Application Note 7Long-term 3D Cultureof Human Primary Hepatocytes
in GrowDex®
Lauri Paasonen, Ph.D.1, Anne-Mari Moilanen, Ph.D.2, Minna Komu, Ph.D.2
1UPM
2Admescope
INTRODUCTION

Long-term primary hepatocyte cultures are needed for repeated dose drug toxicity
studies. It is well-known that hepatocytes in 2D culture lose their metabolic competence
as well as viability around 1-2 weeks in culture. In this study 35 days culture of humanprimary hepatocytes on GrowDex is described. The viability of the cells was examined bymeasuring the lactate dehydrogenase (LDH) from cell culture medium collected at time points
throughout the cultivation of the cells, and the cell functionality assessed by measuring thelevel of albumin secretion.

MATERIALS• Human primary hepatocytes (LiverPoolTM, BioreclamationIVT)• 96-well Ultra Low Attachment plate (Corning)• Hepatocyte cell culture medium (CP, Bioreclamation IVT) supplemented with 10ng/ml
hepatocyte growth factor (HGF) and 20 ng/ml epidermal growth factor (EGF)• CytoTox-ONE LDH assay kit (Promega)• Human Albumin ELISA kit (Abcam®)• GrowDex 1.5 % (UPM)

METHODS

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0.5% GrowDex was prepared by diluting the stock hydrogel with hepatocyte cellculture medium supplemented with 10 ng/ml HGF and 20 ng/ml EGF.
100 μl of 0.5% GrowDex was transferred to each well of a 96-well plate.
50,000 cells were plated on top of 0.5% GrowDex in 100 μl volume
The microplate was incubated at 37°C, 5% CO2 for a total of 35 d (7d pre-culture
to form spheroids + 28d spheroid culture). The medium was changed every second orevery third day. Starting from the first medium change, medium volume was increased
to 150 μl, and half of the amount was renewed to avoid the disruption of the sample.
Medium samples were taken from each microplate well and aliquots analysed for LDH
and albumin release.
Viability of the cells was examined by analysing the LDH release during the culture.
Functionality of the cells was studied by analysing albumin secretion during the culture
period.

RESULTS

LDH release from the cells indicates the viability of primary hepatocytes throughout theexperiment (Fig.1). Most of the cell death occurred during the 7 d pre-culture and during
the following 7 d culture period (spheroid days 1-7). Albumin secretion was used as abiomarker for liver-like function of the primary hepatocytes. The albumin secretion observed
throughout the experiment indicates good liver cell-like functionality (Fig.2).

Fig.1. The observed cytotoxicity
measured as LDH leakage from thespheroids shows the cell viability during
the 28 days spheroid culture.
7 day pre-culture = spheroid day 0.

Fig. 2. Albumin secretion from primary
hepatocytes during the 28 d spheroid
culture (after 7d pre-culture) onGrowDex indicates thefunctionality of the cells.
7 d pre-culture = spheroid day 0.

CONCLUSIONS

It was shown that primary hepatocytes can be cultured in GrowDex for 35 days. The cells
form 3D spheroid structures with good viability and liver cell-like functionality. Culturingof primary hepatocytes in GrowDex enables their use in extended applications, such as
repeated dose toxicity studies.
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