Long-Term Expansion of Functional Human Pluripotent Stem Cell-Derived Hepatic Organoids

ABSTRACT

A human cell-based liver model capable of long-term expansion and mature hepatic function is a fundamental requirement for pre-clinical drug development. We previously established self-renewing and functionally mature human pluripotent stem cell-derived liver organoids as an alternate to primary human hepatocytes. In this study, we tested long-term prolonged culture of organoids to increase their maturity. Organoid growing at the edge of Matrigel started to deteriorate two weeks after culturing, and the expression levels of the functional mature hepatocyte marker ALB were decreased at four weeks of culture. Replating the organoids weekly at a 1：2 ratio in fresh Matrigel, resulted in healthier morphology with a thicker layer compared to organoids maintained on the same Matrigel and significantly increased ALB expression until three weeks, although, it decreased sharply at four weeks. The levels of the fetal hepatocyte marker AFP were considerably increased in long-term cultures of organoids. Therefore, we performed serial passaging of organoids, whereby they were mechanically split weekly at a 1：3∼1：5 ratio in fresh Matrigel. The organoids expanded so far over passage 55, or 1 year, without growth retardation and maintained a normal karyotype after long-term cryopreservation. Differentiation potentials were maintained or increased after long-term passaging, while AFP expression considerably decreased after passaging. Therefore, these data demonstrate that organoids can be exponentially expanded by serial passaging, while maintaining long-term functional maturation potential. Thus, hepatic organoids can be a practical and renewable cell source for human cell-based and personalized 3D liver models.