundation and Villum Foundation’s Center for Intelligent Drug Delivery and Sensing Using Microcontainers and Nanomechanics (IDUN), Department of Health Technology, Technical University of Denmark, Ørsteds Plads 345C, 2800 Kgs. Lyngby, Denmark.

Introduction: Orally administered drugs will often be absorbed across the small intestine followed by transport to the liver through the portal vein. Hereby, they are passing the small intestinal epithelium and endothelium. Simulating these processes in vitro with cell models spare numerous of laboratory animals. The aim of this study was to 3D print a three compartment in vitro cell system simulating tissue involved in the first pass metabolism. For the three compartments, Caco-2 cells imitate the small intestinal barrier, HUVEC cells the endothelium, and HepG2 cells the liver (Figure 1A).

Methods: 3D printed compartments were printed in Dental LT resin on a Formlabs 2 3D printer. Subsequently, the 3D prints were washed in isopropyl alcohol and cured in a Form Cure UV oven at 60°C for 2 h. Hydrogels of 5 % (w/v) gelatin, crosslinked by microbial transglutaminase, were casted into inserts by crosslinking the hydrogel at 37°C for 1 h. Subsequently, 500 µL of 105

cells/mL Caco-2 or HUVEC cells were seeded on top of the hydrogels or 105 HepG2 cells were seeded into the 100 µL hydrogels during the crosslinking step. The Caco-2 and HepG2 cultures were grown for three weeks at 37°C with 5 % CO2, whereas the HUVEC cells were seeded and settled overnight. For staining cells, they were fixed with 3 % paraformaldehyde for 30 min and stained with either phalloidin conjugated with Alexa-448 for 20 min or by primary ZO-1 rabbit antibody for 1 h and secondary goat anti-rabbit antibody for 1 h. Followed by 10 min incubation with Hoechst 33342. Fluorescent photomicrographs were obtained with a laser scanning confocal microscope.

Results: After three weeks, the Caco-2 cells on hydrogels had grown to confluence with expression of tight junctions (Figure 1B). In addition, elongated HUVEC cells covered most of the gel after settling overnight (Figure 1C), and HepG2 cells had grown into large spheroids over three weeks (Figure 1D). Growth conditions were close to in vivo, since the cell layer had a gelatin hydrogel as a growth matrix. Once the three cell compartments were individually fully grown, they were assembled for transport and metabolism studies (Figure 2). Studies were made by sampling from each compartment and subsequently, cutting out the hydrogel for microscopy.

Conclusion: The presented system can be utilized to study drug metabolism from the small intestine throughout the liver. Furthermore, the system allows for sampling at each compartment to investigate where the drug modification take place.

Figure 1: A): Setup of twelve plate insert of the three compartment system. B) Stain of tight junctions (ZO-1) in Caco-2 cells on a hydrogel. C) F-actin-green (Phalloidin) and nucleus-blue (Hoechst 33342) stain of HUVEC cells on a hydrogel. D) F-actin-green (Phalloidin) and nucleus-blue (Hoechst 33342) stain of HepG2 cells grown in a hydrogel. Scalebar: 100 µm.

Figure 2: Assembled setup with the three cells showing how a drug travels through the system. A) Intestinal compartment (light blue) with hydrogel (purple) seeded with Caco-2 cells (dark blue) on top. B) Vascular compartment (light yellow) with hydrogel (purple) and HUVEC cells (dark yellow) on top. C) Hepatic compartment (light red) with hydrogel (purple) and HepG2 cells (dark red) in the gel.

Acknowledgements: The authors would like to acknowledge the Danish National Research Foundation (DNRF122) and Villum Fonden (Grant No. 9301) for Intelligent Drug Delivery and Sensing Using Microcontainers and Nanomechanics (IDUN).

Learning objectives:Describe how each cell line can grow and the system can be assembled once the cell lines are fully grown.Evaluate how results on transport and metabolism can be obtained from sampling in the presented in vitro system.Discuss how the presented in vitro system can be used to obtain metabolic and transport related information relevant for their own research.

Presenter biography: Morten is a PhD student at Department of Health Technology at Technical University of Denmark. Before starting his PhD, Morten achieved a bachelor and master of science in Molecular Medicine from Aarhus University, Denmark.