Background The drug development process is widely hampered by the lack of human models that recapitulate liver functionality and efficiently predict toxicity of new chemical compounds. Moreover, liver failure is a global medical problem, with transplantation being the only effective treatment currently available. The bipotent liver progenitor cell line HepaRG can be differentiated into cholangiocyte and hepatocyte-like cells that express major functions of mature hepatocytes, representing a valuable tool to model hepatic function [1]. Current two-dimensional (2D) protocols for the differentiation into mature hepatocyte-like cells fail to recapitulate the complex cell-cell interactions, which are crucial for maintaining polarity and inherent mature hepatic functionality. Herein, we present a three-dimensional (3D) strategy for the culture of HepaRG cells based on the encapsulation of aggregates. The effect of matrix stiffness on expansion and differentiation was evaluated through encapsulation with different concentrations of alginate (1.1% and 2%). Further characterization of the hepatic features will reveal the extent of the hepatic functionality of the generated spheroids. Materials and methods HepaRG cells were routinely propagated in static conditions as previously described [2]. Briefly, culture medium Williams E was supplemented with 1% (v/v) Glutamax, 1% (v/v) pen/strep, 5 μ g/ml insulin and 50 μ M hydrocortisone hemissuccinate and 10% (v/v) FBS and cultures were maintained at 37 ° C, 5% CO2 .S pinner vessels with ball impeller (Wheaton) were inoculated with inoculums ranging from 5 to 8 × 10 5 cell/mL and an agitation ranging from 35 to 45 rpm to attain the desired aggregation conditions. Aggregate size was determined by measuring Ferret’s diameter using the Image J software (NIH). After 3 days of aggregation, spheroids were encapsulated in 1.1% and 2% (w/v) of Ultra Pure MVG alginate (UP MVG NovaMatrix, Pronova Biomedical) in NaCl 0.9% (w/v) solution. Encapsulation was performed in an electrostatically driven microencapsulation unit VarV1 (Nisco) and cultures were maintained for 14 days in stirred culture conditions. Viability was determined by the double stain viability test - alginate beads were collected from stirred cultures, incubated with fluorescein diacetate (10 μg/ mL) and TO-PRO3 ® (1 μM) and observed on a fluores