Qianyue Zhang¹, Annie S.P. Yang¹, Joost M. Lambooij^1,2, Loes Verkade¹, Maarten E. Tushuizen², Arnaud Zaldumbide³, Noortje de Haan^4*, Bruno Guigas^1*

^1.Leiden University Center for Infectious Diseases, ^2.Department of Gastroenterology and Hepatology, ^3.Department of Cell & Chemical Biology,^4. Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden.

*: shared authorship

q.zhang@lumc.nl

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects one third of the world’s population and is characterized by a complex and progressive pathophysiology, ranging from simple steatosis to steatohepatitis (MASH), fibrosis and hepatocellular carcinoma. Emerging evidence suggests that glycans, which are complex carbohydrates attached to both proteins and lipids, may play a role in MASH development by promoting liver inflammation. Primary human hepatocytes (PHHs) offer a physiologically relevant model for studying glycan-mediated mechanisms underlying MASLD-MASH progression. However, it remains unclear whether PHHs maintain a stable glycome during long-term in vitro culture, and whether their glycosylation can be modulated to investigate the role of specific glycan types in the crosstalk between hepatocytes and immune cells. PHHs were isolated from human liver tissue and cultured using a specific media preventing cell de-differentiation. Two distinct culture models were used: 2-dimentional (2D) monolayer and 3-dimentional (3D) spheroids. Glycosylation was modulated using specific chemical inhibitors of the cellular machinery. N- and O-glycan profiles were analyzed using LC-MS/MS-based glycomics. When compared to freshly isolated cells, PHH in 2D culture exhibited stable N-glycan and O-GalNAc profiles for at least 5 days. In contrast, O-GlcNAc diversity levels declined significantly during prolonged culture. Interestingly, treatment with various inhibitors targeting key enzymes involved in sialylation, fucosylation and complex branching formation effectively and stably altered glycan profiles in both 2D PHHs and 3D spheroids, confirming the feasibility of glycome modulation in primary cells. Of note, targeted glycome modulation using genetic tools in order to overexpress or knockdown key enzymes involved in the glycosylation machinery is also currently explored. Altogether, PHHs represent a robust and versatile in vitro model for investigating the role of glycans in intercellular crosstalk and MASLD-MASH progression.