Entrapment of primary human hepatocytes in extracellular matrix gels for long term toxicity studies

Introduction: During the last decade pharmaceutical companies aimed to develop strategies to reduce animal tests for drug screenings. Many of these new methods are based on the in vitro culture of primary human hepatocytes (hHeps), which rapidly lose their function under conventional culture conditions (2D). 3D cultures, mandatory to obtain their function, cannot be used for drug-testings due to their huge need for cells. Therefore, aim of this work was to develop culture system for hHeps that allows down-scaling for screening purposes.Materials and methods: Collagen was isolated from rat tails. hHeps were isolated, by a two step collagenase perfusion technique, from liver sections according to the ethical guidelines of the MRI. Immediately after isolation hHeps were resuspended in collagen-gels and seeded into 96-well-plates. Viability was measured by resazurin conversion and life-dead-staining. CYP and hepatic transporter channels expression were observed by PCR. Results: Entrapment of cells into collagen-gels did not affect viability of the cells. The highest viability was observed with a cell density of 5.000 cells per µl gel. With this concentration the cells could interact with each other, as in the organotypic culture conditions of a bioreactor. Both hHeps cultures (2D and 3D) showed expression of CYP and hepatic transporter channels during the performance of PCR after 48 hours of culture. However, hHeps cultured in collagen-gels respond to Acetaminophen and Diclofenac stimulation with cellular damage with their viability reduced to one fifth. This is a difference to hHeps in conventional cultures which barely respond to these well known in vivo toxins. Conclusion: The present work demonstrates with a simple technique the importance of a 3D structure for hHeps to maintain their differentiated state for longer time permitting more accurate cell studies.

collagen - primary human Hepatocytes - toxicity