Hepatic trans-differentiation of pancreatic hepatocyte-progenitor cells (B-13) in a 3D high-density culture system

A rat pancreatic progenitor cell line (B-13) is of interest since it has been shown to trans-differentiate into functional hepatocyte-like cells (B-13/H) when treated with glucocorticoids.

Since the use of primary hepatocytes for clinical application in liver support systems is problematic due to their limited availability, B-13 cells are under investigation within the EU project d-LIVER. Further, there is a need for a culturing system, which enables sufficient cell numbers to be generated in a closed environment to support liver disease patients. Our group used an experimental bioreactor system offering dynamic 3D culture conditions to examine B-13 trans-differentiation and B-13/H functionality. Additionally, the project aims to integrate different types of sensors to monitor cell culture in the bioreactor.

Two approaches were investigated. Approach 1: B-13 cells were inoculated and trans-differentiated in the bioreactor for 15 days; Approach 2: trans-differentiated B-13/H cells were maintained over 15 days in bioreactors. The efficacy of trans-differentiation and the cell performance were assessed by determination of liver-specific marker expression and functional parameters, including glucose, lactate, urea, albumin and CYP activity testing (ethoxyresorufine metabolism). Optical pH and oxygen sensors were used to monitor and control culture conditions.

The experiments showed successful growth and trans-differentiation of B-13 cells in bioreactors. Both approaches showed similar performances for urea, CPS-1 and albumin as hepatocyte specific markers with slightly higher values in approach 2. Different CYP isoforms were expressed in both approaches and CYP activity testing resulted in constantly increasing formation rates in approach 1, whereas approach 2 showed four times higher values on day 8 compared to approach 1 but declined till day 15 of culture. Amylase release characteristic for pancreas exocrine cells increased over the first week and declined slowly thereafter in approach 1. In approach 2 amylase values were lower and declined almost to zero. RT PCR and Western blotting analysis showed similar performance. Oxygen concentrations in the perfusate indicated increasing oxygen consumption in approach 1. Online monitoring of the pH allowed to support constant culture conditions. With respect to clinical application in extracorporeal liver support, first successful test runs using up-scaled versions of the technology were undertaken.

The results show that the B-13 cell line has a high potential. These experiments showed that the bioreactor system is able to provide a closed environment suitable for culturing high cell numbers. If a cell line of human origin with similar characteristics as B-13 could be generated, this could provide an attractive cell source for bio-artificial liver support.

The project received funding from the European Union's Seventh Framework Programme (FP7/2007 – 2013) under grant agreement no. 287596.