Heterotypic spheroids as a strategy for 3D culture of cryopreserved primary human hepatocytes in stirred-tank systems.

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Primary human hepatocytes (PHHs) are the preferred cell source to address liver function. Despite originating from the native tissue, one of the bottlenecks when using primary material is the donor-to-donor variability. Cryopreserved PHHs offer a high number of cells from the same donor and standardization of cell isolation and cryopreservation procedures, mitigating some of the inter-donor variability. Still, PHHs from different commercial sources present variability in vitro in several parameters, including viability post-thawing, plating capacity, aggregation potential and culture longevity. Here we combine stirred-tank culture systems, which allow robust aggregation processes, and co-culture approaches with the HepaRG cell line to generate spheroids from cryopreserved PHHs. By employing small-scale stirred-tank culture systems we could cope with the scarce availability and high cost of primary material. In the optimized co-culture conditions we could generate PHH:HepaRG spheroids from 12 donors acquired from 4 different commercial sources. All PHHs showed similar aggregation profiles, forming small compact heterotypic spheroids as early as 3 days in co-culture and were maintained for at least 5 weeks in culture. The heterotypic spheroids maintained the hepatocyte polarization and identity and showed metabolization capacity for 5 main phase I metabolizing enzymes, namely CYP3A4, CYP2C9, CYP1A2, CYP2D6, and CYP2C8. Moreover, the heterotypic spheroids showed the capacity to metabolize a novel compound under clinical development, showing their potential to be employed in drug discovery applications. Overall, we present a robust aggregation strategy for cryopreserved PHHs from different suppliers, applicable for pharmacological and toxicological in vitro research.