(L-467) Human Hepatocyte Organoid Expansion for in vitro testing

Introduction::
The liver is a remarkable organ that performs over 500 different functions in the human body and can fully regenerate after injury or resection. Unfortunately, the primary parenchymal cell of the liver, the hepatocyte, rapidly dedifferentiates ex vivo, limiting human liver function and regeneration studies to expensive humanized mouse models or cancer cell lines that do not accurately represent healthy hepatocytes. Our lab has developed human hepatocyte organoids. We merge the advancing world of tissue engineering techniques with in vitro-grown human hepatocyte organoids to present a novel technology for liver regeneration. We are actively optimizing the new organoids to represent the phenotype and function of the liver accurately—further advancements include hepatocyte proliferation in vitro and hepatocyte functionality with long-term culture and purification techniques.




Materials and Methods::
Human hepatocyte organoids were generated from cryopreserved single cells by encapsulating them into matrigel domes. The growth and size of these organoids were monitored and quantified over time using minimum-intensity projections of brightfield z-stacks captured on a high-resolution widefield microscope. Human albumin levels were measured from the media samples obtained from the organoids to assess their functionality. Additionally, the organoids were harvested and immunostained with antibodies against Hoechst, cytokeratin 18 (CK18) and cytokeratin 19 (CK19), EdU. Our experiments demonstrate the successful in vitro growth of functional human hepatocyte organoids.




Results, Conclusions, and Discussions::
Results and Discussion: The present study demonstrates that human hepatocyte organoids can self-organize into densely packed spheroids with enlarged cells and a low nuclear to cytoplasmic ratio, like hepatocytes, in contrast to biliary organoids that typically form cystic structures. These organoids exhibit essential hepatic functionality following prolonged cultivation under diverse conditions (Figure 1B). Using human hepatocyte organoids as a substitute for conventional liver transplantation procedures may eliminate the need for invasive and costly interventions. Moreover, our findings suggest that the culture of human hepatocytes as organoids in vitro represents a superior method compared to conventional tissue culture techniques (Figure 1A). Our study underscores the potential of tissue engineering approaches utilizing human hepatocyte organoids and establishes a basis for further investigations in this field.



Conclusion: We have demonstrated the potential of our organoids to self-assemble into liver-like human tissues that mimic human physiological responses. We emphasize achieving sustained functionality and growth to enhance direct hepatocyte maturation. Our findings represent a significant milestone toward constructing clinically relevant human liver tissues and developing personalized medicine strategies. Future investigations will concentrate on directing the differentiation of cells to generate more intricate and fully functional engineered liver tissues in vivo for therapeutic applications.


Acknowledgements (Optional): :
This work was supported in part by the UW NIEHS sponsored




Environmental Pathology/Toxicology Training Program (EP/T) Training Grant (NIEHS




T32ES007032).




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