Differentiation of liver progenitor cell line to functional organotypic cultures in 3D nanofibrillar cellulose and hyaluronan-gelatin hydrogels

Melina M Malinen,Liisa K Kanninen,Anne Corlu,Helena M Isoniemi,Yan-Ru Lou,Marjo L Yliperttula,Arto O Urtti

doi:10.1016/j.biomaterials.2014.03.020

Abstract

Physiologically relevant hepatic cell culture models must be based on three-dimensional (3D) culture of human cells. However, liver cells are generally cultured in two-dimensional (2D) format that deviates from the normal in vivo morphology. We generated 3D culture environment for HepaRG liver progenitor cells using wood-derived nanofibrillar cellulose (NFC) and hyaluronan-gelatin (HG) hydrogels. Culture of undifferentiated HepaRG cells in NFC and HG hydrogels induced formation of 3D multicellular spheroids with apicobasal polarity and functional bile canaliculi-like structures, structural hallmarks of the liver tissue. Furthermore, hepatobiliary drug transporters, MRP2 and MDR1, were localized on the canalicular membranes of the spheroids and vectorial transport of fluorescent probes towards the biliary compartment was demonstrated. Cell culture in 3D hydrogel supported the mRNA expression of hepatocyte markers (albumin and CYP3A4), and metabolic activity of CYP3A4 in the HepaRG cell cultures. On the contrary, the 3D hydrogel cultures with pre-differentiated HepaRG cells showed decreasing expression of albumin and CYP3A4 transcripts as well as CYP3A4 activity. It is concluded that NFC and HG hydrogels expedite the hepatic differentiation of HepaRG liver progenitor cells better than the standard 2D culture environment. This was shown as improved cell morphology, expression and localization of hepatic markers, metabolic activity and vectorial transport. The NFC and HG hydrogels are promising materials for hepatic cell culture and tissue engineering.

Highlights

We investigated hydrogels of native wood-derived nanofibrillar cellulose (NFC) and hyaluronan-gelatin (HG) as supporting materials in the 3D culture of HepaRG liver progenitor cells
The size of the spheroids exceeded the size of a single HepaRG cell already during the first day (Fig. 1A and B), and they reached the full size within a week
The plasticity of HepaRG liver cells was exploited to evaluate the impact of naturally derived hydrogels, native nanofibrillar cellulose (NFC) and hyaluronan-gelatin (HG), on the phenotype of both undifferentiated HepaRG progenitors and differentiated HepaRG cells

Summary

Introduction

Pharmaceutical industry, regulatory authorities, and academic investigators need liver cell cultures to predict and estimate metabolism, excretion and toxicity of drugs and other chemicals in the human liver. Due to the inter-species differences animals and animal cells lead frequently to misleading, and sometimes hazardous, estimates of pharmacokinetics and toxicity in humans. Human liver microsomes are used to study xenobiotic metabolism, but the microsomes do not have drug transporters or transcription machinery. This limits seriously their usefulness in pharmacokinetics and toxicology.

Methods

Results

Discussion

Conclusion