A perfusion incubator liver chip for 3D cell culture with application on chronic hepatotoxicity testing

Fang Yu,Ng Chan Way,Ciprian Iliescu,Li Huan,Wen Hao Tong,Rensheng Deng,Hanry Yu,Anik Islambadhan

doi:10.1038/s41598-017-13848-5

Abstract

Liver chips have been developed to recapitulate in vivo physiological conditions to enhance hepatocyte functions for assessing acute responses to drugs. To develop liver chips that can assess repeated dosing chronic hepatotoxicity, we need to ensure that hepatocyte functions be maintained at constant values over two weeks in stable culture conditions of sterility, temperature, pH, fluidic-flow of culture media and drugs. We have designed a perfusion-incubator-liver-chip (PIC) for 3D cell culture, that assures a tangential flow of the media over the spheroids culture. Rat hepatocyte spheroids constrained between a cover glass and a porous-ultrathin Parylene C membrane experienced optimal mass transfer and limited shear stress from the flowing culture media; maintained cell viability over 24 days. Hepatocyte functions were significantly improved and maintained at constant values (urea, albumin synthesis, and CYP450 enzyme activities) for 14 days. The chip act as an incubator, having 5% CO2 pressure-driven culture-media flow, on-chip heater and active debubbler. It operates in a biosafety cabinet, thus minimizing risk of contamination. The chronic drug response to repeated dosing of Diclofenac and Acetaminophen evaluated in PIC were more sensitive than the static culture control.

Highlights

Contamination of bacteria, mycoplasma or fungi is a common problem in long-term cell culture
To investigate whether the PIC can support chronic hepatotoxicity testing of drugs, we studied whether the hepatocyte functions can be maintained over 14 days
We found that hepatocytes cultured in spheroid model shows enhanced CYP1A2 activity; the amount of acetaminophen produced by collagen sandwich cultured hepatocytes was measured at 49.2ng/106 cells/90 min, while hepatocytes in PIC produced 195.1ng/106 cells/90 min on Day 8

Summary

Introduction

Contamination of bacteria, mycoplasma or fungi is a common problem in long-term cell culture. Two stages are critical in ensuring sterility in long-term cell culture: 1) initial chip assembly and cell seeding, 2) exposure to contaminants during the extended cell culture over weeks. For monitoring cell responses under a microscope, we frequently move the chips in and out of the incubator To minimize such frequent movements and connect/ disconnect in the non-sterile incubators, we integrate all the perfusion chip accessories into a single perfusion incubator chip (PIC) such that the entire cell culture process is performed in the sterile biosafety cabinet. In long-term microfluidic culture systems, dissociated gas from the culture media often results in bubble accumulation in the microfluidic channels. This leads to clogging and dead volumes. PIC addressed the various issues affecting the robustness and performance of liver chips for chronic drug testing

Methods

Results

Conclusion